PH.D. THESIS - 52.172.27.147:8080

LIPID PEROXIDATION AND ANTIOXIDANT

DEFENCE STATUS IN LEPROSY

PH.D. THESIS

SUBMITTED TORAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE

BY

MR.C.V.BALASUBRAHMANYA PRASAD.M.Sc.

RESEARCH GUIDE

DR.M.V.KODLIWADMATH.M.D.

PROFESSOR AND HEAD.

DEPARTMENT OF BIOCHEMISTRY,J. N. MEDICAL COLLEGE,

NEHRU NAGAR, BELGAUM-590010.

JUNE 2005

PH.D.THESIS

LIPID P

EROXIDATION AND A

NTIOXIDANT

DEFENCE S

TATUS IN L

EPROSY

JUNE2 0 0 5

PH.D .THESIS



JUNE2 0 0 5



PH.D. THESISSubmitted to Rajiv Gandhi University of Health

Sciences, Karnataka, Bangalore,in partial fulfilment of the requirement for

the award of the degree of

Doctor of Philosophyin

Medical Biochemistry(Under The Faculty of Medicine)

By

MR.C.V.BALASUBRAHMANYA PRASAD.M.Sc.

Under The Guidance Of

DR.M.V.KODLIWADMATH.M.D.

PROFESSOR AND HEAD.DEPARTMENT OF BIOCHEMISTRY,

J. N. MEDICAL COLLEGE,NEHRU NAGAR, BELGAUM-590010.

JUNE 2005

JAWAHARLAL NEHRU MEDICAL COLLEGE,BELGAUM - 590 010.

CertificateCertificateCertificateCertificateCertificate

I certify that this thesis entitled "LIPID

PEROXIDATION AND ANTIOXIDANT DEFENCE STATUS IN LEPROSY" is abonafide record of research work carried out byMr. C. V. BALASUBRAHMANYA PRASAD under my guidancefor the degree of DOCTOR OF PHILOSOPHY IN MEDICAL BIOCHEMISTRY

of Rajiv Gandhi University of Health Sciences,Karnataka. The results presented in this thesis havenot previously formed the basis for the award of anydegree or fellowship.

I forward this thesis with great pleasure.

Dr. M.V. KODLIWADMATH. M.D.Research Guide,Professor and Head,Department of Biochemistry,J.N.Medical College,Belgaum - 590 010.Karnataka.

Place : Belgaum.Date :


CertificateCertificateCertificateCertificateCertificate

This is to certify that Mr. C. V.

BALASUBRAHMANYA PRASAD is a Ph.D. Student in theDepartment of Biochemistry at J.N. MedicalCollege, Belgaum and has completed his Thesis titled"LIPID PEROXIDATION AND ANTIOXIDANT DEFENCE STATUS IN LEPROSY"underthe guidance of Dr.M.V. KODLIWADMATH. M.D. Professorand Head, Department of Biochemistry,J.N.Medical College, Belgaum. He has undergonethe prescribed course of Research Work in accord-ance with the University regulations.

I have a great pleasure in forwarding it toRajiv Gandhi University of Health Sciences,Karnataka, Bangalore.

Dr. V.D. PATIL.M.D., D.C.H.

Principal,J.N.Medical College,Belgaum - 590 010.Karnataka.



DeclarationDeclarationDeclarationDeclarationDeclarationIn agreement with ordinance governing Ph.D.

Degree course (Notification No. ACA/ORD-23/97-98 Dt. 24-10-

1997) I hereby declare that the thesis entitled "LIPID

PEROXIDATION AND ANTIOXIDANT DEFENCE STATUS IN LEPROSY" submittedby me for the degree of DOCTOR OF PHILOSOPHY IN MEDICAL

BIOCHEMISTRY of the Rajiv Gandhi University of HealthSciences, Karnataka, Bangalore, is the result of myoriginal and independent work done at J. N. MedicalCollege, Belgaum, during the year 2001-2004 underthe supervision of Dr.M.V. KODLIWADMATH. M.D. Profes-sor and Head, Department of Biochemistry,J.N.Medical College, Belgaum, and has not formedthe basis for the award of any Degree, Diploma,Associateship, Fellowship or other similar title pre-viously.

