Chapter 1: Review of literature

1. Introduction

1.1. Origin of research problem

Several advancements made in the field of medical sciences like, the

introduction of antibiotics, newer chemotherapeutic option in treatment of

cancer organ transplantation were intended to make tremendous impact on

general public health. Paradoxically, some of these stipulated opportunity to

infections, mainly those of fungal origin to establish themselves in the host. In

fact, human subjects have become more susceptible to a range of opportunistic

infections for last two or three decades. Besides HIV infection, individuals who

have undergone organ transplantation or remain on treatment with anticancer

drugs are prime targets of such infections (Vivier et. al. 2011). Similarly, patients

receiving broad-spectrum antibacterial therapy or those subjected to invasive

procedures are also highly prone to infection by fungal pathogens (Church et. al.

2006)

The association of the most of the opportunistic infections in patients with AIDS

background significantly exacerbated the situation both in developing as well as

developed countries. For example, candidiasis occurs in as many as 90% of AIDS

patients (Buchacz et. al. 2010). In fact, the incidence of invasive candidiasis in

such patients has increased several folds to become among the first five most

common blood culture isolates including it. The recents upsurge in incidence of

candidiasis certainly needs serious attention, since india is poised to register the

highest number of HIV infection cases in the world (Leena et. al. 2005).

Like most of the other microbes, the fungal pathogens have also found the ways

of thwarting escape mechanism against various antifungal agents (Duffy et. al.

2003,). Already vexatious with the availability of limited number of antifungal

agents, the recent emergence of drug-resistant candidiasis infections against the

gold standard Amp B leaves little choice but to opt either for its appropriates

replacement by some potent substitute or to rely on an altogether different

prophylactic strategy by developing vaccines against the deadly disease.

Efforts will be made to evaluate the fully biocompatible and biodegradable

delivery system made up of fibrin microsphere for sustained delivery of anti

fungal drugs/immunomodulators for efficient elimination of the fungal

pathogens from systemic circulation. Besides, a novel strategy for immunization

of animals against pathogenic Candidiasis spp is also envisaged in the proposed

study. Candida cytosolic proteins with immunogenic potential will be trapped in

the fibrin network directly or after their encapsulation in appropriate liposome.

Finally, the potential of the vaccine will be evaluated against experimental murine

candidiasis in balb/c mice.

1.2. Interdisciplinary relevance

The function of the immune system becomes more pertinent in case of

opportunistic fungal pathogens that targets immunodebilitant subjects mainly.

The pathogens that inflict such patients do not respond to conventional

chemotherapy presumably because of collapsing physiological conditions as well

as fragile immune system of the host. Concomitant delivery of anti-fungal agents

and immunomodulators, using delivery systems that facilitate specific targeting to

the pathogen harbouring immune cells (macrophages and neutrophils) is expected

therefore to be beneficial in eliminating the pathogens from such host.

The proposed study envisages the development of a new strategy for imparting

protection against highly pathogenic fungal disease viz. candidiasis. We wish to

attempt the development of sustained delivery system using a combination of

immunomodulators tufsin/picroliv along with antifungal drugs and incorporating

them in fully physiological and biodegradable fibrin microspheres. A parallel

study involving incorporation of C. Albicans immunogenic proteins

(cytosolic/membrane proteins) in fibrin microspheres as candidate vaccine will be

undertaken. Both free as well liposome encapsulated antigen will be incorporated

in the fibrin microspheres and evaluated for its potential to impart protection

against experimental murine candidiasis.

1.3. Nanotechnology in medicine (Nanomedicine)

Nanotechnology is the science and engineering involved in the design, synthesis,

characterization, and application of materials and devices whose smallest

functional organization in at least one dimension is on the nanometer scale or one

billionth of a meter (Moghimi et al. 2005). Structures constructed using

nanotechnology, given the general term ‘nanoparticles’, are being increasingly

used in various applications ranging from industry and diagnostics to medicine.

One of the most promising applications of nanotechnology is in the realm of

medicine and involves the development of nanoscale tools and machines

designed to monitor health, deliver drugs, cure diseases, and repair damaged

tissues, at the molecular level in cells and organelles. The National Institutes of

Health, USA has coined the term ‘nanomedicine’ to refer to the mushrooming

innovations in nanotechnology that find applications in diagnosis, treatment,

monitoring, and manipulation of biological systems. In other words,

nanomedicine is the medical application of nanotechnology (Freitas 1999).

Initially nanotechnology was used in the form of passive structures (such as in

cosmetics), then as active structures in the form of new, more effective delivery

systems. Research in nanomedicine is chiefly focused on understanding the issues

related to toxicity, environmental impact of nanoscale materials and the rational

delivery and targeting of pharmaceutical, therapeutic, and diagnostic agents with

nano-sized particles.

Nanomedicine is now within the realm of reality starting with nanodiagnostics

and drug delivery. In the last 30 years there has been an explosive growth of

nanotechnology in the purview of medicine which has ushered in challenging

innovations in pharmacology. These discoveries have been highly instrumental in

revolutionizing the temporal and spatial site-specific delivery of biologically active

compounds (Couvreur and Vauthier 2006). Nanoengineered materials can be

designed to exhibit very specific and controlled bulk chemical and physical

properties as a result of the control over their molecular synthesis and assembly.

1.3.1. An introduction to nanostructural carrier systems

Therapeutic and preventive strategies, in order to be successful, depend not only

on the appropriate choice of the active principle but to a large extent on the use

of an appropriate delivery system. This holds true for difficult to deliver

compounds; in particular drugs having poor solubility (hydrophobic) and poor

permeability. In fact, the quest to find the ideal therapeutic strategy will continue

till a drug with maximum efficacy and no side effects is found. Many of the

present day drugs are plagued by narrow therapeutic window, and their effective

use is limited by problems of dose limiting toxic effect. These bottlenecks can be

circumvented and the therapeutic effectiveness of existing drugs can be improved

through the use of an appropriately designed delivery system which can modify

the distribution of the drug in the body by targeting it to desired site and by

controlled release. In the present scenario these ideas are being brought to reality

through the usage of nanotechnology to develop nanoparticulate delivery systems

for drug and antigen delivery.

Nanotechnology is being increasingly used by the pharmaceutical and

biotechnology industries and nanoparticle-based delivery strategies are beginning

to make a significant impact on global pharmaceutical planning and marketing

(market intelligence and lifecycle management). Nanotechnology will be applied

at all stages of drug development–from formulations for optimal delivery to

diagnostics applications in clinical trials. Many of the assays based on

nanotechnology will enable high-throughput screening. The most important

pharmaceutical applications of nano-particles are in drug delivery. Besides

offering a solution to solubility problems, nanoparticulate systems provide

efficient means of intracellular delivery.

The age of nanostructural delivery systems began with the development of

liposomes by Bangham (Bangham et al. 1965). Since then a large number of

nanoparticulate systems have been developed and as of this day the sheer number

and types of nanoparticulate structures that have been already developed or being

researched upon is tremendous. The concept of the 'magic bullet' proposed a

century ago by Nobel laureate Paul Ehrlich came to reality with the recent

appearance of several approved forms of drug-targeting systems for the treatment

of certain cancer and serious infectious diseases. A recent example is the launch

of the FDA-approved breast-cancer drug Abraxane (American Pharmaceuticals,

U.S.A). Furthermore many other nanomedicinal formulations for treatment of

skin disorders and infections are in the pipeline.

1.4. Drug delivery system

The primary objective of a drug delivery system is to release therapeutics at a

predetermined anatomical site and maintain circulating drug concentration within

a therapeutic band for the required duration. Drugs administered orally,

parenterally or by other means enter the blood stream and gain access to almost

all tissues of the body. The drugs disseminate from the blood to most tissues by

crossing the endothelial barriers or by entering through the endothelial gaps in

tissues with leaky vasculature. This evidently leads to unwanted delivery to non-

target tissues, necessitating high dosages and at times results in serious side

effects. Controlled drug delivery systems have therefore acquired seminal

significance in pharmaceutical research. Drug delivery systems offer numerous

advantages including minimization of peak-valley fluctuations, reduction in the

drug dosage, lowering of dosage frequency, decrease in side effects and improved

patience compliance.

The earliest drug delivery systems investigated were polymer carriers for

accomplishing spatiotemporal release of therapeutics, both pulsatile dose delivery

products and implanted reservoir systems. Various polymers including cellulose

derivatives, poly(ethylene glycol) (PEG) and poly (N-vinyl pyrrolidone) are in use

for over five decades and their early applications include use in drug sprays, dip

coating and encapsulation (Liechty et al., 2010). A large number of other natural

and synthetic drug delivery systems have also been investigated with varying

success. From the drug delivery perspective, polymer devices can be classified

into i. Diffusion controlled (monolithic devices) ii. Solvent activated (swelling or

osmotically controlled devices) iii. Chemically controlled (biodegradable) and iv.

