INDEX

CHAPTERNO.

CONTENTS PAGE NO.

1. INTRODUCTION

1.1 CONCEPT OF TARGETING

1.1.1 RATIONALE OF DRUG TARGETING

1.1.2 CLASSIFICATION OF DRUG TARGETING

1.1.2.1 PASSIVE TARGETTING

1.1.2.2 INVERSE TARGETTING

1.1.2.3 ACTIVE TARGETTING

1.1.2.4 DUAL TARGETTING

1.1.2.5 DOUBLE TARGETTING

1.1.2.6 COMBINATION TARGETTING

1.2 DRUG TARGETTING FOR RECEPTOR

1.2.1 ENDOCYTOSIS

1.2.2 LIGAND MEDIATED TARNSCYTOSIS

1.3 CHEMICAL DRUG TARGETTING

1.3.1 CDS FOR KIDENY

1.3.2 LUNG AS A TARGET ORGAN

1.3.3 CDS FOR LIVER TARGETTING

1.3.4 LYMPHATIC TARGETTING

2. REVIEW OF LITERATURE

2.1 LIPOSOMES

2.1.1 LIPOSOME FORMATION

2.1.2 METHOD OF LIPOSOME PREPERATION

2.1.3 CHEMICAL CHARACTERIZATION

2.2 NANOPARTICLES

2.2.1 PREPEARTION TECHNIQUES

2.2.2 CHARACTERIZATION OF NANOPARTICLES

2.2.2.1 SIZE AND MORPHOLOGY

2.2.2.2 SPECIFIC SERFACE

2.2.3 APPLICATIONS OF NANOPARTICLES

2.3 RELEASED ERYTHROCYTES

2.3.1 DRUG TARGETTING

2.3.2 DRUG TARGETTING TO RES ORGANS

2.3.3 RES IN OXYGEN DEFICIENCY THERAPY

2.3.4 NOVEL SYSTEM(NANOERTHROSOMES)

2.4 MAGNETIC DRUG TARGETTING SYSTEM

2.4.1 PRINCIPLES OF MAGNETIC TARGETTING

2.4.2 APPLICATIONS

2.5 MICROSPHERES

3. DRUG DELIVERY TO BRAIN

4. APPLICATION TO TARGETED DRUG DELIVERY TO BRAIN

5. CONCLUSION

6. REFERENCES

12

CHAPTER -1

INTRODUCTION:

Because the brain is tightly segregated from the circulating blood by a unique

membranous barrier, the blood-brain barrier (BBB), many pharmaceuticals cannot be

efficiently delivered to, or sustained within the brain; hence, they are ineffective in

treating cerebral diseases. Therefore, drug delivery methods that can provide brain

delivery, or eventually preferential brain delivery (i.e. brain targeting), are of

particular interest.

To achieve successful delivery, an understanding of the major structural, enzymatic, and

active transport aspects related to the BBB, and of the issues related to lipophilicity and its

role in CNS entry, is critical. During the last years, considerable effort was focused in the

field of brain-targeted drug delivery. Various more or less sophisticated approaches, such as

intracerebral delivery, intracerebroventricular delivery, intranasal delivery, BBB disruption,

nanoparticles, receptor mediated transport (vector-mediated transport or ‘chimeric’

peptides), cell-penetrating peptides, prodrugs, and chemical delivery systems, have been

attempted. These approaches may offer many intriguing possibilities for brain delivery and

targeting, but only some have reached the phase where they can provide safe and effective

human applications. Site-target indexing and the use of targeting enhancement factors can

be used to quantitatively assess the site-targeting effectiveness from a pharmacokinetic

perspective of chemical delivery systems.

Drug targeting is the delivery of drugs to receptors or organs or any other specific part of the 

body to which one wishes to deliver the drug exclusively.  The drug therapeutic index(TI)

as measured by its pharmacological response and safety. Drugs minimizing its interaction

with non target tissue. The desired differential distribution of drug by its targeted delivery 

would spare the rest of the body and thus significantly reduce the over all toxicity while ma

intaining it's therapeutic benefits. The targeted or site specific delivery of drugs is indeed a 

very attractive goal because this provides one of the most potential ways to improve the the

rapeutic index of the drug. The need for targeted delivery of drugs is best illustrated with

peptide drugs where failure in the clinic may not be due to a poor intrinsic activity, but

rather due to transport factors including widespread disposition,rapid catabolism and excre

tion, variable or in efficient extravasations, and the subsequent high dosing levels required 

13

to obtain a therapeutic effect (Tomlinson etcal.1986). Earlier work done between late 1960s

and the mid 1980s'stressed the need for drug;-carrier systems primarily to alter the

pharmacokinetics of the already proven drugs whose efficacy might be improved by alter-

ing the rates of metabolism in liver or clearance by the kidneys (Pozanski&Juliano, 1984).

These approaches generally were not focused to achieve site-specific or targeted delivery

such as getting a cytotoxic drug to cancerous tissue while sparing other normal, though 

equally sensitive tissue (Papahadjopoulos, 1978)1.

1.1 CONCEPT OF TARGETING:

1.1.1 RATIONALE OF DRUG TARGETING (CARRIERS):

Carrier is one of the most important entities essentially required for successful

transportation of the loaded drug (s) Delivery systems developed and exploited in the

last decade for ligand directed receptor mediated targeting are mainly focuses on

liposome's and microparticulates, bioconjugates (drug-antibody conjugate, drug-

polymer conjugates, drug-immunotoxin conjugates), fusogenic proteins and peptides

and certain polymeric and macromolecular delivery systems. An ideal drug carrier

engineered as a targetable device should have the following features.

It must be able to cross anatomical barriers and in case of tumour

chemotherapy tumour vasculature.

It must be recognized specifically and selectively by the target cells and must

maintain the avidity and specificity of the surface ligands.

The linkage of the drug and the directing unit (ligand) should be stable in plasma,

interstitial and other biofluids.

Carrier should be non-toxic, non-immunogenic and biodegradable particulate or

macromolecule and after recognition, and internalization, the carrier system

should release the drug moiety inside the target organs, tissues or cells.

The bimolecular used for carrier navigation and site recognition should not be

ubiquitous otherwise it may cross over the sites, defeating the concept of

targeting2.

1.1.2 CLASSIFICATION OF DRUG TARGETING:

The various approaches of vectoring the drug to the target site can be broadly classified as

1. Passive targeting

2. Inverse targeting

14

3. Active targeting (Ligand mediated targeting and Physical targeting)

4. Dual targeting

5. Double targeting

6. Combination targeting

1.1.2.1 PASSIVE TARGETING:

Systems that target the systematic circulation are generally characterized as" passive"

targeting delivery systems (i.e. targeting occurs because of the body's natural response

to the physicochemical characteristics of the drug or drug -carrier system. It is a sort of

passive process that utilizes the natural course of attributed to inherent characteristics)

biodistribution of the carrier system, through which, it eventually accumulate in the organ

compartment (s) of body. The ability of some colloids to be taken up by the RES

especially in liver and spleen has made them as ideal vectors for passive hepatic

targeting of drugs to these compartments.

1.1.2.2 INVERSE TARGETING:

One strategy applied to achieve inverse targeting is to suppress the function of RES by

a pre-injection of a large amount of blank colloidal carriers or macromolecule like

dextran sulphate. This approach leads to RES block-ade and as a consequence

impairment of host defense system Alternative strategies include modification of the size,

surface charge, composition, surface rigidity and hydrophilicity of carriers for desirable

biofate.

1.1.2.3 ACTIVE TARGETING:

The natural distribution pattern of the drug carrier composites is enhanced using chemical,

biological and physical means, so that it approaches and identified by particular biosites.

The facilitation of the binding of the drug-carrier to target cells through the use of ligands or

engineered homing devices to increase receptor mediated (or in some cases receptor

independent but epitope based ) localization of the drug and target specific delivery of drug

(s) is referred to as active targeting.. The targeting approach can further be classified it into

three different levels of targeting.

15

(1)First order targeting (organ compartmentalization).

(2)Second order targeting (cellular targeting).

(3)Third order targeting (intracellular targeting).