Mr. C. V. BALASUBRAHMANYA PRASADPh.D. Reg. No. RGUHS/Ph.D/M8/2001-02Dept. of Biochemistry,J. N. Medical College,Belgaum - 590 010.Karnataka.


Acknowledgement

It is a great pleasure to utilize this unique opportunity to express my

deep sense of gratitude and offer my most sincere and humble regards to my

esteemed teacher and guide Shri. Rajendra C. Doijad, Assoc. Professor.

Dept of pharmaceutics, KLE’S college of pharmacy Belgaum, for his

unparalleled and excellent guidance, continuous encouragement and

support in completion of my course and dissertation successfully. His

discipline, principles, simplicity and fearless work environment was

cherished during the course.

I am thankful to Dr. F. V. Manvi, Principal K.L.E. Society’s College

of Pharmacy, for providing the facilities required for my dissertation work.

I sincerely thank Shri S.K. Krishnan Sr. Manager, Analytical

Research (HINDALCO), Belgaum, for providing me the facility of scanning

electron microscopy (SEM) and X- ray diffractrometry study at his

respective organization.

I heartily thank Mr.Suchit Chaudhary and Mr. Viond Nayak in

helping me to procure gift samples of Cisplatin from sun pharmaceutical

Mumbai & Cipla Ltd. Banglore, respectively.

I am also grateful for the invaluable guidance and kind co-operation

provided by Dr. A. R. Bhatt, Shri. C. R. Patil, Mr. Tippeswamy, Mr.

Banappa, Shri. M. B. Palkar, Shri. S. Bhongade, Shri. Noolvi and Ms.

Talath.

I extend my heartiest and dearest gratitude to my close friend

Jacqueline, Prajakta, mithra, Manish, Uday Bolmal sir and Sujata who

stood by me in every aspect. I shall cherish all the moments spent with them

throughout my life.

i

I take this opportunity to thank my seniors Deepak Kapoor, Prasun,

Ravindra singh, Shailesh, Ashok and Swati for their kind co-operation.

I extend my thanks to my colleagues Jayprakash, Biren, Nagesh,

Sallauddin, Shailju, Rupal, Gulshan, Keyur, Sriram, Basavraj, Kapil,

Navneet and Lakshman who stood by me in every walks of life.

I extend my heartiest & dearest gratitude to my roommates Sunil,

Sampat, rupesh & all my juniors. I shall cherish the excellent moments

spent with them throughout my life, Special thanks to Jiten, Gopal, Asif,

Mehboob for their constant support and help in my thesis work.

I am also thankful to all the technical and non-teaching staff,

K.L.E.Society’s College of Pharmacy, for their co-operation in various

capacities.

I express my deep sense of love and affection to my beloved and

respected parents, my brothers and sisters and all other family members,

without whose encouragement, co-operation and good wishes this task

would have been impossible.

Last… but not the least, I wish to express my gratitude towards “God

– Almighty”, who gave me the strength and courage to fulfill my dream and

has showered upon me his choicest blessings.

Amber Vyas.

Date:

Place: Belgaum

ii

Affectionately Dedicated To My

Late Grand Father,

Father,

Mother,

And

My Brothers & Sisters

||| Om Shree Ganeshaya Namaha |||

LIST OF ABBREVIATIONS USED

DDS - Drug delivery system

RES - Reticulo endothelial system

MRM - Magnetically responsive microspheres

PSEP - Phase separation emulsion polymerization

CSE - Continuous solvent evaporation

CDDP - Cis-Diammine-dichloroplatinium (II)

BSA - Bovine serum albumin

HCC - Hepatocellular carcinoma

NER - Nucleotideexcision repair

DMSO - Dimethyl sulphoxide

DMF - Diethyl formamide

DMA - N,N-dimethylacetamide

PBS - Phosphate buffer saline

IR - Infrared

DDTC - Diethyldithiocarbamic acid

UV-Vis - Ultra violet visible

T - Tesla

i

TABLE OF CONTENTS

CHAPTER TITLE PAGE NO.

1 INTRODUCTION ……………………………...…… 1

2 OBJECTIVES ….…………………………………… 30

3. REVIEW OF LITERATURE ………………………. 33

4. METHODOLOGY ………………….……………… 46

5 RESULTS AND DISCUSSION……………………. 59

6 CONCLUSION …………………………………….. 102

7 SUMMARY…………………………………………. 105

8 BIBLIOGRAPHY……………………………………. 108

9 ANNEXURE…………………………………………. 117

iii

LIST OF FIGURES

Sl. No. Title of Figure Page

No.