Triggered systems (Liechty et al., 2010).

1.5. Use of Nanoparticulate delivery systems in Nanomedicine

The use of nanotechnology approaches to drug delivery focuses on developing

nanoscale particles to maximize bioavailability of a drug both at specific places in

the body and over a period of time. Nanoparticulate systems can be designed at

the molecular level to favorably interact with cells and tissues. The small size of

these particles and the capacity to engineer their surface with synthetic polymers

or appropriate ligands allow nanoparticulate carriers to be targeted to specific

cells, tissues and extracellular elements in the body. These approaches are aimed

at enhancing detection sensitivity in medical imaging, improving therapeutic

effectiveness, and decrease side effects. Such nanoscale engineering may also

enable specialized structures that can be activated by changes in the

environmental pH, chemical stimuli, by the application of a rapidly oscillating

magnetic field, or by application of an external heat source (Moghimi et al. 2001,

Panyam et al. 2002). Such modifications offer control over particle integrity,

delivery rates, and the location of load release, for example within specific

organelles. These carriers can also be made to target cell receptors and

simultaneously deliver drugs and biological sensors (Quintana et al. 2002). These

strategies are applied to deliver drugs, antigens, genetic materials, and diagnostic

agents (encapsulated, covalently attached, or adsorbed to such nanocarriers)

selectively to specific locations via intravenous and interstitial routes of

administration (Allen and Cullis 2004, Moghimi et al. 2001, Sahoo and

Labhasetwar 2003, Sudimack and Lee 2000). These approaches can easily

overcome drug solubility issues, particularly with the view that large proportions

of new drug candidates emerging from high-throughput drug screening initiatives

are water insoluble. In the case of drug delivery these novel concepts have already

been shown to cause fewer side effects and be more effective than traditional

therapies in terms of controlled delivery and specific targeting of tissues and

organs.

Apart from drug molecules, nanoparticulate carriers can be useful to stabilize

immunogens and protein drugs, such as various cytokines, against enzymatic and

chemical degradations and to increase their bioavailability. Such systems can also

be employed in the targeting of nucleic acids in gene therapy. It has also been

proved that nanoparticulate systems provide a useful tool in the transdermal and

pulmonary drug administration. In cancer chemotherapy, these nanostructural

carriers can target drugs to cancer cells and limit the side effects of many overtly

cytotoxic anticancer drugs. Moreover many new antiviral drugs and

immunosuppressive agents, which require long-term, even lifelong,

administration, benefit much from being packaged in nanoparticulate systems.

Nanoparticulate delivery systems play a critical role in the development of

treatment for various life-threatening diseases due to the versatility, control, and

future potential of the design of these systems.

1.6. Types of Nanoparticulate delivery systems

A vast array of different types of nanoparticles, of different shapes and sizes and

composed of an assortment materials, and with various chemical and surface

properties, have already been constructed. These nanoparticulate systems

comprise a variety of constructs: nanospheres, nanocapsules, lipid nanoparticles,

microemulsions, macromolecular complexes, ceramic nanoparticles and vesicular

carriers like liposomes and niosomes. Progress in nanotechnology is very dynamic

and new systems continue to be developed. Some more common general classes

of nanoparticulate delivery systems and their functions are listed below:

1) Bucky balls and Carbon tubes

These are carbon based lattice like, potentially porous molecules and are grouped

as fullerene class of structures. Bucky balls are spherical while carbon tubes are

cylindrical. In nanomedicine, carbon tubes have been used as carriers for

vaccines, drugs and other molecules.

2) Nanoshells

Nanoshells also called core-shells are few nanometers thick spherical cores

composed of a particular compound surrounded by a shell or outer coating of

another compound. Their ability to absorb at biologically useful wavelengths,

depending on the shell thickness justifies thier use in nanomedicine. For example

silica is used to form the core and some sticky compound to adhere gold particles

as the outer shell. Such nanoshells can be injected into a tumor, followed by

application of radiation whereby nanoshells heat up enough to kill the tumor

cells.

3) Dendrimers

Dendrimers are highly branched structures having hook-like structures on

their surfaces that can be used to attach cell-identification tags, fluorescent dyes,

enzymes and other molecules. Dendrimers are of two basic structural types-

a) Globular with branches radiating from a central core.

b) A series of highly branched polymers with no central core.

Nanomedical applications for dendrimers are many and include nanoscale

catalysts and reaction vessels, micelle mimics, imaging agents and chemical

sensors, and agents for delivering drugs or genes into cells.

4) Quantum dots

Also known as nanocrystals, quantum dots behave as semiconductors emitting

light in the entire visible light spectrum. These nanostructures confine conduction

band electrons, valence band holes, or excitons in all three spatial directions.

Examples of quantum dots are semiconductor nanocrystals and core-shell

nanocrystals, where there is an interface between different semiconductor

materials. They are used for cell labelling and imaging, particularly in cancer

imaging studies.

5) Superparamagnetic nanoparticles

These nanoparticles are attracted to a magnetic field but do not retain residual

magnetism after the field is removed. Nanoparticles of iron oxide with diameters

in the 5-100 nm range have been used for selective magnetic bioseparations.

Typical techniques involve coating the particles with antibodies to cell-specific

antigens, for separation from the surrounding matrix. Used in membrane

transport studies, superparamagenetic iron oxide nanoparticles (SPION) are

applied for drug delivery and gene transfection. Targeted delivery of drugs,

bioactive molecules or DNA vectors is dependent on the application of an

external magnetic force that accelerates and directs their progress towards the

target tissue. They are also useful as MRI contrast agents.

6) Nanorods

Nanorods are usually 1-100 nm in length, and are most often made from

semiconducting materials like small cylinders of silicon, gold or inorganic

phosphate. They find use as imaging and contrast agents.

7) Cross-linked micelles

Micelles are self-forming particles that are prepared from individual surfactant

molecules that have a water-loving and a water-hating component. The surfactant

molecules orientate themselves to form spheres where the water-loving

component is on the outside and in contact with the water. The water-hating

component remains inside the sphere, preferring to interact with itself rather than

with the water. This hydrophobic core makes them ideal carrier systems for

water-hating small molecules. Crosslinked micelles are similar. However, they are

held in their configuration much more tightly and are therefore more robust.

Typically prepared in the size range 5–50nm, they offer more protection than

conventional micelle systems do. Size and physico-chemical properties can be

manipulated by varying the composition or molecular weight of the surfactant.

8) Solid lipid nanospheres

Prepared by homogenisation of a melted lipid in an aqueous surfactant solution,

these colloidal carrier systems have good bioavailability, stability and low toxicity.

The inclusion of an active or other small molecule does not affect the particle

stability or particle size, which can be between 10nm–2mm. The matrix can

release the incorporated active/chemical entity either by heating the matrix or by

rubbing the nanospheres on to a material such as human skin or fabric.

9) Nanocapsules

Liquid-filled capsules can be prepared with an aqueous or organic core to enable

solubilization, stability and protection of compounds in a cross-linked polymer

matrix. These compounds can be released by the rupture of the nanocapsule

membrane in a burst profile.

10) Microgels

Materials that respond to subtle changes in external stimuli may be described as

“intelligent” or “smart”, for example, colloidal microgels. Acting like

microsponges, when held in dispersion they undergo a conformational change as

a function of temperature, pH, ionic strength and solvency. They can exhibit as

much as a fourfold change in particle volume as they shrink and swell in response

to environmental conditions. These microsponges, with their responsive porous

network, can be used for a variety of applications, including drug adsorption

(poorly soluble drugs) and drug protection (sensitive to pH extremes), as well as

drug delivery. These nano-particulate systems provide a vehicle to deliver a

number of actives or volatile components. The range of commercial applications

of these materials is expanding, as is this area of research.

11) Vesicular systems

Vesicular systems are highly ordered assemblies of one or several concentric

bilayers that are formed when certain amphiphilic building blocks are dispersed in

water. The commonly used vesicular systems include liposomes, niosomes,

transferosomes, pharmacosomes, ISCOMS, etc. These vesicles can be formed

from a diverse range of amphiphilic building blocks. Vesicular carriers are used

extensively in the pharmaceutical and cosmetic industries because of their

capacity for entering into and breaking down inside cells. Liposomes have the

distinction of being the first engineered nanoparticles used for drug delivery.

However their affinity to fuse together in aqueous environments and release

entrapped material has lead to devising of measures to stabilize them or replacing

them with alternative nanoparticles made from more stable materials. Figure 1

displays comparative size ranges of various nanoparticulate carriers. Figure 2 is a

diagrammatic representation of various nanoparticles used in drug delivery.