(1) FIRST ORDER TARGETING:

It refers to restricted distribution of the drug-carrier system to the capillary bed of a

predetermined target site, organ or tissue. Compartmental targeting in lymphatic,

peritoneal cavity, plural cavity, cerebral ventricles, lungs, joints, eyes, etc., represents first

order targeting it could also be categorized as a level of passive targeting. Large liposome

(10u or above ) are rapidly removed via mechanical filtration of lungs and from this size

range down upto 150nm are removed by tissue macrophages originated in the liver and

spleen, which are the natural target for these vesicles.

(2) SECOND ORDER TARGETING:

The selective delivery of drugs to a specific cell type such as tumour cells and not to the

normal cells is referred as second order drug targeting.

(3) THIRD ORDER TARGETING:

The third order targeting is defined as drug delivery specifically to the intracellular site of

target cells. An example of third order targeting is the receptor based ligand-mediated

entry of a drug complex into a cell by endocytosis, lysosomal duration of carrier followed

by release of drug intracellular or gene delivery to nucleolus.

(4) LIGAND MEDIATED TARGETING:

Targeting components, which have been studied and exploited are pilot molecules

themselves (bioconjugates) or anchored as ligands on some delivery vehicle (drug -carrier

system). All the carrier systems, explored so far, in general, are colloidal in nature. They

can be specifically functionalized using various biologically relevant molecular ligands

including antibodies, polypeptides, oligosaccharides (carbohydrates), viral proteins and

fusogenic residues. Ligand mediated activity targeting could be achieved asing specific

uptake mechanisms such as receptor dependent uptake of natural low density lipoproteins

(LDL) particles and synthetic lipid microemulsions of partially reconstituted LDL particles

coated with the apoproteins .

16

(5) PHYSICAL TARGETING (TRIGGERED RELEASE):

The selective drug delivery programmed and monitored at the external level (ex vivo) with

the help of physical means is referred to as physical targeting; In this mode of targeting,

some characteristics of the bioenvironmental are used either to direct the carrier to a

particular location or to cause selective release of its contents . The first such approach

reported is the temperature sensitive liposomes, which were developed and applied to

tumour by (Weinstein and co-workers, 1979).

Table-1.1 : Passive Hepatic Targeting for Macrophage Associate Diseases

Macrophage associated Drugs proposed for encapsulation

Leishmaniasis; brucellosis; candidiasis Antimalarial and antiinfective

Intracellular fungal infections Histoplasmosis:

systemic Mycoses

i Antifungal (Amphotericin B)

Histiocytes medullar Reticulosis; monocyte &

hairy Cell leukemia; Hodgkin's disease Viral

infected diseases Hepatitis

Cytotoxic drugs

Anti-viral drugs

Enzyme storage diseases Gaucher's disease,

mucoliposes Type II & III!

Glucocerebroside and Other enzymes

1.1.2.4 DUAL TARGETING:

This classical approach of the drug targeting employs carrier molecules, which have their

own intrinsic antiviral effect thus synergies the antiviral effect of the loaded active drug.

Based on this approach, drug conjugates can be prepared with fortified activity Profile

against the viral replication.

1.1.2.5 DOUBLE TARGETING:

For a new future trend, idrug targeting may be combined with another methodology, other

than passive and active targeting for drug delivery systems. The combination is made

between spatial control and temporal control of drug delivery.

1.1.2.6 COMBINATION TARGETING:

17

Combination targeting for the site- specific delivery of proteins and peptides these

targeting systems are equipped with carriers, polymers and homing devices of molecular

specificity that could provide a direct approach to target site. Modification of proteins and

peptides with natural polymers, such as polysaccharides, or synthetic polymers, such as

poly (ethylene glycol), may alter their physical characteristics and favour targeting the

specific compartments, organs or their tissues within the vasculature 3.

1.2 DRUG TARGETING FOR RECEPTOR:

LIGAND DRIVEN RECEPTOR MEDIATED DRUG DELIVERY

Design and development of potential carriers for cell specific delivery of therapeutics are

immensely dependent on the selectivity of the carrier to the cellular receptors distributed

variable at intracellular sites and on the surface of cellular systems. Other crucial factors

include the anatomical and pathological barriers that have to be circumvented, enroute

before recognition site(s) are arrived. Intracellular mesogenic constraints as well as

physiologic constraints are also encountered following receptor recognition, similarly,

cellular internalization is equivocally critical for intracellular rounding4.

1.2.1 ENDOCYTOSIS:

Endocytosis (phagocytosis and pinocytosis) has been defined as the internalization of

plasma membrane with concomitant engulfment of plasma membrane with concomitant

engulfment of extracellular cargo/fluM. The process serves to selectively retrieve and

assimilate various macromolecules from extracellular fluid for a variety of cellular

18

functions. It is the main cellular activity involved in the internalization of the extracellular

cargo and their vesicular coat proteins, which are subsequently processed via different

pathways to appropriate intracellular targets. Phagocytosis is the engulfment of the

endogenous and exogenous particulate materials, such as bacteria, erythrocytes, latex beads,

colloidal particles and immunoglobulin molecules. It is performed by the phagocytic cells of

the hepatic sinusoids, the tissue fixed macrophages (histocytes) and the blood macrophages

or monocytes that the fluid phase and receptor mediated pinocytosis are not separate cellular

events, but they are different facets of the same event. Non specific adsorption piocytosis is

responsible for the uptake of many non-glycosylated proteins particularly following cellular

damage or protein denaturation.

1.2.2 LIGAND MEDIATED TRANSCYTOSIS:

Transcytosis is the process by which intracellular ligand or extracellular cargo

internalized at one plasma membrane domain of a polarized cell which is transported via

vesicular intermediates to the contra lateral plasma membranes. Much of the

characterization of transcytosis in the basolateral to apical direction. Accordingly, the

polymeric immune globulin receptor (plgR), a protein specialized for basal to apical

transcytosis that recycles at the apical membrance as part of its transcytotic sojourn, has

been used apparently as an apical endocytic carrier to introduce vector DMA into cells

that express the PlgR. The apical to basal transcytosis has been however identified at

molecular levels in Cacao 2 cells. This protein as well as additional ones should provide

powerful tools for future characterization of transcytosis pathway. Ideally some of these

proteins may represent better putative carriers for the apical to basal transcytosis of

therapeutic agents. Transcytosis pathway has been well established for the protein sorting

an trafficking of transferrin, polymeric Ig, and viral pathogens. With respect to the

delivery and transport of pharmaceuticals, characterization of this pathway should lead to

the advances in the development of transcellular drug delivery.

1.3 CHEMICAL DRUG TARGETING:

1.3.1 CDS FOR KIDNEY:

Kidney possesses high concentrations of L-glut amyl transpeptidase and L-amino acid

decarboxylase. Selective delivery of dopamine in kidney has been obtained after

administration of L-Y glutamyl dopa produces almost 5 times higher

concentrations of dopamine in kidney compared with equivalent dose of L-dopa.

19

1.3.2 LUNG AS A TARGET ORGAN:

The lung possesses the next highest levels of nearly all the metabolic enzymes found in

liver an in some cases even higher specific activities in certain cell types. Additionally, in

contrast to all other tissues, the lung receives total venous return first, so it in an ideal

position to regulate the concentration of substrates in the blood before they reach the

arterial circulation hence avoiding problems that may be associated with a hepatic first pass

and permitting a more efficacious sequestration of a drug entity.

1.3.3 CDS FOR LIVER TARGETING:

Liver is an important organ and is considered to be focal point of metabolic ativities in

body. Targeted drug delivery to liver is achieved using bile acid transport system

associated with the sinusoidal membrane of the hepatocytes developed bile acid prodrug

of chlorambucil for liver targeting. et. al. 1992. Hepatic asialoglycoprotein

receptor mediated endocytosis has been exploited for antiviral drug targeting to liver

parenchymal cells.

1.3.4 LYMPHATIC TARGETING:

The lymphatic system is regarded as an integral and necessary part of the vascular system.

Its main function is to collect the excessive tissue fluid and return it back to the blood.

Lymphatics are numerous in number and distributed throughout the body. Their major

physiological function is to maintain the body's water balance, thus acting as body's

drainage system. The intestinal lymphatic system consists of a network of vessels

distributed throughout the small and large intestine. They play a major role in the

absorption of variety of nutrients, lipids including long chain fatty acids, triglycerides,

cholesterol esters fluids, lipid soluble vitamins some xenobiotics (e.g. DOT). The

potential advantage of transporting drug through the intestinal lymphatic system includes.