1. METHODS OF MICROSPHERE TARGETING 11

2. CONCEPT OF MAGNETIC DRUG TARGETING 12

3. PRINCIPLE OF MAGNETIC DRUG TARGETING 12

4. APPARATUS FOR IN VITRO MAGNETIC RESPONSIVENESS

STUDY

54

5. STANDARD CALIBRATION CURVE OF CISPLATIN 83

6. DRUG ENTRAPMENT EFFICIENCY OF MAGNETIC

MICROSPHERES

84

7. PERCENT MAGNETITE CONTENT 85

8. IN VITRO MAGNETIC RESPONSIVENESS OF MAGNETIC

MICROSPHERES

86

9. PLOT OF CUMULATIVE % DRUG RELEASED Vs. TIME FOR

PURE CISPLATIN

87

10. PLOTS OF CUMULATIVE % DRUG RELEASED Vs. TIME FOR

DIFFERENT FORMULATIONS OF CISPLATIN MAGNETIC

MICROSPHERES (IN VITRO RELEASE PROFILE) [ZERO ORDER

PLOTS]

88

11. PLOTS OF CUMULATIVE % DRUG RETAINED Vs. TIME FOR


MICROSPHERES (IN VITRO RELEASE STUDIES) [FIRST ORDER

KINETICS]

89

12. PLOTS OF CUMULATIVE % DRUG RELEASED Vs. ROOT TIME

FOR DIFFERENT FORMULATIONS OF CISPLATIN MAGNETIC

MICROSPHERE (IN VITRO RELEASE STUDIES) [HIGUCHI

MATRIX]

90

vi

13. PLOTS OF LOG CUMULATIVE % DRUG RELEASED VS. LOG

TIME FOR DIFFERENT FORMULATIONS OF CISPLATIN

MAGNETIC MICROSPHERES (IN VITRO RELEASED STUDIES)

[PEPPAS PLOT]

91

14. PLOTS OF CUBE ROOT OF % DRUG RETAINED Vs. TIME FOR


MICROSPHERES (IN VITRO RELEASE STUDIES) [HIXSON

CROWELL]

92

15. COMPARISION BETWEEN AMOUNT OF DRUG DISTRIBUTED

FROM MAGNETIC MICROSPHERES WITH AND WITHOUT

MAGNETIC FIELD IN VARIOUS ORGANS

(IN VIVO TISSUE DISTRIBUTION STUDIES)

93

16. PERCENT DRUG CONTENT Vs. TEMPERATURE FOR


MICROSPHERES AFTER 60 DAYS STORAGE

94

17. PLOTS OF CUMULATIVE % DRUG RELEASED Vs. TIME OF F-3

FORMULATION AFTER 60 DAYS STORAGE

95

vii

A

LIST OF SPECTR

Sl. No. Title of Spectrum Page

No.

1.

2.

3.

4.

5.

6.

IR SPECTRUM OF CISPLATIN

IR SPECTRUM OF BOVINE SERUM ALBUMIN (BSA)

IR SPECTRUM OF MAGNETITE

IR SPECTRUM OF CISPLATIN + BSA + MAGNETITE

X-RAY DIFFRACTROGRAM OF MAGNETITE

X-RAY DIFFRACTROGRAM OF FORMULATION (F-3)

96

97

98

99

100

101

viii

LIST OF TABLES

Sl. No. Title of Table Page No.

1. FORMULATION PLAN OF CISPLATIN MAGNETIC

MICROSPHERES.

51

2. ABSORBANCE VALUES OF CISPLATIN STANDARD

SOLUTIONS AT 210 nm.

69

3. PERCENTAGE PRACTICAL YIELD OF BOVINE SERUM

ALBUMIN MAGNETIC MICROSPHERES OF CISPLATIN.

70

4. DRUG ENTRAPMENT EFFICIENCY OF MAGNETIC

MICROSPHERES.

71

5. PERCENT MAGNETITE CONTENT. 71

6. IN VITRO MAGNETIC RESPONSIVENESS OF MAGNETIC

MICROSPHERES.