Figure 1. Size ranges of various nano-particles

Figure 2. Various types of nanoparticles

1.7. Introduction to vesicular carrier systems

A wide variety of lipids and surfactants can be used to prepare vesicular carriers

(Sinico et al., 2009). The composition of the vesicles influences their physico-

chemical characteristics such as, size, charge, thermodynamic phase, lamellarity

and bilayer elasticity. These physico-chemical characteristics have a significant

effect on the behaviour of the vesicles and hence on their effectiveness as a drug

delivery system.

Vesicular systems have been able to address the problems of drug insolubility,

instability, and rapid degradation. These systems delay elimination of rapidly

metabolizable drugs, and function as sustained release systems. Encapsulation of

a drug in vesicular structures can be predicted to prolong the existence of the

drug in systemic circulation, and reduce the toxicity if selective uptake can be

achieved (Biju et al., 2006). Vesicular drug delivery reduces the cost of therapy by

improved bioavailability of medication, especially in case of poorly soluble drugs.

They can incorporate both hydrophilic and lipophilic drugs. Hydrophilic drugs

can be entrapped into the internal aqueous compartment, whereas amphiphilic,

lipophilic and charged hydrophilic drugs can be associated with the vesicle bilayer

by hydrophobic and/or electrostatic interactions (Martin et al., 1992).

1.7.1. Structure and nomenclature of Vesicular systems

The basic structure of vesicular systems is exemplified by liposomes and

niosomes. These vesicles comprise a bilayered membrane enclosing an aqueous

core. The walls of the vesicles consist of amphiphilic molecules in a bilayer

conformation. In an excess of water these amphiphilic molecules can form one

(unilamellar vesicles) or more (multilamellar vesicles) concentric bilayers.

Hydrophilic drugs can be entrapped into the internal aqueous compartment,

whereas amphiphilic, lipophilic and charged hydrophilic drugs can be associated

with the vesicle bilayer by hydrophobic and/or electrostatic interactions. The

thickness of the membrane (phospholipid bilayer) measures approximately 5 to 6

nm. Liposomes and niosomes can be categorized on the basis of their size

(shown in Table 1) which is also the most widely accepted terminology. Figure 3

is a representative diagram showing relative sizes of these vesicles.

Table 1

Types of vesicular systems in terms of size

Type Specifications Diameter

MLV Multilamellar large vesicles >0.5 m

MVV Multivesicular vesicles 0.1-20 m

OLV Oligolamellar vesicles 0.1-1.0 m

SUV Small unilamellar vesicles 20-100 nm

LUV Large unilamellar vesicles > 100 nm

GUV Giant unilamellar vesicles > 1 m

Figure 3. Relative sizes of vesicular systems

1.7.2. Fate of vesicles inside host body

When vesicles such as liposomes and niosomes are administered into host they

may either disintegrate in the bloodstream or may wander in the systemic

circulation until picked up, mostly by macrophages. The endothelial lining of

healthy blood vessels usually prevents escape of these vesicles from the

circulation. In case inflammation occurs at a site, the endothelium becomes more

permeable thus allowing extravasations of small vesicles. This leads to their rapid

clearance from the blood circulation and their capture by the organs of reticulo-

endothelial system (Vollmar, 2009), corresponding to the tissue distribution

pattern of some pathogenic microorganisms responsible for intracellular

infections. Passive site-specific drug targeting with vesicles, as it is called, may

facilitate lowering of the drug dosage relative to the amount of the free drug used,

thereby reducing the potential toxic side effects exerted by higher doses of the

drugs. However for some purposes fast clearance is undesirable. To this end

‘stealth’ liposomes or niosomes can be prepared by coating their surfaces with

polyethylene glycol (PEG) whereupon the resulting vesicles are endowed with

long half-life in blood circulation and may act as a reservoir for sustained release

of the therapeutic agents for longer duration. Moreover, antibodies or other

ligands may be covalently attached to the surface of vesicles allowing uptake of

the vesicles by specific tissues or cells.

The applications of vesicles in drug delivery are based on physicochemical and

colloidal characterization such as composition, size and loading efficiency and the

stability of the carrier, as well as their biological interactions with the cells. A

major interaction is lipid exchange whereby liposomal lipids are exchanged with

the lipids of various cell membranes. This depends on the mechanical stability of

the bilayer and can be reduced by the addition of cholesterol (which gives rise to

greatly improved mechanical properties, such as increased stretching elastic

modulus, resulting in stronger membranes and reduced permeability). The second

major interaction is adsorption onto cells, which occurs when the attractive forces

(ele ctrostatic, electrodynamic, Van der waals, hydrophobic interaction and

hydrogen bonding etc.) exceed repulsive forces (electrostatic, steric, hydration,

ondulation, protrusion etc.). Adsorption onto phagocytic cells is normally

followed by endocytosis or rarely, by fusion. Fusogenic vesicles mimic the way by

which several viruses (HIV, Sendai virus) bind and merge with cell membranes at

neutral pH and subsequently release their content into the cytoplasm.

In the perspective of treating intracellular infections (where the pathogen resides

inside host cells, mainly the macrophages), once the drug-carrying vesicle is

endocytosed by the macrophage, the acidic environment of the phagolysosome

ruptures the membrane and releases the drug therein. The released drug then also

reaches the cytosol. This delivery of drug inside the pathogen-harboring

macrophage significantly increases the chance of the drug interacting directly with

the pathogen leading to efficient killing.

1.7.3. Vesicular carriers in treatment of intracellular infections:

Escheriosomes

Vesicular carriers are regularly used to deliver drugs, immunogens, genetic

materials and diagnostics agents to specific desired locations. The most

commonly used vesicles are liposomes and niosomes that are composed of

phospholipids and non-ionic surfactants respectively. Initial and most extensive

work has been on lipid vesicles (liposomes) which have been utilized in

immunology, membrane biology, diagnostic techniques, and most recently,

genetic engineering (Singh, 2011; Copland 2005; Semple, 2005)

Several studies have shown that Escheriosomes have been able to overcome the

major problem in treating intracellular infections; that is low lipid permeation of

drugs into cells. Phagocytic uptake of drug-loaded vesicular delivery system from

the systemic circulation provides an efficient method for delivery of drug directly

to the site of infection, leading to reduction of drug toxicity with much reduced

adverse effects.

1.8. Liposomes/Escheriosomes

Liposomes are vesicular structures of colloidal nature assembled when

amphipathic lipids are dispersed in water. The vesicular structures can be thought

of as artificial lipid bilayer membranes enclosing aqueous inner core. These were

first described by Alec D. Bangham in 1965 (Bangham et al., 1965).

Escheriosomes emulate biological membranes that form the ubiquitous delimiting

structures that surround and compartmentalize all cells and organelles. Infect

liposomes were initially used as models for studying dynamic properties such as

fluidity, phase transitions etc. of biomembranes. However, potential of these

vesicles was soon realized as versatile models, reagents and tools in various

scientific disciplines including, biology, biochemistry, and biophysics. Over the

years lipid vesicles have evolved successfully as vehicles for controlled delivery.

E.coli membrane comprises a great majority of anionic phospholipids that play a

pivotal role in membrane-membrane fusion (Syed et al. 2003). In the present

study, we report that the liposomes made of E.coli lipid vesicles (escheriosomes)

readily fuse with the plasma membrane, and successfully deliver the encapsulated

antigen to cytosol of the target cells. In vivo administration of escheriosomes

encapsulated antigen (cAg) induced antigen specific strong CTL responses in the

immunized mice. In contrast, the antigen encapsulated in egg PC-liposomes, in a

manner similar to the antigen-IFA emulsion, had limited access to the cytosolic

pathway of MHC-I dependent antigen presentation and failed to generate antigen

specific cell mediated immune response.