Avoidance of hepatic first-pass metabolism.Selective treatment of diseases and infections

of the mesenteric lymphatic system.Directing the delivery of appropriate agent to various

sites of intestinal and thoracic lymphatic system.Enhancement of the absorption of large

macromolecules such a peptides an particulates.Inhibition of cancer cell

metastasis.Lymphocytic targeting and receptor mediated targeting via the low density

lipoproteins receptor to regions of the lymphatic that are directly supplied by lymph from

mesenteric lymphatic’s. These regions are often poorly perfused by the systemic

20

circulation making it difficult to attain adequate drug concentration at the target organ/cell

after the compound has absorbed via the portal blood Reduction in local gastrointestinal

irritation and toxicity .An overall modulation in the rate of drug input thus providing a

sustained delivery5.

21

CHAPTER-2

REVIEW OF LITERATURE:

2.1 LIPOSOMES:

Liposomes have attracted a considerable amount of intevest for potential use as a drug

delivery system owing to their suitable characteristics. They consist of one or more

concentric phospholipid bilayers enclosing an aqueous space. They are biocompatible,

biodegradable, and normally nonimmunogenic. More importantly, they are capable of

loading both hydrophilic and hydrophobic drugs in the aquesous and bilayer phase

respectively. Drugs encapsulated in liposomes are protected from enzymatic degradation

and other inactivation processes. There are basically two different modes in liposome

targeting passive and active targeting. The former takes advantage of the fact that

systemically injected liposomes are rapidly and efficiently taken up by phagocytic

cells of the reticuloendothlial system (RES) located Mainly the live and the spleen Some

of the advantage of liposome are as follows:

• Provides selective passive targeting to tumour tissue.

• Increased efficacy and therapeutic index.

• Increased stability via encapsulation

• Reduction in toxicity of the encapsulated agent.

• Site avoidance effect.

• Improved pharmacokinetic effects (reduced elimination, increased circulation life

times)

•     Flexibility to couple with site-specific lignads to achieve active targeting

2.1.1 MECHANISM OF LIPOSOME FORMATION:

In order to understand why liposomes are formed when phospholipids are hydrated it

requires a basic understanding physicochemical features of phospholipids.

Phospholipids are amphipathic (having affinity for both aqueous and polar moieties)

molecules as they have a hydrophobic tail and a hydrophilic or polar head. The

hydrophobic tail is composed of two fatty acid chains containing 10-24 carbon atoms

and 0-6 double bonds in each chain. The polar end of the molecule is mainly

phosphoric acid bound to a water soluble molecule. The hydrophilic and hydrophobic

domains/segments within the molecular geometry of amphiphilic lipids orient and self

orgainzie in ordered supramolecular structure when confronted with solvents.In aqueous

22

medium the molecules in self assembled structures are oriented in such a way that the

polar portion of the molecule remains in contact with the polar environment and at the

same time shields the non-polar part. Among the amphiphiles used in the drug delivery

viz. soaps, detergents, polar lipids, the latter (polar lipids) are often employed to form

concentric bilayered structures. However, in aqueous mixture these molecules are able

to form various phases, some of them are stable an others remain in the metastabel

state. At high concentrations of these polar lipids, liquid-crystalline phases are formed

that upon dilution with an excess of water can be dispersed into relatively stable

colloidal particles. The macroscopic structures most often formed include lamellar,

hexagonal or cubic phases dispersed as colloidal Nanoconstructs (artificial

membranes) referred to a liposomes, hexasomes or cubosomes , respectively.The most

common natural polar phospholipids are phosphatidylcholine (PC). These are amphipathic

molecules in which a glycerol bridge links to a pair of hydrophobic acyl hydrocarbon

chains with a hydrophilic polar head group, phosphocholine explains that the fatty chains

are embedded in the hydrophobic inner region of the membrane, oriented at an angle to

the plane of the membrane surface, the hydrophilic head group, including the phosphate

portion. Points out towards the hydrophilic aqueous environment. Molecules of PC are not

soluble (rather dispersible) in aqueous media in the physical chemistry sense, as in

aqueous media they align themselves closely in planer bilaryer sheets to minimize the

unfavorable interactions between the bulk aqueous phase and long hydrocarbon fatty acyl

chain. Such interactions are completely eliminated when the sheets fold over themselves

to form closed, sealed and concentric vesicles. The large free energy change between an

aqueous and hydrophoble environment explains the most favored orientation of lipids to

assemble as concentric bilayer structures that exclude confrontation between aqueous and

hydrophobic domains.Thus the amphipathic (amphiphilic) nature of Phospholipids and

their analogues render them the ability to form closed concentric bilayers in the presence of

water. Liposomes (lipid vesicles) are formed when thin lipid films or lipid cakes (of

amphiphilic nature) are hydrated and stacks of liquid crystalline bilayers become fluid and

swell.

2.1.2 METHODS OF LIPOSOME PREPARATION:

Liposomes are manufactured in majority using various procedures in which the water

soluble (hydrophilic) materials are entrapped by using aqueous solution of these materials

as hydrating fluid or by the addition of drug/drug solution at some stage during the

manufacturing of liposomes (Ostro, 1987, 1989; Talsma and Cromunelin, 1992). The

lipid soluble (lipophilic) materials are solubilized in the organic solution of the constitutive

lipid (s) and then evaporated to a dry drug containing lipid film followed by its hydration.

These methods involve the loading of the entrapped agents before or during the

manufacturing procedure (passive loading ) However, certain types of compounds with

ionizable groups,

Fig-2.1:liposome drug delivery

then membrane phospholipids are disrupted, they can reassemble themselves into tiny

spheres, smaller than a normal cell, either as bilayers or monolayers. These are liposomes.

The lipids in the plasma membrane are chiefly phospholipids like phosphatidyl

ethanolamine and cholesterol. Phospholipids are amphiphilic with the hydrocarbon tail of

the molecule being hydrophobic; its polar head hydrophilic. As the plasma membrane faces

watery solutions on both sides, its phospholipids accommodate this by forming a

phospholipid bilayer with the hydrophobic tails facing each other.

Liposomes can be composed of naturally-derived phospholipids with mixed lipid chains

(like egg phosphatidylethanolamine), or of pure surfactant components like DOPE

(dioleoylphosphatidylethanolamine). Liposomes, usually but not by definition, contain a

core of aqueous solution; lipid spheres that contain no aqueous material are called micelles,

however, reverse micelles can be made to encompass an aqueous environment.

2.1.3 CHEMICAL CHARACTERIZATION OF LIPOSOMES:

Various chemical analysis methods used for quantitative and qualitative tests of liposomal

components prior to and after the preparation are critical characteristics of liposomes

(Barenholz and Cromellin 1994). These methods become more essential to characterize

liposomes, which require lipid stability cropping up from oxidatin, lipid peroxidation,

hydrolysis and degradation ni various environment used in their manufacturing6.

Fracture electron microscopy, photon

correlation spectroscopy. Laser light

scattering, gel permeation and gel exclusion.

Surface charge Free- flow electrophoresis.

Phase behaviour Freeze-fracture electron microscopy.

Drug release Diffusion cell/ dialysis

3. Bioligical Chractcrization

Sterility Aerobic or anaerobic cultures

Pyrogenicity

Animal

Rabbit fever responses test

Monitoring survival rates. Histology and

pathology.

TABLE-2.1: Liposome Characterization with their Quality Control

Assays –

Characterization parameters Analytical method/ Instrumentation

1. Chemical Characterization

Phospholipid concentration Lipid phosphorus content using Barlett assay

Stewart assay, HPLC

Cholesterol concentration Cholesterol oxidase assay and HPLC

Phospholipid peroxidation UV absorbance. TBA ( for endoperoxidase),

iodometric (for hydroperoxidase) and GLC.