72

7. IN VITRO RELEASE PROFILE FOR PURE CISPLATIN 73

8. IN VITRO RELEASE PROFILE OF CISPLATIN FROM MAGNETIC

MICROSPHERES FORMULATION-1.

74



75



76



77

12. KINETIC VALUES OBTAINED FROM IN VITRO RELEASE DATA

OF DIFFERENT MAGNETIC MICROSPHERE FORMULATIONS

OF CISPLATIN.

78

13. KINETIC VALUES OBTAINED FROM IN VITRO RELEASE DATA

OF DIFFERENT MAGNETIC MCROSPHERE FORMULATIONS

OF CISPLATIN.

79

14. IN VIVO TARGETING STUDIES OF MAGNETIC

MICROSPHERES OF CISPLATIN.

80

15. STABILITY STUDIES FOR PERCENT DRUG CONTENT [AFTER

STORAGE AT 4ºC, AMBIENT TEMPERATURE AND HUMIDITY

& AT 30ºC /65% RH].

81

16. STABILITY STUDIES- IN VITRO RELEASE STUDY OF A

SELECTED FORMULATION (F-3) AFTER ONE MONTH

STORAGE AT 4°C, AMBIENT TEMPERATURE AND HUMIDITY

AND 30ºC /65% RH.

82

v

ABSTRACT

The capability to deliver high effective dosages to specific sites in the human

body has become the holy grail of drug delivery research. Drugs with proven

effectiveness under in vitro investigation often reach a major roadblock under in vivo

testing due to a lack of an effective delivery strategy. In addition, many clinical scenarios

require delivery of agents that are therapeutic at the desired delivery point, but otherwise

systemically toxic.

Magnetically responsive albumin microspheres containing Cisplatin were

prepared by PSEP technique and were evaluated with respect to Particle size analysis by

SEM, entrapment efficiency, magnetite content, in vitro magnetic responsiveness in a

7000 Oe magnetic field, in vitro drug release studies, in vivo drug targeting studies and

stability studies.

Spherical particles of average 3-12 µm in diameter and incorporation efficiency

up to 56.37% were obtained. Result of X-ray diffractrometry confirms the presence of

magnetite in prepared Cisplatin magnetic microspheres. Using chemical analysis, it was

found that total percentage of Fe2O3 in the microspheres was between 42.53%-55.48%.

Cumulative percent drug release after 24 hours was 89.60%, 82.22%, 78.41%, and

76.35% for F-1-F-4 respectively. Results of in vitro magnetic responsiveness and in vivo

targeting demonstrated that the retention of microspheres in presence of magnetic field

was significantly more than those in the absence of the magnetic field. Stability studies

showed that maximum drug content and closest in vitro release to initial data was found

in the samples stored at 4°C. Overall, this study shows that the magnetic albumin

microspheres can be retained at their target site in vivo, following the application of

magnetic field, and are capable of releasing their drug content for an extended period of

time. This would make them a suitable depot for delivering chemotherapeutic agent(s)

in vivo.

ii

Chapter 1 Introduction

Benjamin Franklin: "If everyone is thinking alike, then no one is thinking."

INTRODUCTION

The capability to deliver high effective dosages to specific sites in the human

body has become the holy grail of drug delivery research. Drugs with proven

effectiveness under in vitro investigation often reach a major roadblock under in vivo

testing due to a lack of an effective delivery strategy. In addition, many clinical scenarios

require delivery of agents that are therapeutic at the desired delivery point, but otherwise

systemically toxic. This project proposes a method for targeted drug delivery by applying

high magnetic field gradients within the body to an injected super paramagnetic colloidal

fluid carrying a drug, with the aid of modest uniform magnetic field.1

The nonspecific distribution of drugs is wasteful and hampers the clinical

usefulness of most of these agents after their systemic administration in the body. It

increases the incidence of undesirable reaction (toxic reactions), thereby narrowing down

the therapeutic index of a drug.2

Another problem associated with systemic drug administration is the inability to

target a specific area of the body. So systemic drug therapy is an undesirable way to

attack a local disease, hence localization of chemotherapeutic agent to diseased area (i.e.

drug targeting to desired site) is more suitable and rational answer to this problem.