1.9. Fibrin beads

Fibrin is a critical blood component responsible for hemostasis, which has been

used extensively as a biopolymer scaffold in tissue engineering. Fibrin can be

prepared from autologous plasma, and is available as glue or as

engineeredmicrobeads. Fibrin alone or in combination with other materials has

been used as a biological scaffold for stem or primary cells to regenerate adipose

tissue, bone, cardiac tissue, cartilage, liver, nervous tissue, ocular tissue, skin,

tendons, and ligaments. Thus, fibrin is a versatile biopolymer, which shows a

great potential in tissue regeneration and wound healing (Ahmed et. al. 2008)

Fibrin is a biopolymer of the monomer fibrinogen. The fibrinogen molecule is

composed of two sets of three polypeptide chains named Aa, Bb, and g, which

are joined together by six disulfide bridges (Mosesson et. al. 2005). Fibrin is

formed after thrombin-mediated cleavage of fibrinopeptide A from the Aa chains

and fibrinopeptide B from the Bb chains (Mosesson et. al. 2001), with subsequent

conformational changes and exposure of polymerization sites. This generates the

fibrin monomer that has a great tendency to self-associate and form insoluble

fibrin. Further, the blood coagulation factor XIIIa is a transglutaminase that

rapidly cross-links g chains in the fibrin polymer (Horan et. al. 2001) by

introducing intermolecular x-(g-glutamyl) lysine covalent bonds between the

lysine of one g-chain and glutamine of the other (Doolittle et. al. 1971). This

covalent cross-linking produces a stable fibrin network that is resistant to

protease degradation (Schense et. al. 1999). This effect can be reinforced by

introducing chemical cross-linker such as genipin.

1.9.1. Preparation of Fibrin beads

FMBs are small spherical dense beads with a diameter ranging from 50 to 250

microns that consist of highly condensed and cross-linked fibrin. FMBs are

produced from plasma fibrinogen obtained by fractionation (Gorodetsky et. al.

1999). The fibrinogen is mixed with thrombin, and the activated fibrin is

immediately and quickly stirred in a heated oil emulsion (corn oil: isooctane, 1:1;

758C) to yield spherical droplets that are further cross-linked into solid beads

(Fig. 4) (Marx et. al. 2008). Fibrinogen denatures above 508C due to the instability

of the D-domain, whereas factor XIIIa is much more stable and can cross-link

proteins at higher temperatures. Denatured fibrinogen in the FMB is greatly

haptotactic to mesenchymal-type cells, such as endothelial cells, smooth muscle

cells, and fibroblasts (Marx et. al. 2008). FMBs have been used widely to isolate

and grow mesenchymal stem cells from both bone marrow and blood

(Gorodetsky et. al. 1999, Shimony et. al. 2006). Kidney gene and cell therapy has

been tested in vitro using FMBs as a threedimensional platform, since a variety of

transduced renal cells grow and differentiate in this material (Shimony et. al.

2006). Further, FMBs in combination with the appropriate cell source can be used

in bone regeneration and wound healing (Gurevich et. al. 2002).

Figure 4: Prepration of fibrin beads

1.9.2. Fibrin as a Delivery System

The use of fibrin for tissue engineering has been a popular field of research over

the past number of years. The ability of fibrin to achieve homeostasis during

healing and to naturally act as a scaffold for tissue repair has resulted in a vast

field of research aimed at investigating fibrin as a delivery system for cells and

biomolecules in tissue engineering (Fig. 5).

Drug delivery

Fibrin has been used as an antibiotic delivery system for some years (Redl et. al.

1983, Tsourvakas et. al. 1995). However, the challenge has been to sustain the

delivery of the drug for adequate periods of time. Senderoff et al. have

investigated different forms of fibrin for sustained drug delivery (Senderoff et. al.

1991). Fibrin microparticles, fibrincoated drug particulates, and fibrin sheets with

entrapped dexamethasone were assessed for drug release behavior. It was seen

that fibrin microparticles prepared by oil emulsion, with entrapped

dexamethasone sustained the release of the drug, but only for 4 h. Release

kinetics of the drug from fibrin films were evaluated and showed a diffusion

coefficient indicative of near-zero-order release kinetics. Yoshida et al. evaluated

the release of different anti-cancer drugs, and it was noted that sustained release

correlated with the hydrophobicity of the drug (Yoshida et. al. 2000). Similarly,

Woolverton et al. investigated tetracycline release, as an example of a poorly

soluble drug (Woolverton et. al. 2001). Tetracycline was delivered within an

implanted fibrin scaffold to the peritoneal wall of a mouse model and

subsequently infected with 108 cfu of S. aureus. A single treatment of fibrin

containing 500mg/mL tetracycline provided 100% protection against infection in

mice for 7 days compared to control mice treated with fibrin only. However, it

has been noted that addition of the drug to the fibrin during polymerization

results in fragile scaffolds and poor retention of the drug. Therefore, researchers

have used freeze-dried fibrin as a possible fabrication method for preventing

spontaneous release of the drug (Kumar et. al. 2004). Results showed a sustained

release of tetracycline for 12 days with no initial burst release (Kumar et. al. 2004),

and of antibiotic for 18 days (Itokazu et. al. 1997). Fibrin has been used to deliver

chemotherapy agents, such as carboplatin to the retina (Simpson et. al. 2002, Tusi

et. al. 2000, Pardue et. al. 2004), sustaining delivery for up to 2 weeks. In the

treatment of brain tumors, fibrin has been investigated as a delivery system for b-

emitting radioisotopes in microspheres (Hafeli et. al. 2007). Results showed that

fibrin adhered to the brain tissue, showing superior adherence than all other

tested mucoadhesive materials, thereby preventing migration of microparticles to

nearby tissues. Survival data proved the efficacy of this treatment. Recently, a

tissue engineering strategy to treat bone fractures suffering infection involved the

co-delivery of an antibiotic system in addition to bone marrow mesenchymal

stem cells via a fibrin scaffold (Hou et. al. 2008). Vancomycin alginate beads were

embedded in a fibrin scaffold, to localize antibiotic exposure at the site of the

defect and combat infection. Additionally, bone marrow mesenchymal stem cells

were delivered in the scaffold to encourage bone regeneration. In vitro results

showed that cell morphology did not change with exposure to antibiotic beads,

and cells showed a tendency to proliferate more in fibrin scaffolds containing

antibiotic beads. A possible reason for this enhanced proliferation was suggested

to be due to the more porous fibrin scaffold resulting from inclusion of the

beads, thereby facilitating cellular proliferation.

Figure 5: Schematic of the common cells and molecules currently delivered via fibrin

scaffolds.

1.10. Biodegradable Polymeric Systems

Biodegradable polymers have emerged as one of the most important polymeric

systems with remarkable drug delivery potential. Although molecules that

undergo bio-erosion are also used interchangeably with biodegradation, the two

differ; erosion occurs by the dissolution of chain fragments in non crosslinked

systems without chemical alterations to the molecular structure, while

biodegradation occurs through the covalent bond cleavage by a chemical reaction.

Both biodegradation and erosion can occur as surface or bulk process. In surface

degradation the polymeric matrix is progressively remove from the surface but

the polymer volume fraction remains almost unchanged. In contrast, during bulk

degradation no significant changes occur in the physical size of the polymer until

it is nearly completely degraded or eroded.

To be biodegradable, a polymer requires hydrolysis, usually enzymatic, of a

susceptible bond either in the backbone or crosslinker. Most of the biodegradable

carriers rely on the cleavage of ester bond, ester derivatives like poly

(lactic/glycolic acid) and poly (ε-caprolactone) or peptide bond. Other

hydrolysable bonds include those of poly(anhydrides), poly(orthoesters),

poly(phophoesters).

Biodegradable polymers include polymers of diverse origin such as

polysaccharide, protein and synthetic polymers. Among the polysaccharide

polymers alginate, dextran, hyaluronic acid, chitosan and cellulose have been

studied extensively. Gelatin, collagen, albumin and silk fibroin are proteinaceous

studied in detail. Synthetic polymers investigated for drug delivery include

polyphosphazene, polyurethane, polycaprolactone, polyorthoester. The polymers

and their applications in delivery of pharmaceuticals are discussed in some detail

below.

1.10.1. Polysaccharide Polymers

1.10.1.1. Alginates

Commercial alginates are extracted from brown algal species which include

Laminaria hyperborean, Ascophyllum nodosum, and Macrocystis pyrifera. It is a linear

polysaccharide containing varying amounts of 1, 4-linked β-D-mannuronic acid

and α-L-guluronic acid (Fig.6a.). The exact composition and residue sequence of

alginates vary depending on the source. Due to its biocompatibility, low toxicity,

nonimmunogenic nature, relatively low cost and ready gelation with divalent

cations such as Ca,2+ it has been widely used in drug delivery and tissue

engineering applications (George et al., 2007). Cross-linking with various types of

molecules has been carried out to control the mechanical and/ or swelling

properties of alginate gel. The high porosity of alginates makes it more suitable

for the entrapments of cells and large molecular weight pharmaceuticals

(Smidsrod et al., 1990). Alginate has also been studied as an injectable delivery

vehicle of cells (Atala et al., 1994), proteins (Wee et al., 1998), hormones (Silva et

al., 2006) and various growth factors (Kolambkar et al., 2010).