Phospholipid hydrolysis HPLC and TLC and fatty acid concentration

Cholesterol auto-oxidation HPLC and TLC

Anti- oxidatant degradation HPLC and TLC

pH pH meterr

Osmolarity Osmometer

2. Physical Characterization

Vesicle shape and surface morphology Transmission electron microscopy , freeze-fricture

electron microscopy

Vesicle size and size distribution

Submicron range

Micron range

Dynamic light scattering, transmission electron

microscopy, zetasizer Transmission electron

microscopy,

2.2 NANOPARTICLES:

The colloidal earners based on biodegradable and biocompatible polymeric systems have

largely influenced the controlled and targeted drug delivery concepts. It was realized that

the nanoparticles loaded bioactives could not only deliver drug (s) to specific organs within

the body but delivery rate in addition could be controlled as being by standers, burst,

controlled, , pulsatile or modulated. The possibilities and potentials further prompted the

work and as a result a great deal of related information covering preparation

methodologies, characterization. Engineering, bio-fate and toxicology has been gathered.

The understanding that relates to the biodistribution in particular has propelled and

motivated the development of functionally designed nonoparticulates6.

2.2.1 PREPARATION TECHNIQUES OF NANOPARTICLES:

The selection of the appropriate method for the preparation'of nanoparticles depends on

the physicoehemical characteristics of the polymer. Various Proteins and Polysaccharides

used for the prepartion of Nano particles:

Proteins Polysaccharides

Gelatin Alginate

Albumin Dextran

Lectins Chitosan

Legunin Agarose

Vicilin Pullulan

The drug to loaded. on the contrary, the preparation techniques largely determine the inner

structure, in vitro release profile and the biological fate of these polymeric delivery systems

.Two types of systems with different inner structure are apparently possible matrix type

system consisting of entanglement of oligomeror polymer units

(nanoparticles/nanoshpheres). A reservoir type of system comprised of an oily core

surrounded by an embryonic polymeric shell (Nanocapsules). The Polymers are strictly

structured to a nanometric size range particle (s) using appropriate methodologies.

FIG-2.2: Flowchart of preparation of nanoparticles

These methodologies are conveniently classified as follows:

1. Amphiphilic macromolecule cross-linking.

a. Heat cross-linking

b. Chemical cross-linking

2. Polymerization based methods

a.  Polymerization of monomers in situ

b. Emulsion(micellar) polymerization

c.Dispersion polymerization

d.Interfacial condensation polymerization

e.Interfacial complexation

3. Polymer precipitation methods

a. Solvent extraction/evaporation

b. Solvent displacement (nanoprecipitation)

c. Salting out

FIG-2.3:NANOPARTICLE

2.2.2 CHARACTERIZATION OF NANOPARTICLES:

The nanoparticles are generally characterized for size, density, electrophoresis mobility,

angle of contact and specific surface area.

2.2.2.1 SIZE AND MORPHOLOGY:

The particle size is one of the most important parameters of nanoparticles. Particles size

and sizing of sub-optical particulates is a different procedure, as it involves not only

procedural variability, but some of the surface associated properties may even change

during sizing procedure. Two main techniques are being used to determine the particle size

distribution of nanopartitcles and include photon correlation spectroscopy (PCS) and

electron microscopy. The latter (TEM) and freeze -fracture techniques. The size evaluation

of nanoparticale dispersion demonstrates better results with freeze-fracturing microscopy

and photon correlation spectroscopy as quantitative methods. The freeze-fracturing with

poly (methyl methacrylate) is confronted with and interrupted by in process

particles aggregation which only yields a few discrete particles for size measurement of

analysis. The electron microscopy however, could be adopted as an alternative option,

which measures individual particles for size and its distribution. It is relatively less time

consuming. Additionally, freeze fracturing of particles allows for morphological

determination of freeze fracture procedures, TEM permits differentiation among

nanocapsules, nanoparticles and emulsion droplets. Similarly, scanning electron

microscopy is much less time consuming. However, since particles are based on organic

and non-conductive material, they require from 30-50 nm. Thus determined size should be

denoted as gold- coated particle size rather than as particle size.

Table2.2: Different Parameters and Characterization Methods for

Nanoparticles:

Parameter. Characterization Method (S)

Particle size and size distribution Photon correlation Spectroscopy (PCS)

laser defractometnnry transmission electron microscopy 

scannming Electron Microscopy.

Charge determination Laser doppIcr Anemomclry Zeta potentiometer

Surface hydrophobicity Water contact angle measurements rose bengal binding

hydrophobic interaction chromatography X- ray

photoelectron spectroscopy

Chemical analysis of surface Static secondary ion mass spectrometry Sorptometer .

Carrier- drug interaction Differential scanning calorimetry.

Nanoparticle dispersion stability Critical flocculation temperature (CFT)

Release profile In vitro release characteristic under physiolgic and sink

conditions

Drug stability Bioassay of drug extracted from nanoparticles Chemical

analysis of drug.

2.2.2.2 SPECIFIC SURFACE:

The specffic surface area of freeze-dried nanoparticles is generally determined with the

help of sorptometer (Kreuter, 1983). The equation can be used in the calculation of

specific surface area.

Inmost of the cases, the measured and calculated specific surface areas fairly compare

while in some cases the residual surfactant could affect deviation in measured values.

The surfactant coating apparently reduces the specific surface area.

2.2.3 APPLICATION OF NANOPARTICLES :

Nanoparticles with different composition and characteristics have been formulated and

investigated for various therapeutic applications sevral different types of biodegradable

polymers including biopolymers (e.g. gelation, albumin, casein, polysaccharide, lectin etc.)

and synthetic polymers (polycaprolactone, polyesters, polyanhydrides, polycyanoacrylates)

with various drug release characteristics ranging from several hours to several months

have been used to formulate sustained release nanoparticles. It is the submicron size of

this delivery system, which makes it more efficient in certain drug therapy applications,

such as in intracellular localization of therapeutic agents. These systems, in addition to

sustained drug delivery have been investigated for various therapeutic applications7.

2.3 RESEALED ERYTHROCYTES:

These are prepared by placing RBC's in hypotonic media which leads to rupturing of cell

membrane & formation of pores (dia 200-500A) through which intracellular & erxtracellular

exchange takes place. In this way a drug compound in extra cellular media entrees the RBC's are

loaded into RBC's.. Resealed erythrocytes are biodegradable, non-immunogenic. Resealed

erythrocytes can be used to target drug to liver & spleen.

Desirable properties of released erythrocytes:

Biodegrability Circulate throughout the circulatory system.

Large quantities of material can be encapsulated within small volume of cells.

Can be utilized for orgn targeting within RES.

A wide variety of bioactive gents can be encapsulated within them. Erythrocytes

biocompatible provided that compatible cells are used in patients there is no possibility of

triggered immunological response8. 

2.3.1 DRUG TARGETING:

A drug delivery should ideally be site-specific & target oriented in order to exhibit maximal

therapeutic index & minimum side & toxic effects. It has been observed that osmotically

loaded erythrocytes can act as drug carriers in systematic circulation, whereas chemically

surfaces modified erythrocytes are targeted to organs of the mononuclear phagocytic

system/ reticuloendithelial system (MPS/RES) because of change incorporated in the

membranes that are recognized by macrophage cells.

FIG -2.4: RESEALED ERYTHROCYTES

2.3.2 DRUG TARGETING TO RES ORGANS:

The damaged erythrocytes are quickly removed from circulation by phagocytic kupffer cells

located in liver & spleen. Though, released erythrocytes have been proposed for passive auto-

vectorization to MPS/RES system where modified surfaces characteristics lend them

selectivity & specificity towards target cells (mainly liver & spleen).

2.3.3 RES IN OXYGEN DEFICIENCY THERAPY:

Released erythrocytes are also used in cases of oxygen deficiency where an improved oxygen

supply is required as in the following cases:

1. High altitude conditions (Where partial pressure of oxygen is low).

2. Small number of alveoli (Where lung exchange surface is low).

3. Increased resistance to oxygen diffusion in the lungs.

2.3.4 NOVEL SYSTEM (NANOERYTHROSOMES):

An erythrocyte based new drug carrier, named nanoerythrosome has been developed which

is prepared by extrusion of erythrocyte ghosts to produce small vesicles having an average

diameter of 100 nm. Daunorubicin was covalently conjugated to the nEryt using glutraledhyde as

homobifunctional liking arm. This given has a higher antoneoplastic index than free drug 9.

2.4 MICROSPHERES :

The term microsphere is defined as a spherical particle with size varying from 50 nm to 2

nm, containing a core substance. Microspheres are,in strict sense, a spherical empty

particles. However, the term microcapsules and microsphere are often used synonymously.

The microsphere are characteristically free flowing powders consisting of proteins or

synthetic polymers, which are biodegradable in nature and ideally having particle size less

than 200 micrometer.

2.4.1 TECHNOLOGY AND APPLICATIONS:

2.4.1.1 POWDERS AND GRANULATES :

Free-flowing powders and granulates are needed for a variety of industrial processes. These,

however, do not always meet the exacting standards which modern manufacturing demands

of them, due to their varying grain size distribution and odd shapes. These properties are

detrimental to efficient processing and lead to agglomeration, inexact dosage, abrading

with loss of material, or low reproducibility of castings.

Pharmaceutical applications require highly reproducible dosage and the controlled release of

active agents, which can not be achieved with conventional powders and granulates.

The use of small and perfectly round Microspheres with exactly the same size circumvents

all of the disadvantages that are encountered while using powders and granulates. These

Microspheres are free-flowing and roll with practically no friction, that means there is no

abrasion,  guaranteeing a dust-free environment.

Fig- 2.5: Flow chart of microsphere synthesis

2.4.2 PROCESSING CHARACTERISTICS:

Microsphere production units have a minimal space requirement (15 to 40 ft2), the energy

consumption is very low and they are noiseless during operation. These units operate at

atmospheric pressure or slightly above and can be designed to be explosion-proof and/or

according to the GLP/ GMP guidelines. Microsphere production units from BRACE need

practically no maintenance, therefore only a minimal staff is required. Metal oxide spheres

as molded (yellow), dried (yellow transparent), calcined (black) and sintered (black,

smallest). The shrinkage in diameter corresponds to their solid content during sintering.

Units are delivered with automated controls and can be delivered as remote controlled and

enhanced10.

2.4.3 TYPES OF MICROSPHERES:

There are very few restrictions on the types of Microspheres than can be produced. With the

right combination of liquid precursor, solidification process, and subsequent treatments, a

wide range of Microspheres can be produced.

Dry metal oxide Microspheres produced on the basis of a sol (Al2O3, ZrO2, HfO2,

TiO2, CeO2, SiO2, and mixed oxides) can be used as highly sinteractive press-feed

for the production of high-tech ceramics.

Through calcining, the pore size and surface area of the Microspheres can be

tailored to exacting specifications. These Microspheres make excellent

catalystcarriers, homogeneous catalysts, or filtering materials. Unusually effective

and abrasion resistant Microspheres for grindingther materials are made from

sintered Al, Zr, and Hf-oxides.

Fig -2.6:Comparison of size of microsphere

Monodispersive alumina Microspheres.

Ultra Spherical Microspheres .

Microspheres with a monodisperse grain sizedistribution and the smallest

divergence are manufactured by BRACE.

perfectly spherical Microspheres .

monodisperse grain size, narrow sizedistribution with diameters between

50µm and 5000µm nonabrading, therefore dust-free, free flowing, porous,

large surface area, soft or rigid for embedding pharmaceuticals, biomass (e.g.,

yeast or enzymes) or other heterogeneous catalysts with or without coating.

2.4.4 USES OF MICROSPHERE:

Microspheres produced from molten materials (inorganic, organic, alloys, and

polymers) can be used for dosing, proportioning, compounding, coloring, and light

stabilization. Microspheres with dissolved or embedded active agents, with or

without coating, are used for numerous pharmaceutical and cosmetic products.

Soluble chemical compounds can be incorporated into Microspheres by precipitation

for use in the agricultural, food, pharmaceutical, and cosmetics industries.

Suspensions are used to produce Microspheres with embedded enzymes or bacteria.

With our special double nozzle systems, Microspheres with encapsulated materials

can be obtained. Especially for the encapsulation of water, aqueous solutions or cells,

a microsphere with a liquid core and a solidified shell can be produced. The shell and

the core material can be chosen as appropriate: alginate, PVA, PEI, PEG, wax, metal

oxides, gelatin, hydroxylcelluloses, etc11.

CHAPTER-3

DRUG DELIVERY SYSTEM IN BRAIN

Drug delivery to specific locations in the brain is a challenging task in the treatment of

diseases related to Central Nervous System (CNS) such as brain tumors, epilepsy,

Parkinson's, Alzheimer's and Huntington's diseases owing to the blood-brain barrier

(BBB). There is an interest from medical community in delivering Glial-Derived

Neurotrophic Factor (GDNF) and Brain-Derived Neurotrophic Factor (BDNF) drugs at

specific locations to CNS [1]. It is also known that these drugs consisting of big molecules

cannot overcome the BBB. A promising solution to this problem is to deliver the required

drug into the targeted location by invasive techniques as the convection enhanced

delivery (CED).

In this work, computational fluid dynamics (CFD) techniques are utilized to study

invasive drug delivery in multi-dimensional brain geometries with the consideration

of the chemical interactions that the drug undergoes while it diffuses into the brain

tissue. A challenge is to accurately reconstruct the three-dimensional structure of the

human brain. We are able to resolve very accurately the brain geometry and render

physiologically consistent the distribution of the complex brain inner organization.

We distinguish between gray and white matter and assign transport properties of

relevance according to the data obtained by MR images or histological data. We will

quantify with numerical simulations the diffusive and convective transport

phenomena in the porous brain tissues and the effectiveness of the drug release to a

desired region. This approach will help to evaluate precisely the penetration depth of

the drug and the concentration profiles need to surpass set thresholds in order to

ensure proper efficacy of the drug. We rigorously examine the variables that influence

CED and pose constraints to the treatment. These include effect of infusate (bulk)

flow rate, concentration of the infusate, drug diffusivity, effect of molecular weight of

the drug, and effect of white matter anisotropy, infusate leak-back by considering

metabolic uptake by the parenchyma cells and re-absorption of the bulk fluid [2].

Understanding the parameters that could possibly influence the convective delivery of

drugs in the CNS is very important because, it will improve the current medical

approaches. The proposed methodology will provide a systematic approach to

optimally choose catheter dimensions, infusion rates, drug concentrations etc. The

information obtained from these accurate simulations could be used to model inverse

kinetic problems capable of predicting the mass diffusivity of the drug and the kind of

metabolism that actually takes place.

Brain targeted transcranial route of drug delivery of diazepam

The term transcranial route means the brain targeted transfer of drug molecules across

the cranium through the layers of the skin and skin appendages of the head, arteries

and veins of the skin of the head, the cranial bones along with the diploe, the cranial

bone sutures, the meninges and specifically through the emissary veins. The

administration of drugs through the scalp in ayurvedic system for the diseases

associated with the brain was evaluated with a view to develop a novel targeted route

for central nervous system drugs. It is expected to circumvent the systemic side

effects of oral route. Diazepam was dissolved in an oil medium and applied on scalp

as practiced in the ayurvedic system. Thirty rats were tested on the rotating rotarod for

muscle relaxant effect of diazepam. Five groups of rats tested were the control,

diazepam i.v. injected (280 µg/0.1 ml) group, two groups treated with transcranial

diazepam oil solution (1.5 mg/0.2 ml) and the transcranial blank vehicle treated

groups. Holding time in triplicate for each rat on the rotating rotarod was measured.

The holding times following each treatment was statistically compared (one-way

ANOVA). The pooled average times for the control, diazepam i.v. injected, diazepam

oil solution transcranial treated two groups and the blank vehicle treated groups were

35.45, 4.73, 16.5, 15.39 and 33.23 seconds respectively. The two groups subjected to

the brain targeted transcranial route showed a statistically significant decrease (50%

drop) in the holding time against the control group indicating the centrally acting

muscle relaxant effect due to absorption of diazepam into the brain through the

proposed route.

Man entertained a special care in all matters relating to the head because the head

housed the brain. The effects of a bath are remarkably different from that of a body

wash. A head injury, however trivial, is considered a precarious situation and a pimple

on the face may be fatal. The apprehensions mentioned above and the drug delivery

route that is being undertaken in the present study is incidental to a special anatomical

feature of the skull. The emissary veins draining blood from extracranial sites into the

intracranial sinuses pierce a series of foramina present in the cranial bones. Seven

major sinuses within the skull are interconnected by a number of anastomosing veins,

which finally drain intracranially into the jugular veins giving ample scope for the

diffusion of the drug molecules into the nerve tissue of the brain. There are thirteen

emissary veins connecting extracranial sites of the head with the intracranial

sinuses[1]. The emissary veins are present in all higher animals starting with aves[2]

and their presence in the horse is well established.