1.10.1.2. Dextrans

Dextrans are a complex, high molecular weight, branched polymers of D-glucose

(Fig.6b), available in a wide range of molecular dimensions and types. Dextrans

have been investigated for numerous applications, including as plasma expanders

and blood substitutes in addition to their use as delivery vesicles of drugs.

Dextran based microparticles have been used for the delivery of recombinant

human bone morphogenetic protein-2 (rhBMP2) (Chen et al., 2005). Acetylated

dextran micro particles are potent in vitro delivery platforms for vaccine adjuvants

(Bachelder et al., 2010). Qi et al. (2010) prepared composite BSA-dextran

nanoparticles for the delivery of an anticancer drug doxorubicin for the treatment

of murine ascites hepatoma H22 tumor-bearing mice. The nanoparticles

decreased the toxicity of doxorubicin and significantly increased the survival of

the tumor-bearing mice.

Figure 6: Partial structure of alginate (a), dextran (b), hyalouronic acid (c) and chitosan (d).

1.10.2. Proteinic Polymers

1.10.2.1. Collagen

Collagens are a group of naturally occurring proteins found exclusively in animals,

especially in the muscle and connective tissues of mammals (Muller and Werner,

2003). Collagens are the main components of connective tissue, and the most

abundant proteins in mammals, making up to about 25-35 percent of the whole-

body protein content (Di Lullo et al., 2002). Collagen gels are visco-elastic thereby

semi-solid, which makes them conveniently injectable and biocompatible drug

delivery matrices. They can even be induced to flow under stress (e.g., extrusion

from a syringe) (Wallace et al., 1988; Wallace et al., 1989; Rosenblatt et al., 1993;

Chow et al., 1985). In addition, they exhibit good cell and tissue compatibility

(Wallace et al., 1988; Kligman, 1988; Stegman et al., 1988; Stegman et al., 1987) and

thus do not interfere with normal body functions. When injected into the site of

interest, collagen matrices release the entrapped pharmaceuticals in their

surroundings in a controlled manner (Wallace and Rosenblatt, 2003). The

available forms of injectable collagen gels include suspensions of collagen fibers

(Wallace et al., 1988; Rosenblatt et al., 1993) and non-fibrillar, viscous aqueous

solutions (Chow et al., 1985). Small molecular weight drugs and therapeutic

proteins have also been used with the fibrillar suspensions or viscous collagen

solutions (Wallace and Rosenblatt, 2003). The collagen network readily retains

various cells by physical entrapment (Wallace and Rosenblatt, 2003) and have also

been employed as scaffolds in tissue engineering, delivery matrices for cells and in

gene therapy (Wallace and Rosenblatt, 2003).

1.10.2.2. Serum albumin

Albumin is the most abundant protein in blood plasma of higher animals and

constitutes up to 50% of total mass of plasma. Albumin plays an important role

in binding and transport endogenous and exogenous substances, including those

which are toxic in unbound state. Albumin has the ability to bind to a variety of

molecules including fatty acids and a variety of drugs (Koch-Weser and Sellers,

1976) and serves as a circulating depot of several compounds (Kragh-Hansen,

1990). Nearly all of human body cells degrade albumin making the protein a

highly suitable for biomedical applications (Prinsen and de Sain-van der Velden,

2004). Serum albumin can be easily processed into variety of forms including

microsphere, membranes, nanofibres, nanospheres (Nair and Laurencin, 2007).

Human serum albumin microspheres have potential as a slow release drug

delivery system. They offer advantages over other type of polymeric microspheres

in being nonantigenic, readily metabolizable with potential to bind a variety of

drugs in relatively non specific fashion. Albumin conjugated with heparin was

used in preparation of microsphere for some interesting drug delivery application.

Adriamycin-loaded albumin-heparin conjugate microspheres (AHCMS) show an

increased antitumour efficacy in L1210 tumour-bearing mouse and the CC531

tumour-bearing rat model and reduce its acute toxicity (Cremers et al., 1994).

1.11. Biodegradable Microspheres As Drug Delivery System

A key factor in the design of injectable protein /drug delivery systems is the

choice of an appropriate biodegradable polymer. PLGA is one of the polyester

largely used as drug delivery system because of availability of toxicological and

chemical data, biocompatibility/histocompatibility, predictable biodegradation

kinetics, ease of fabrication, versatility in properties, chemical integrity,

commercial availability, variety in copolymers ratios and molecular weights, lastly

and most important is its regulatory approval. Microsphere, composed of

chemicals like biodegradable polymer polyvinyl alcohol (PVA) and PLGA, is one

of the primary candidates that can be used as a carrier for sustained release of

drugs and antigens administered either by oral or parenteral routes (Eldridge et

al, 1991. O'Hagan et al, 1993). These polymers have demonstrated excellent

tissue compatibility. Moreover, the restorable synthetic polymers are non-toxic

and have already been used for other biochemical applications including drug

delivery (Pistner et al, 1993). PVA, obtained from poly(vinyl acetate) by

alcoholysis, hydrolysis, or aminolysis (Marten et al, 2002), is useful to deliver

proteins in a controlled manner (Lee et al, 2007). The PVA is also used to

increase the loading content of water-soluble drug molecules into porous PLGA

microcapsules (Mandal et al, 2002), as well as for long-term delivery of proteins

(Yeo et al, 2001). DL-PLGA induces only a minimal inflammatory response and

biodegrades through the hydrolysis of its ester linkages to yield biocompatible

lactic and glycolic acids.

1.11.1. Time of degradation

The parameters such as co-polymer ratios, different crystallinities, glass transition

temperature (T) and hydrophilicity affect biodegradation profile of PLGA

microspheres. Homogeneous degradation involves bulk erosion which is the case

in aliphatic polyesters, erosion occurs throughout the device and rate of water

penetration is greater than its conversion to water soluble fragments. Initially

there is random cleavage of hydrogen bonds due to hydration followed by

cleavage of covalent bonds. The molecular weight decreases due to continuous

cleavage and solubilization of low molecular weight components and complete

absorption. The carrier in such situations retains its original shape and mass until

significant degradation has occurred (~90%). At a given time point it attains

critical molecular weight that ultimately unsued in solubilization and mass loss.

The PLA and PLGA chains are cleaved to monomeric acids, i.e., lactic and

glycolic acids that are eliminated from the body through Kreb’s cycle as CO2 and

water. Role of enzymatic involvement in biodegradation is not clear. It varies

depending upon other properties which include: molecular weight of the polymer,

sequencing and cross-linking within the polymer backbone, surface area of the

device, porosity of the matrix, hydrophobicity of matrix and reactive groups

present. 50/50 poly(DL-lactide-co-glycolide) degrades in approximately 1–2

months, 75/25 in 4–5 months and 85/15 in 5–6 months (Middleton et al,

2000).

1.11.3. Preparation of microspheres

The biodegradable polymer based microspheres can be prepared by number of

methods. Each of them has its own advantages and disadvantages. In fact the

method of preparation plays important role on the properties of microspheres

and therefore the desired properties should be kept in mind during the selection

of a particular method of preparation. To formulate microspheres from the

biodegradable polymer matrix, it is essential to select an encapsulation process,

which fulfils the requirements of an ideal controlled release system. The attributes

of the encapsulating material are optimal drug loading, high yield of

microspheres, stability of the encapsulated drug, batch uniformity and inter-batch

reproducibility, adjustable release profiles, low burst effect, free flowing

microspheres. The encapsulation efficiency of the drug molecule should be high.

The ratio of the drug molecule to the polymer should be such that the largest

amount of drug is encapsulated in the minimal amount of polymer. This reduces

the mass of the material to be administered. The biological activity of the

encapsulated drug should be maintained during the process of microsphere

formulation. It is desirable to use a process where the exposure of the labile drug

into strong denaturing solvent is low. The method of encapsulation should be

such that by manipulating the formulation conditions, different types of release

profiles of the encapsulated material can be produced. The particles should be

formulated in such a way that minimum of the encapsulated drug is released

during the burst phase. This will help in extending the release of the drug for a

longer period of time. The encapsulation method used should always produce

free-flowing microspheres which do not aggregate. As with all parenteral

products, microspheres need to be sterile. This can be ensured by a terminal

sterilization step or through aseptic processing. Further, in relation to safety

requirements, the excipients and various solvents used in the processing should

either be nontoxic or removed from the final product. Many procedures are there

for the preparation of lactide-glycolide microspheres for protein delivery like

phase separation–coacervation, double emulsion solvent technique, spray drying,

interfacial deposition, phase inversion microencapsulation, in situ polymerization,

and thermal cross linking, etc, but widely used techniques for microsphere

preparation of proteins/drugs are:

(i) Spray drying;

(ii) Double emulsion;

(iii) Phase separation–coacervation.