The arteries of the scalp send small twigs to the underlying bones of the scalp. The

spongy diploe within the flat skull bones is also well supplied by numerous small

diploic branches from arteries both on external and internal surfaces of the skull[3].

These anatomical arrangements of the vascular system are made use of in the

investigations to develop the brain targeted transcranial route (abbreviated TCR) of

drug delivery.

In the ayurvedic system there are five methods, namely Shirodara, Shiroabyanga,

Shiropichu, Shirovasthi and Shiropralepa in which drugs are delivered by the

transcranial route[4]. Most of these ayurvedic preparations are oil based. There are

many household ayurvedic medicinal head oils for minor ailments such as headaches,

sinusitis, vertigo and migraine. An important dosage design feature in this study is the

use of the essentially non polar active diazepam drug moiety dissolved in an oil

medium as against the use of polar salt forms in aqueous media that are popular with

the modern formulations. Diazepam was selected as the screening agent since it is a

prototypical benzodiazepine acting in the central nervous system. Diazepam has

central depressant and centrally acting skeletal muscle relaxant effects[5]. The skin

area of the head which drains venous blood through emissary veins into the

intracranial locations is probably the region lying above the circular contour drawn

through the angles of the mouth and the ears. Therefore the venous blood draining the

eyes, ears and the nose are also drained by this route.

The proposed transcranial route is intended to circumvent the side effects encountered

during treatment by the oral route. Some of the central nervous system diseases such

as epilepsy need long term therapy. There are prospects of adopting the transcranial

route for several groups of drugs. They include antiepileptics, antipsychotics,

tranquilizers, analeptics, antiparkinsonian drugs, those acting on the endocrine glands

located in the brain, drugs related to diseases of the labyrinth, glaucoma,

anticoagulants and those employed in the treatment of drug addiction. CNS side

effects of other drugs could be counteracted by administering the specific antagonists

through transcranial route. There is a good prospect in adopting the transcranial route

in veterinary practice as well. Development of subscalpal injections is another

possibility. The proposed brain targeted transcranial route of drug delivery could be

viewed as a parallel drug delivery system to that of metered dose inhalers in diseases

of the respiratory system.

Ethical clearance for animal experiments was granted by the Ethical Review

Committee of the Medical Faculty, Colombo. Both male and female inbred Sprague-

Dawley rats weighing between 165-230 g were used. They were divided into five

groups of six rats including three from either sex. In the animals that were subjected

to the treatment by transcranial delivery route the hair of the scalp was trimmed with

scissors without injuring the skin. It was done within the trapezoidal area of the head

bound by the pair of eyes and ears, closer to the line joining the ears than the eyes.

They were kept at room temperature (around 30°) and fed with adult rat meal pellets

and water without any restriction. They were appropriately numbered with picric acid.

Rotating rotarod, constant rpm:

The equipment was devised by having an electrically driven horizontal circular rod 18

mm in diameter, 29 cm in length rotating constantly at 15 rpm, mounted on two side

panels 43 cm above the base. The rod was covered with no. 300 silicon carbide

abrasive paper to provide roughness for the rats to grip the rod firmly.

Reformulation of human diazepam i.v. injection for rats:

Diazepam i.v. injection 5 mg/ml, 2 ml ampoules, manufactured by Lab Renaudin,

France was reformulated into i.v. injection for rats. Adult human reference i.v. dose of

diazepam 10 mg/60 Kg[6] is equivalent to 35 µg/210.6 g rat on a weight basis.

Considering the ten fold metabolic rate in rats as against man an eight fold increase in

the dose was considered, i.e., 35 µg×8=280 µg of diazepam per rat. Since the strength

of the commercial diazepam injection is 5 mg/ml or 5 µg/µl the number of microlitres

required for 280 µg of diazepam from the original commercial injection is 280/5 µl =

56 µl. Volume to be injected into a rat was restricted to 100 µl. Therefore the volume

of the diluting vehicle needed was 100 µl-56 µl=44 µl. The diluting vehicle was a 1:2

cosolvent of propylene glycol:50% ethyl alcohol. On the above basis the i.v. injection

formula for rats was prepared aseptically using twenty times the following amounts to

yield 2 ml of the injectable solution. 56 µl of diazepam i.v. injection 5 mg/ml

(equivalent to 280 µg of diazepam), propylene glycol 14 µl and 50% ethyl alcohol 30

µl.

Formulation of diazepam oil solution 1.5 mg / 0.2 ml for transcranial route

(TCR):

It was prepared by dissolving 30 mg of diazepam (250 mesh powder) in 1 ml

isopropyl alcohol by shaking for 15-30 min and then adding 3 ml of sesame oil to the

above solution and kept mixing until a clear solution formed. The product was left

overnight for complete dissolution. The dose and the volume (1.5 mg in 0.2 ml) for

TCR administration was decided based on the results of preliminary trials. The trials

indicated that the doses which are a few multiples of the intravenous dose did not

elicit marked central effects on the animals by the TCR. The volume 0.2 ml was the

amount that could be accommodated in the restricted scalp area of the rat without

undue spreading.

Blank oil vehicle 0.2 ml for TCR administration was prepared by dissolving 1 ml of

isopropyl alcohol in 3 ml of sesame oil. Disposable insulin syringes of 100 units/1 ml

capacity with 29 gauge needle were used for rat dorsal tail vein i.v. injection as the

positive control and also to deliver the oil based formulation drop wise onto the scalp

of the animals.

The animals were divided into the following five groups. Group 1, untreated animals

(blank). Group 2, reformulated diazepam tail vein i.v. injected animals (positive

control). Group 3, transcranial diazepam oil solution treated animals tested 15 minutes

after drug application (test group A). Group 4, same as Group 3 but tested 45 minutes

after drug application (test group B). Group 5, transcranial blank vehicle treated

animals tested 15 minutes after the application (control group).

One animal at a time was placed on the rotating rod. Rats fell off the rod when the

grip was lost. The holding time on the rod in seconds was observed for each animal.

Each rat was subjected to three rotarod trials, five minutes apart.

Group 2 was tested on the rotarod 15 minutes after the i.v. injection. Group 3 (test

group A) and Group 4 (test group B) were tested as follows. Each rat was treated with

0.2 ml of the transcranial diazepam oil formulation containing 1.5 mg of diazepam

using insulin syringe. The oil solution in 1/3 quantities were delivered drop wise on to

the hair trimmed area of the scalp leaving a gap between the needle end and the skin

in three stages as follows. First application at 00:00 time, 2nd application at 00:15

minutes and 3rd application at 00:45 minutes. The first application was followed by

gentle rubbing on the scalp with a gloved finger previously smeared in the diazepam

oil formulation by stroking ten times in a cranial to caudal direction. This was to

facilitate dispelling any air pockets and to bring the oil solution into intimate contact

with the skin of the scalp.

Each animal was tested thrice 5 minutes apart on the rotating rotarod starting 15

minutes after the 3rd application in Group 3 and starting 45 minutes after the 3rd

application in Group 4, respectively. Accordingly animals were tested one hour after

the first application in Group 3 and one and a half hours after the first application in

Group 4. In testing Group 5 the animals were treated with blank oil solution similar to

Group 3 and tested 15 minutes after the 3rd application of the vehicle. Statistical data

analysis was done using SPSS software package. One-way ANOVA and Dennett T 3

Post Hoc test was done to compare mean holding times between the groups.

Holding time on the rotating rotarod:

Mean value of three trials were calculated for each animal in all five groups. The

individual means of time in seconds on the rotating rotarod of six animals in each

group were pooled to get the mean holding time on the rod for each group [Table - 1]

Statistical comparison of mean holding times between groups showed significant

difference between the groups (1-way ANOVA, P < 0.05). 1-way ANOVA followed

by multiple comparisons with Dunnett T3 Post Hoc test showed statistically

significant difference between group 1 and groups 2, 3 and 4, while there was no

significant difference between groups 1 and 5. There was no statistically significant

difference between the mean holding times between group 3 and 4 (test groups A and

B) while both groups 3 and 4 showed a statistically significant difference from group

2 and 5.

The mean holding times on the rotarod for group 1 (untreated) and the transcranial

blank vehicle treated group (Group 5) being not significantly different (35.45 and

33.23 s, respectively) suggests that there are no effects of either isopropyl alcohol or

sesame oil or scalp stroking on the holding time and therefore on the muscle

tone/grip. The diazepam i.v. injected group 2 as expected had a significant effect on

the mean holding time, bringing it down to 4.73 seconds, further proving the known

muscle relaxant effect of diazepam.

The two groups subjected to transcranial route treatment with diazepam showed

statistically significant reduction in mean holding times from that of the untreated and

the vehicle treated groups and this reduction was almost by 50% [Figure - 1]. These

results suggest that the diazepam drug molecules have been conveyed transcranially

in to the nerve tissue of the rat brain under the experimental conditions described

here. However the mean holding times of the transcranially treated groups were

longer than in the i.v. treated group and these differences were statistically significant,

suggesting that the amount of diazepam delivered by the transcranial route to the CNS

is significantly lower than that delivered by the i.v. route. This difference in diazepam i

delivery may be due to the novel experimental route and the properties of the oily

formulation of diazepam prepared for transcranial application.

The results of the experiments further indicate that after a certain point irrespective of

the concentration of the drug in the oil solution, the volume applied and the time

allowed before testing the response to the drug tend to even out unlike in the

conventional oral or parenteral routes. This is evident by the fact that the holding

times are nearly the same for two transcranially treated groups despite the

substantially longer time allowed for the group 4 to effect the diffusion of the drug.

There appear to be a wide therapeutic window for diazepam administered by the

transcranial route.

On these findings we suggest further experiments with improved formulations of

diazepam and other CNS drugs to establish transcranial route as an effective and

convenient route of drug administration.

  

   

CHAPTER-4

APPLICATION OF TARGETED DRUG DELIVERY

SYSTEM IN BRAIN:

Scientists began to study targeted drug delivery, because the traditional drug delivery

system had many disadvantages, such as high toxic effect and high minimum

effective dose. In traditional drug delivery system, after the patient takes some drugs,

the drugs will distributed throughout his body through the systemic blood circulation.

Only a small amount of drugs can reach the affected organ which it needs to act on.

Since many drugs have some toxicity, they can kill some helpful bacteria or normal

cells in some normal organs.

The targeted drug delivery system can overcome these shortcomings and deliver the

drugs right to the specific organ, without having any adverse effects on other healthy

organs and tissues. Actually, the targeted drug delivery can be used to cure many

diseases, such as the cardiovascular diseases and diabetes. However, the most

important application of the targeted drug delivery is to treat the cancerous tumor.

There are two kinds of targeted drug delivery. The first one is active targeted drug

delivery, such as some antibody drugs. The second one is passive targeted drug

delivery, such as the Enhanced Permeability and Retention effect (EFR-effect). Some

Important applications are given below.

(1) Magnetic drug targeting: Tumor targeting:

Magnetic drug targeting allows the concentration of drugs at a defined target site

generally and importantly, away from the reticular endothelial system (RES) with

the aid of a magnetic field. Site-directed drug targeting is one way of local or

regional antitumor treatment. The drug & an appropriate Ferro fluid are formulated

into a pharmaceutically stable formulation which is usually injected through the

artery that supplies the target organ or tumor in the presence of an external

magnetic field. Prolonged retentions of the magnetic drug carrier at the target site

alleviate or delay the RES clearance & facilitates extra vascular uptake. For

effective retaining of magnetic drug carrier, the magnetic forces must be high

enough to counteract liner flow rates within the organ or tumor tissue (between 10

& 0.05 cm/s depending on vessel size & branching pattern . There is increase in

drug concentration in the target tissue after administration of the drug dose has

been observed.The efficiency of chemotherapy treatment may be enhanced to a

great extent by magnetically assisted delivery of cytotoxic agent to the specific

site. There are a large number of magnetic carrier systems which demonstrates

increasing drug concentration efficiency at the tumor site.

Magnetism can play very important role in cancer treatment. The first clinical

cancer therapy trials using magnetic microspheres were performed by Lubbe et al.

in Germany for the treatment of advanced solid tumor while current preclinical

research is investigating use of magnetic particles loaded with different

chemotherapeutic drugs such as mitoxantrone, paclitaxel. Non invasive permanent

magnetic field for one hour way found to induces lethal effects on several rodent &

human cancers. Anticancer drugs reversibly bound to magnetic fluids & could be

concentrated in locally advanced tumors by magnetic field that or arranged at

tumor surface outside of the subject.

In case of brain tumors, the therapeutic ineffectiveness of chemotherapy is mainly

due to the impervious nature of the blood-brain barrier (BBB), presence of drug

resistance and lack of tumor selectivity. Various novel biodegradable magnetic

drug carriers are synthesized and their targeting to brain tumor is evaluated in vitro

and in animal models. New cationic magnetic aminodextran micro spheres

(MADM) have been synthesized. Its potentiality for drug targeting to brain tumor

was studied. this particles were retained in brain tissue over a longer period of

time.

A magnetic fluid has been reported to which the drugs, cytokines & other molecule

can be chemically bound to enable that agent to be directed within subject under

the influence of high energy magnet. In one of such examples magnetic

doxorubicin in liposome, significant anticancer effect in nude mice bearing colon

cancer .

(2) Magnetic bioseparation:

Bioseparation is an important phenomenon for the success of several biological

processes. Therefore, prospective bioseparation techniques are increasingly

gaining importance. Amongst the different bioseparation techniques, magnetic

separation is the most promising. The development of magnetically responsive

microspheres has brought an additional driving force into play. Particles that are

bound to magnetic fluids can be used to remove cells and molecules by applying

magnetic fields and-in vivo-to concentrate drugs at anatomical sites with restricted

access. These possibilities form the basis for well-established biomedical

applications in protein and cell separation. Additional modifications of the

magnetic particles with monoclonal antibodies, lectins, peptides, or hormones

make these applications more efficient and also highly specific.

The isolation of various macro molecules such as enzymes, enzyme inhibitors,

DNA, RNA, antibodies and antigens etc. from different sources including nutrient

media, fermentation broth, tissues extracts and body fluids, has been done by using

magnetic absorbents. In case of enzyme separation, the appropriate affinity ligands

are immobilized on polymer coated magnetic carrier or magnetizable particles.

Immobilized protein A or protein G on silanized magnetite and fine magnetotactic

bacteria can be used for isolation and purification of IgG.  Monosized super

paramagnetic particles, Dynabeads, have been used in isolation of mRNA,

genomic DNA and proteins.

(3) Magnetically induced Hyperthermia for treatment of

cancer:

Heat treatment of organs or tissues, such that the temperature is increased to 42–46

C and the viability of cancerous cells reduces, is known as hyperthermia. It is

based on the fact that tumor cells are more sensitive to temperature than normal

cells. In hyperthermia it is essential to establish a heat delivery system, such that

the tumor cells are heated up or inactivated while the surrounding tissues (normal)

are unaffected.

a)Intracellular hyperthermia:The alternative approach is to use fine particles as

heat mediators instead of needles or rods such that hyperthermia becomes

noninvasive. When fluids containing submicron-sized magnetic particles(typically

1–100nm) are injected, These particles are easily incorporated into the cells, since

their diameters are in the nanometer range. These magnetic particles selectively

heat up tissues by coupling AC magnetic field to targeted magnetic nano particles.

As a result, the whole tumor can be heated up uniformly This is called intracellular

hyperthermia. It has been shown that malignant cells take up nine times more

magnetic nano particles than normal cells. Therefore the heat generated in

malignant cells is more than in normal cells. Also, as blood supply in the

cancerous tissues is not normal, the heat dissipation is much slower. Hence, the

temperature rise in the region of tumor is higher than in the surrounding normal

tissues. It is therefore expected that this therapy is much more concentrated and

localized.

b) Magnetic fluid hyperthermia (MFH):Magnetic fluids can be defined as fluids,

consisting of ultramicroscopic particles. (~100Å) of magnetic oxide. Magnetic

fluid hyperthermia is based on the fact that sub domain magnetic particles produce

heat through various kinds of energy losses during application of an external AC

magnetic field. If magnetic particles can be accumulated only in the tumor tissue,

cancer specific heating is available, various biocompatible magnetic fluids.