1.11.4. Release kinetics from microspheres

The release from the microspheres is dependent both on diffusion through the

polymer matrix and on polymer degradation. If during, the desired release time,

polymer degradation is considerable, then the release rate may be unpredictable

and erratic due to breakdown of microspheres. However, the release of case,

microspheres are covered by one or several core material from such systems is

dependent diffusivity through the polymer barrier, solubility of core in bulk

phase, size of drug molecule and distribution of core throughout the matrix, etc.

Nature of polymer plays a major role in release process. Route of administration

of injectable microspheres may also alter the duration of release. Release from

PLA (poly-lactic acid) and PLGA (polylactic–glycolic acid) is dependent both

diffusion and polymer degradation (Makadia et al, 2011). The possible

mechanisms of drug release are:

(i) initial release from microsphere surface;

(ii) release through the pores dependent on spheres structure;

(iii) diffusion through the intact polymer barrier which is dependent on

intrinsic polymer properties and core solubility;

(iv) diffusion through a water swollen barrier dependent on polymer

hydrophilicity, which in turn depends on polymer molecular weight;

(v) polymer erosion and bulk degradation, release affected by the rate of

erosion and hydrolysis of polymer chains, leading to pore formation in

matrix.

All these mechanisms together play a part in release process. Nature of

core also influences release kinetics either by increasing polymer degradation or

by physically binding with the polymer chain. Drug–polymer interaction leads to

decreased release. Additives such as plasticizers decrease Tg (glass transition

temperature) which leads to decreased diffusion rates.

1.11.5. Microspheres–immune system interaction

Microspheres are capable of forming protein depots from which protein is slowly

released at the injection site. Interestingly, microsphere size is an important design

parameter. Small particles, with sizes smaller than 10 µm can be directly taken up

to macrophages by phagocytosis, whereas larger microspheres (greater than 10

µm) need to undergo biodegradation before phagocytosis can occur. In this case,

microspheres are covered by one or several layers of macrophages as a

consequence of wound healing response to injected particles. Consequently,

degradation, antigen release, location and antigen presentation of microspheres

larger than 10 µm are to be different from smaller ones. Upon administration of

the microspheres, a foreign-body response occurs, resulting in an acute initial

inflammation. This initial inflammation is followed by the infiltration of small

foreign body giant cells and neutrophils (Visscher et al, 1987). These immune

cells could consume the released protein and produce an immune response.

However, if protein is recognized as a self protein (e.g., homologous), the

probability of an immune response by these cells is reduced. It is therefore always

essential to release the protein in native conformation. The release of denatured

protein from the microspheres may result in an unwanted immune response

(Cleland et al, 1993).

1.11.6. Applications of microspheres

Microspheres are used in wide range of therapeutics and pharmaceutical

applications. Various types of drugs, oligo-nucleotides, anti-tumor agents,

proteins, peptides and vaccines have been encapsulated in these biodegradable

polymers which are as follows:

1.11.6.1. Vaccines

1.11.6.1.1. Group B Streptococcus vaccine (GBS)

Group B Streptococcus (GBS) is the leading bacterial cause of neonatal sepsis

and meningitis. Although antibiotic prophylaxis has decreased the infection rate,

the best long-term solution lies in the development of effective vaccines. The

GBS capsular polysaccharide (CPS) is a major target of antibody mediated

immunity. The feasibility of producing a GBS having the ability to produce both a

local IgA immune response at the mucosal surface and humoral IgG response

having capability of transplacental passive immunization was investigated (Sinha

et al, 2003). Immunization of female mice with normal immune systems was

done with these PLG microparticles containing GBS type III polysaccharide and

CpG adjuvant (PLG/GBS/CpG), and results indicated a significantly higher GBS

antibody response as compared to nonencapsulated GBS antigen or PLG

encapsulated GBS PS vaccine without the addition of the CpG.

1.11.6.1.2. Tetanus toxoid (TT)

Tetanus is considered as a major health problem in the developing and under-

developed countries, with approximately one million new cases occurring each

year. Tetanus is an intoxication manifested primarily by neuromuscular

dysfunction. TT was encapsulated using PLGA with different molar

compositions (50:50, 75:25) by w/o/w multiple emulsion technique and protein

integrity was evaluated during antigen release in vitro in comparison to Al

adsorbed TT for in vivo induction of tetanus-specific antibodies (Jung et al,

2002). TT microspheres elicited antibody titers as high as conventional Al

adsorbed TT which lasted for 29 weeks leading to the conclusion that TT

microspheres can act as potential candidates for single shot vaccine delivery

systems.

1.11.6.1.3. Japanese encephalitis virus ( JEV)

Japanese encephalitis is a disease that is spread to humans by infected mosquitoes

in Asia. It is one of mosquito-borne viral diseases that can affect central nervous

system and cause severe complications and even death. Vaccination is one of the

ways of treating it. JEV vaccine was encapsulated in PLGA microspheres by

w/o/w technique and influence of various process variables such as stirring rate,

types and concentration of emulsifier, polymer concentration were studied on

size, size distribution and biodegradation Rate of biodegradation of non-porous

microspheres was slower than that of porous microspheres leading to the

conclusion that PLGA microspheres can be used to apply oral vaccination

through Payers patches across GIT (Khang et al, 1996).

1.12. Opportunistic fungal infections

Over the last several years, the frequency of life-threatening fungal infections has

increased dramatically, particularly among cancer, diabetic and

immunocompromised patients (Anaissie et al., 1992; Pfaller and Webzel, 1992;

Richardson et al., 1991; Walsh et al., 1992). On one hand, the observed upsurge

could be attributed to the advancement in the field of medical sciences that made

possible improved recognition and diagnosis of fungal infections. Besides

prolonged survival of patients with defects in their defense mechanisms, more

invasive surgical procedures, the use of prosthetic devices and indwelling

catheters, increased administration of parenteral nutrition, development of

resistance fungal strains to currently available antifungal drugs, the increase in the

number of patients contracting AIDS and the use of peritoneal dialysis and

hemodialysis are some of the potential opportunities offered to microbes for their

establishment leading to full blown disease. In these patients invasive fungal

infections may account for as many as 30 % of deaths.

Although the occurrence of fungal drug resistance is far below in comparison

antibacterial agents, historical precedence warns that it is only a matter of time

before selective pressures will lead to population shifts resulting in more

widespread resistance. Moreover, overuse and inappropriate prescription of

antifungal agents has contributed significantly to the situation when pathogens do

not respond to the chemotherapy. The emergence of fungi as clinically important

pathogens has been well documented, although their role in pathogenesis of

human infection has only recently been appreciated (Banerjee et al., 1991; Emori

et al., 1993). Perhaps more worrisome predicament is the increased occurrence of

drug-resistant Candida sp. than their emergence as a pathogen (Goff et al., 1995;

Nolte et al., 1997; Nguyen et al., 1996; Chavanet et al., 1994).

There is a strong suggestion that invasive fungal infections have become more

common in recent years, with a nearly 500% increase in the incidence of blood-

stream infection with Candida spp. since the 1980s (J Hosp Infect 1995 Jun;30

Suppl:329-38). Most systemic fungal infections are caused by Candida spp.,

followed by Aspergillus and Cryptococcus spp. Infections due to Candida species are

the fourth most important cause of nosocomial bloodstream infection. Systemic

fungal infections cause ~ 25% of infection-related deaths in leukaemics. Serious

fungal infections may cause 5-10% of deaths in those undergoing lung, pancreas

or liver transplantation Acquired fungal sepsis occurs in up to 13% of very low

birthweight infants.

1.12.1. Predisposing factors to the establishment of fungal pathogens in host:

1. Neutropenia, T-cell dysfunction, immunosuppression, B-cell dysfunction,

Organ transplantation, AIDS, Diabetes or

2. Cytotoxic chemotherapy or

3. High dose corticosteroids or

4. Long-term antibiotic therapy (broad spectrum) or

5. Prolonged hospitalization or

6. Burns.

1.12.2. Candidiasis: Opportunistic infection by Candida albicans

In the last two decades Candida spp. has emerged as a major human pathogen.