Cationic magnetoliposomes and affinity magnetoliposomes have been used for

hyperthermia treatment.

c) Combination therapy: There also exists the combination therapy which would

induce hyperthermia treatment followed by chemotherapy or gene therapy. A

combination of chemotherapy or radiation therapy with hyperthermia is found

much more effective than hyperthermia itself. The approach involves use of

magnetic carriers containing a drug to cause hyperthermia using the standard

procedure, followed by the release of encapsulated drug that will act on the injured

cells. It is anticipated that the combined treatment might be very efficient in

treating solid tumor. Several reasons are given for the enhanced effect. Tumors are

poorly vascularised and it can be hard for therapeutic agents to reach their target.

Heat increases the perfusion of a tumor and therefore drugs are transported more

effectively into the target tissues. In addition, heat makes blood vessels more

permeable to drugs. This occurs preferentially in tumors where blood vessels tend

to be structurally incomplete. On the other hand, normal blood vessels are

surrounded by a basement membrane and other perivascular cells and not

significantly affected by heat. It has recently been reported that hyperthermia

increases the rate of liposome leakage into tumors by a factor of 2–5 depending on

the type of tumor. In normal tissues however, enhancement of liposome leakage is

not reported.

(4) Magnetic control of pharmacokinetic parameter rand

Improvement of Drug release:

Langer et al.embedded magnetite or iron beads in to a drug filled polymer matrix

and then showed that they could activate or increase the release of drug from the

polymer by moving a magnet over it or by applying an oscillating magnetic field

(Langer et al.,1980; Edelman and Langer,1993 ).The microenvironment with in the

polymer seemed to have shaken the matrix or produced ‘micro cracks’ and thus

made the influx of liquid, dissolution and efflux of drug possible  thereby

achieving magnetically controlled drug release. Macromolecules such as peptides

have been known to release only at a relatively low rate from a polymer controlled

drug delivery system, this low rate of release can be improved by incorporating an

electromagnetism triggering vibration mechanism into the polymeric delivery

devices with a hemispheric design; a zero-order drug release profile is achieved.

(5) Magnetic targeting of radioactivity:

Magnetic targeting can also be used to deliver the therapeutic radioisotopes

(Hafely, 2001).the advantage of these method over external beam therapy is that

the dose can be increased, resulting in improved tumor cell eradication, without

harm to adjacent normal tissues22.

Targeted therapy for brain tumours

Although previously considered untreatable, brain tumours no longer carry the same

prognosis as they did even a decade ago. Recent advances in drug delivery to the

central nervous system have not only bypassed physiological constraints such as the

blood–brain barrier, but have, in fact, changed the course of treatment for patients

with malignant brain tumours. The creation of targeted therapies, which spare normal

tissue and destroy tumour cells, is changing the field of neuro-oncology. In this

article, we review recent developments in the delivery of drugs to tumours of the

central nervous system, discuss current trends and directions in the development of

novel drugs and delivery systems, and present new and cutting-edge strategies for

overcoming the challenges ahead.

FIGURE 1 | Development and progression of astrocytic brain tumours.

Malignant brain tumours can arise in one of two ways. On the one hand, astrocytes

undergo genetic changes accompanied by upregulation of certain receptors, such as

the platelet-derived growth factor (PDGF), endothelial growth factor receptor (EGFR)

or vascular endothelial growth factor (VEGF). These progressive changes culminate

in the formation of a glioblastoma. On the other hand, most primary glioblastomas

arise de novo, without the need for gradual progression from an astrocytoma to a

high-grade astrocytoma to a glioblastoma multiforme.

Brain tumors are one of the most lethal forms of cancer. They are extremely difficult

to treat. Although, the rate of brain tumor incidence is relatively low, the field clearly

lacks therapeutic strategies capable of overcoming barriers for effective delivery of

drugs to brain tumors. Clinical failure of many potentially effective therapeutics for

the treatment of brain tumors is usually not due to a lack of drug potency, but rather

can be attributed to shortcomings in the methods by which a drug is delivered to the

brain and into brain tumors. In response to the lack of efficacy of conventional drug

delivery methods, extensive efforts have been made to develop novel strategies to

overcome the obstacles for brain tumor drug delivery. The challenge is to design

therapeutic strategies that deliver drugs to brain tumors in a safe and effective manner.

This review provides some insight into several potential techniques that have been

developed to improve drug delivery to brain tumors, and it should be helpful to

clinicians and research scientists as well.

CHAPTER-5

CONCLUSION:

The blood brain barrier (BBB) and the systemic toxicity of conventional

chemotherapy present obstacles to the success of future blood-borne drug therapies of

brain tumors. The work with polymer-encapsulated cancer drugs suggests an

alternative and more focused treatment approach. Our experimental strategy integrates

direct intracerebral drug delivery, sustained drug release from liposomes or polymer

implants, and increased targeting of the drug either by chemically modifying the drug

or by using tumor-specific carriers. This review will present some of the recent work

on targeted drug delivery for brain cancer treatment.

Cancer is one of the most challenging diseases today, and brain cancer is one of the

most difficult malignancies to detect and treat mainly because of the difficulty in

getting imaging and therapeutic agents across the blood-brain barrier and into the

brain. Many investigators have found that nanoparticles hold promise for ferrying

such agents into the brain [20-22]. Apolipoprotein E was suggested to mediate drug

transport across the blood-brain barrier [23]. Loperamide, which does not cross the

blood-brain barrier but exerts antinociceptive effects after direct injection into the

brain, was loaded into human serum albumin nanoparticles and linked to

apolipoprotein E. Mice treated intravenously with this complex induced

antinociceptive effects in the tail-flick test. The efficacy of this drug delivery system

of course depends upon the recognition of lipoprotein receptors. Kopelman and

colleagues designed Probes Encapsulated by Biologically Localized Embedding

(PEBBLE) to carry a variety of unique agents on their surface and to perform multiple

functions [22]. One target molecule immobilized on the surface could guide the

PEBBLE to a tumor. Another agent could be used to help visualize the target using

magnetic resonance imaging, while a third agent attached to the PEBBLE could

deliver a destructive dose of drug or toxin to nearby cancer cells. All three functions

can be combined in a single tiny polymer sphere to make a potent weapon against

cancer. Another anti-cancer drug, doxorubicin, bound to polysorbate-coated

nanoparticles is able to cross the intact blood-brain barrier and be released at

therapeutic concentrations in the brain [24]. Smart superparamagnetic iron oxide

particle conjugates can be used to target and locate brain tumors earlier and more

accurately than reported methods [25]. It is known that folic acid combined with

polyethylene glycol can further enhance the targeting and intracellular uptake of the

nanoparticles. Therefore, nanomaterial holds tremendous potential as a carrier for

drugs to target cancer cells.

It is very difficult for the medicine to destruct the target organism at the point of infection

because of complex cellular network of an organism. Target delivery of drugs, as the

name suggests, is to assist the drug molecule to reach preferably to the desired site. The

inherent advantage of his technique has been the reduction in the dose and side effects of

the drug.

By virtue of their size smaller than that of blood capillaries, intravenously administered

particulate drug carriers get accumulated in the liver cells. Among the particulate drug

carriers liposome's are a potential mode of delivery for the treatment of intracellular

infections as the cells of mononuclear phagocytic system easily take these up.

Microparticles may serve as future mode of delivery for the drugs of protein nature .

Orally delivered micro particles (<5 urn in size ) are taken up by the peye's patches. This

leads to induction of immune response against the antigen released from the

microparticles. Also, the antigen is protected from the loss of activity in the Gl tract. A

major limitation is the effective uptake of these particles from the Gl tract which is even

less than 1% However, a combination of biological approach such as incorporation of

specific ligands on the surface of surface of these particles enhances their uptake.

Magnetic Vesicular systems have been realized as extremely useful carrier systems in

various scientific domains. Over the years, magnetic microcarriers have been

investigated for targeted drug delivery especially magnetic targeted chemotherapy

due to their better tumor targeting, therapeutic efficacy, lower toxicity and flexibility

to be tailored for varied desirable purposes

CHAPTER-6

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