Candida albicans (C. albicans) is an opportunistic pathogen, which causes life-

threatening disease in immunocompromised mammalian hosts (Shapiro et al.,

2011; Cannon et al., 2009). C. albicans has been found to account for ~ 52-63% of

all nosocomial fungal infections (Nguyen et al., 1996). It has been proposed that

this occurrence may result from selective pressure induced by the increased use of

antifungal agents. In 1991, Banerjee and colleagues reported rates of candidemia

ranging from 0.28 to 0.61 cases in every 1000 discharges in a hospital

participating in the National Nosocomial Infection Surveillance (NNIS) system

(Banerjee et al., 1991). This represented a five-fold increase in the detection rate of

candidemia in NNIS members between 1980 and 1989 (Banerjee et al., 1991). The

upward trend has continued into the 1990s (Emori et al., 1993). Among NNIS

system centers, Candida species have been reported to be the sixth most

commonly isolated pathogen and the fourth most prevalent bloodstream

pathogen (Banerjee et al., 1991). Factors that are responsible for the exponential

rise in the isolation of the Candida species include the emergence of AIDS,

increased patient exposure to broad spectrum antibiotics resulting in alterations in

normal host flora, increasing number of patients with neutropenia, in

consequence to cancer treatment and transplantation procedures, and expanded

use of intravascular catheter devices. As medical technology progresses, it is likely

that we will see the increased number of opportunistic infections in critically ill

patients. Consequently, the infection by Candida species will probably become

even more predominant in the future.

1.12.2.1. Common types of C. albicans infections

Oropharyngeal Candidiasis (Oral thrush) – This is the most common

infection of the mouth that involves buccal mucous membranes, most commonly

in infants and AIDS patients. It appears as white adherent patches with only

minimal erosion of the membrane.

Vaginal candidiasis (Vulvovaginitis) – It resembles with thrush in feature

except that irritation, intense itching, and discharge accompany it. The

establishment of vaginal candidiasis is facilitated by loss of the normal bacterial

flora (because of antibiotic therapy). The bacteria maintain a low (acid) pH in the

vagina that does not allow growth of C. albicans, and also not allow it to form

pseudohyphae that facilitates establishment of infection.

Skin infection - mostly in moist, warm parts of the body, i.e., axilla, intergluteal

folds, groin, or inframammary folds. Obesity and diabetes are predisposing

conditions for topical fungal infections.

Chronic mucocutaneous candidiasis - Chronic mucocutaneous candidiasis

(CMC) is a primary immunodeficiency disease presenting with debilitating,

persistent and refractory infections with the opportunistic yeast C. albicans

(Kirkpatrick et al., 1989). Infections vary from mild to lethal, are characteristically

localized to the skin, mucous membranes and/ or nails, and as a rule do not usually

progress to disseminated disease or sepsis (Bodey, 1993). The underlying immune

defect is poorly understood, although it has been appreciated that mucocutaneous

infections with Candida other than CMC frequently accompany impairment of T-

cell mediated immunity (HIV, Di George syndrome etc.) (Bodey, 1993).

Secondary infections – Fungemia (presence of fungal pathogens in blood) is

very common in immunosuppressed individuals. The infection is not localized to a

particular site or organ but distributed in whole body including systemic blood

circulation.

1.13. Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast that can live in both plants

and animals. Its teleomorph is Filobasidiella neoformans, a filamentous fungus

belonging to the class Tremellomycetes. It is often found in pigeon excrement.

Cryptococcus neoformans is composed of two varieties (v.): C. neoformans v. neoformans

and v. grubii. A third variety, C. neoformans v. gattii, is now considered a distinct

species, Cryptococcus gattii. C. neoformans v. grubii and v. neoformans have a worldwide

distribution and are often found in soil which has been contaminated by bird

excrement. The genome sequence of C. neoformans v. neoformans was published in

2005 (Loftus et al. 2005). Recent studies suggest that colonies of Cryptococcus

neoformans and related fungi growing on the ruins of the melted down reactor of

the Chernobyl Nuclear Power Plant may be able to use the energy of radiation

(primary beta radiation) for "radiotrophic" growth (Dadachova et al. 2007).

Cryptococcus neoformans grows as a yeast (unicellular) and replicates by budding. C.

neoformans makes hyphae during mating, and eventually creates basidiospores at

the end of the hyphae before producing spores. Under host-relevant conditions,

including low glucose, serum, 5% carbon dioxide, and low iron, among others,

the cells produce a characteristic polysaccharide capsule (Joseph et al. 1998).

When grown as yeast, C. neoformans has a prominent capsule composed mostly of

polysaccharides. Microscopically, the India ink stain is used for easy visualization

of the capsule. The particles of ink pigment do not enter the capsule that

surrounds the spherical yeast cell, resulting in a zone of clearance or "halo"

around the cells. This allows for quick and easy identification of C. neoformans.

C. neoformans grows rapidly on exposure to radiation such as gamma-radiation.

Radiation seems to increase the electron-transfer capability of melanin in the

fungus, increasing its total metabolic activity.

1.13.1. Pathology:

Infection with C. neoformans is termed cryptococcosis. Most infections with C.

neoformans consist of a lung infection. However, fungal meningitis, especially as a

secondary infection for AIDS patients, is often caused by C. neoformans making it a

particularly dangerous fungus. Infections with this fungus are rare in those with

fully functioning immune systems. For this reason, C. neoformans is sometimes

referred to as an opportunistic fungus. It is a facultative intracellular pathogen

(Alvarez et al 2007).

1.13.2. Treatment:

Cryptococcosis that does not affect the central nervous system can be treated

with fluconazole alone. Cryptococcal meningitis should be treated for two weeks

with intravenous Amphotericin B 0.7–1.0 (mg/kg)/day and oral flucytosine 100

(mg/kg)/day (or intravenous flucytosine 75 (mg/kg)/day if the patient is unable

to swallow). This should then be followed by oral fluconazole 200 mg daily for

ten weeks (Saag et al. 2000) and then 200 mg daily until the patient's CD4 count is

above 100 for three months and, if infected, his HIV viral load is undetectable

(Martínez et al. 2000, Vibhagool et al. 2003).

Intravenous Ambisome 4 (mg/kg)/day may be used but is not superior: its main

use is in patients who do not tolerate Amphotericin B. The 200 (mg/kg)/day

dose for flucytosine is not more effective, is associated with more side-effects and

should not be used.

In Africa, oral fluconazole at a rate of 200 mg daily is used. However, this does

not result in cure because it merely suppresses the fungus and does not kill it;

viable fungus can continue to be grown from CSF of patients who have taken

fluconazole for many months. An increased dose of 400 mg daily does not

improve outcomes (Schaars et al. 2006), but preliminary data from Uganda shows

that very high doses of 1200 mg or more per day may be effective. The duration

of this treatment and the post-treatment maintenance dose is not known.

1.14. Antifungal therapy

A number of different classes of antifungal drugs are available for treatment of

fungal infections. These include the following:

1) Allylamines and other non-azole ergosterol biosynthesis inhibitors.

The general mode of action of these compounds is to reduce ergosterol

biosynthesis. One such compound is terbinafine which is an inhibitor of squalene

epoxidase; the enzyme involved in fungal cell wall sterol synthesis.

2) Antimetabolites

Flucytosine (5-fluorocytosine; 5-FC; 4-amino-5-fluoro-2-pyrimidone) is the only

compound of this class. It is a pyrimidine that is activated by deamination within

the fungal cells to 5-fluorouracil.This 5-flurouracil substitutes uracil in fungal

RNA, thus disrupting protein synthesis in the fungi. Flucytosine also inhibits

thymidylate synthetase via 5-fluorodeoxy-uridine monophosphate and thus

interferes with fungal DNA synthesis.

Figure: 7 Different types of Candidiasis

3) Glucan Synthesis inhibitors

These inhibitors prevent synthesis of glucan, an essential fungal cell wall

component by inhibiting the enzyme 1,3-beta glucan synthase. Example are

Caspofungin, Micafungin and Anidulafungin.

4) Azoles

The members of this class inhibit the synthesis of ergosterol (vital for fungal cell

membrane integrity). Basically they block the action of 14-alpha-demethylase by

inhibiting fungal cytochrome P-450 (14-a sterol demethylase). Examples are

Fluconazole, itraconazole, voriconazole, posaconazole, ravuconazole etc.

5) Polyenes:

These very potent agents act by binding to the fungal cell membrane and causing

the fungus to leak electrolytes. Examples are Amphotericin B (Amp B) and

nystatin.

The drug of choice for treatment of severe systemic mycoses is Amphotericin B.

Amp B is considered the 'Gold standard’ of antifungals. It is available in various

forms:

Conventional desoxycholate form

Amp B and lipid complex, named Abelcet (Ann Pharmacother 1997 Oct; 31(10):

1174-86)

Amp B colloidal dispersion with sodium cholesteryl sulphate, named Amphocil

[Drugs 1998 Sep; 56(3):365-83].

Liposomal Amp B (AmBisome), a small unilamellar liposome preparation (45 to

80nm).

1.15. Amphotericin B

Amphotericin B (Amp B), derived from fermentation products of Streptomyces spp.,

is the most effective and widely used drug in the treatment of both pre-systemic

and systemic fungal infections (Meyer et al., 1992) and remains in the forefront of

antifungal therapeutic strategies. However Amp B (Figure 8) is plagued by dose-

dependent toxicity issues, the most prominent of which is renal toxicity. Dose-

dependent nephrotoxicity caused by Amp B results in as much as 60% reduction

in the glomerular filtration rate (Rybak et al. 2009; Tolins et al. 1989).

Renal toxicity due to polyene antimycotics is believed to be caused as a result of

the drug anchoring to the cholesterol moieties within the mammalian cell

membrane, resulting in pore formation, abnormal electrolyte flux, decrease in

adenosine triphosphate (ATP), and eventually a loss of cell viability. Recent

evidences suggest that Amp B can also cause oxidative damage to mammals

(Brajtburg et al., 1990).

Figure 8. Chemical structure of Amphotericin B

However, despite its dose limiting toxicities, Amp B has remained ‘the gold

standard’ among antifungals. Its broad spectrum of activity and relatively low

frequency of mycological resistance to the drug has ensured its long-lasting

acceptance (Ellis et al., 2002). However in an immunocompromised host, Amp B

is found often ineffective. Systemic fungal infections in these patients have very

high lethality, often between 30 % and 100 % depending upon circumstances

(Dupont et al., 2002). In such cases usually a sufficiently high dosage is required

for the drug to be effective. This brings up problems of drug toxicity. Amp B is

known to have severe nephrotoxicity in nearly 50 % of patients.

Conventional Amp B's oral bioavailability is under 5%. Systemic (intravenous)

administration is associated with a long list of adverse effects, including marked

fever (in about 50%), anaphylaxis (about 1 in 100), nausea, vomiting, and

nephrotoxicity. Nephrotoxicity includes lowered GFR, increased loss of

potassium and magnesium, and even distal renal tubular acidosis. Nephrotoxicity

is said to be irreversible if therapy is prolonged (Antimicrob Agent Chemother

1978 13 271-6).

1.15.1. Mechanism of action of Amphotericin B

The polyene antifungal drugs target membranes containing ergosterol. Both Amp

B and nystatin are amphipathic, having both hydrophilic and hydrophobic

moieties. Amp B is speculated to intercalate into membranes, forming a

transmembrane channel consisting of an annulus of eight drug molecules linked

hydrophobically to the membrane sterol. Intermolecular hydrogen bonding

interactions among hydroxyl, carboxyl and amino groups stabilize the channel in

its open form, destroying activity and allowing the cytoplasmic contents,

especially potassium ions, to leak out and thereby destroying the proton gradient

within the membrane (Vanden et al., 1992) and causing fungal death. However,

researchers recently have shown that pore formation is not necessarily linked to

cell death (Angewandte Chemie Int. Ed. Engl. 2004). The actual mechanism of

action of Amp B may be more complex and multi-faceted.

The specificity of the drugs for ergosterol-containing membranes may be

associated with phospholipid fatty acids and the ratio of sterol to phospholipids

(Vanden et al., 1992). Polyenes are less likely to interact with membranes

containing cholesterol. It has been suggested that Amp B causes oxidative

damage to fungal cell membranes (Vanden et al., 1992).

1.16. Herbal drugs in treatment of fungal pathogens

Plants are important sources of therapeutic agents. Most of the modern

medicines originated from active ingredient of plants which were strictly

elucidated and modified for better results. Plant-based drugs are popular since

ancient times due to their effectiveness, easy availability, low cost &

comparatively low toxic side effects or adverse drug interaction. Indiscriminate

use of synthetic drugs and microbe-origin antibiotics has lead to complications

and previously unknown drug-resistance. The demand for plant based or natural

sources-based raw material for pharmaceuticals has increased enormously. The

synthetic drugs and intermediary chemicals are not only expensive they are

hazardous to human body as well as a menace to equilibrium of ecosystem in the

lag run. The World Health Organization (WHO) has been emphasizing the

utilization of indigenous system of medicines based on locally available medicines

from herbal resources as well as other raw material. Nearly sixty percent of all

medicines are of plant origin and rest are modification of less potent natural

active ingredient or their synthesized variety. Only few medicines come under

purely synthetic category.

Since ancient times plants have been the sources of many treatment strategies.

The Neanderthals living 60,000 years ago in present-day Iraq used plants such as

hollyhock for purposes other than food (Cowan et al. 1999). However very few

plant-derived antimicrobials are in scientific use today. This is mostly attributed to

the unavailability of in-depth studies on the pharmacoproperties of these agents.

Since the appearance of microbe-derived antibiotics in the 1950s as the drugs of

choice for treating infections, the use of plant derivatives as antimicrobials has

become almost non-existent. On average, two or three antibiotics derived from

microorganisms are launched each year (Adomi et al., 2006). However, with time,

traditional antibiotics (sourced from microorganisms or their synthesized

derivatives) are losing their efficacy to drug-resistance in pathogens and have,

moreover, always remained wholly ineffective against viral infections. Hence

aggressive research on finding new anti-infective agents (including vaccines) is

progressing apace (Huang et al. 2008). Apart from microbes, fresh insight is being

given into other natural sources, especially plants. Moreover there is an increasing

awareness among people about drug side effects, and problems associated with

the over prescription and misuse of traditional antibiotics. As a consequence

modern medicine is increasingly accepting the use of plant-sourced antimicrobials

as well as other drugs. In fact the scientific discipline of ‘ethnobotany (or

ethnopharmacology)’ is devoted to utilizing the vast repertoire of knowledge

gathered over time by indigenous peoples about the plant products that can be

used to maintain health (Huang et al. 2008; Rojas et al., 1992, Silva et al., 1996;

Vanden et al., 1992).

1.17. Macrophage as a target

Macrophages of the reticuloendothelial system (RES) are very capable of rapid

recognition and clearance of particulate matter has provided a rational approach

to macrophage-specific targeting with nanocarriers (Moghimi et al., 2001).

Intracellular pathogens such as Cryptococcus neoformans, various species of Candida

and Leishmania, Toxoplasma gondii, various species of Leishmania, Mycobacterium

tuberculosis, and Listeria monocytogenes, reside in the lysosome and/or cytoplasm of

the macrophage. Nanoparticulate vehicles containing encapsulated antimicrobial

agents passively targeted to such infected macrophages is a favorable strategy for

combating intracellular microbes (Allen and Cullis, 2004; Moghimi et al., 2001;

Veerareddy and Vobalabonia, 2004; Lavasanifar, 2002; Agrawal and Gupta, 2000).

The endocytic pathway of the macrophages route drug-loaded nanoparticles to

lysosomes where pathogens reside. Lysosomal enzymes then degrade the carrier,

releasing the drug into the phagosome-lysosome vesicle itself or into the

cytoplasm either by diffusion or by specific transporters, depending on the

physicochemical nature of the drug molecule. This strategy has been successfully

applied in FDA approved lipid-based amphotericin B formulations (100–200 nm)

for human use, and are recommended for treatment of visceral leishmaniasis or

confirmed infections caused by specific fungal species (Allen and Cullis 2004;

Veerareddy and Vobalabonia, 2004). This mode of targeting has significantly

reduced the required clinically effective quantity of Amp-B for treatment,

achieving therapeutic drug concentrations in the infected macrophages. Other

beneficial effects include significant reduction in nephrotoxicity, a common side

effect associated with Amp-B administration, and proinflammatory cytokine

release (Shadkchan et al., 2004). For example, intravenous injection of tuftsin-

bearing liposomes to infected animals have not only resulted in delivery of

liposome-encapsulated drugs to the macrophage phagolysosomes, but also in the

nonspecific stimulation of liver and spleen macrophage functions against

parasitic, fungal and bacterial infections (Agrawal and Gupta, 2000).The latter

effect is due to the binding of tuftsin to its receptor, which further incites

macrophage antimicrobial responses.

1.18. Aims and objectives of the thesis

The work incorporated in the thesis deals with the generation of prophylactic

strategies against C. albicans and C. neoformans. Intracellular abode of these

pathogens to avoid antibody onslaughts and to escape neutralizing agents unable

to enter cells combined with apparent lack of vaccines capable of obliterating

these pathogens by CTL response persuaded us to develop alternative

prophylactic strategies against these pathogens. The aims and objectives of the

work are itemised as under:

1. To study escheriosome mediated cytosolic delivery of Candida albicans cytosolic

proteins induces enhanced cytotoxic T lymphocyte response and protective

immunity.

2. To enumerate the xenogenic gama-irradiated pathogen harbouring macrophage

based vaccine: prophylactic potential against intracellular pathogen C.

neoformans.

3. To reveal the evaluation of fibrin beads for effective and sustained delivery of

antigens in combating with experimental murine cryptococcosis.