Drug Targeting- A “State-Of-The-Art Technique”3

Drug Delivery Systems (DDS) are divided into various subsystems. One of these,

targeting DDS, recognizes target cells and tissues of diseases such as cancer and sends

drugs and genes to the target site. Current research in this field is focusing on the

development of nanomaterials for passive targeting. Work is also being done on so-called

Dept. of Pharmaceutics, KLES’s College of Pharmacy, Belgaum. 1


"missile drugs" for active targeting DDS which can enhance the functionality of

targeting. Missile drugs are showing promise as the "wonder drugs" of the 21st century.

The concept of designing specified delivery system to achieve selective drug

targeting has been originated from the perception of Paul Ehrlich, who proposed drug

delivery to be as a “magic bullet”.

Rationale Of Drug Targeting4

The site-specific targeted drug delivery negotiates an exclusive delivery to

specific preidentified compartments with maximum intrinsic activity of drugs and

concomitantly reduced access of drug to irrelevant non-target cells. The controlled rate &

mode of drug delivery to pharmacological receptor and specific binding with target cells;

as well as bioenvironmental protection of the drug en route to the site of action are

specific features of targeting. Invariably, every event stated contributes to higher drug

concentration at the site of action and resultant lowers concentration at non-target tissue

where toxicity might crop-up. The high drug concentration at the target site is a result of

the relative cellular uptake of the drug vehicle, liberation of drug and efflux of free drug

from the target site.

Targeting is signified if the target compartment is distinguished from the other

compartments, where toxicity may occur and also if the active drug could be placed

predominantly in the proximity of target site. The restricted distribution of the parent

drug to the non-target site(s) with effective accessibility to the target site(s) could

maximize the benefits of targeted drug delivery.



Principle & Rationale Of Drug Targeting:4

Levels Of Drug Targeting5,6

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

classified as:

1. Passive targeting.

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

3. Inverse targeting.

4. Dual targeting.

5. Double targeting

6. Combination targeting

1. Passive Targeting:

It is a sort of passive process, which utilizes the natural course of (attributed to

inherent characteristics) ‘homing’ of the carrier system, through which it finally identifies

and eventually approaches the intended cell lines. 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.



2. Active Targeting:

This employs deliberately modified drug-drug carrier molecule capable of

recognizing and interacting with a specific cell, tissue or organ. Modification may include

a change in the molecular size, alteration of the surface properties, incorporation of

antigen-specific antibodies, or attachment of cell receptors-specific ligands. The Active

targeting have further classified it into three different levels of targeting:

a. First order targeting (delivery to a discrete organ).

b. Second order targeting (targeting to a specific cell type within a tissue or organ.

For example, tumor cell Vs normal cells).

c. Third order targeting (delivery to a specific intracellular compartment in the

cells. For example, lysosomes).

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. The ligands afford specific avidity to drug carrier. The engineered

carrier constructs selectively deliver the drug to the cell or group of cells generally

referred to as target. The cascade of events involved in ligand negotiated specific drug

delivery is termed as ligand driven receptor mediated targeting

Physical Targeting:

This refers to a delivery system that releases a drug only when exposed to a

specific microenvironment, such as a change in pH or temperature, or the use of an

external magnetic field.



It requires formulation of the drug using a particulate delivery device, which by

virtue of its physical localization will allow differential release of the drug. The site

specificity is due to the exclusive generation of higher drug concentrations at the site of

localization of the device, while the drug concentration in the rest of the body is very

much diminished due to the simple dilution factor. The carrier systems employed are

either solid particulates such as microspheres, nanoparticles, or liquid colloids such as

liposomes. The particulate carriers may target liver (Kupffer cells and hepatocytes),

endothelial cells, sites of inflammation and lymph nodes. The size or surface of the

particles is crucial factors in targeting. Several anatomical compartments exist where

particles are retained due to either the physical properties of the environment or the

biophysical interactions of particles with the cellular components of the target tissue. The

delivery of drug in this manner yields a persistent and sustained supply of the drug at the

target site.

Physical or Mechanical Approach of Targeting Includes:-

a) Targeting to mononuclear phagocytic system

b) Targeting to the pulmonary region

c) Extravascular delivery

d) Mucosal delivery of antigens

e) Magnetic drug targeting

3. Inverse Targeting:

It is essentially based on successful attempt to circumvent and avoid passive

uptake of colloidal carriers by reticuloendothelial system (RES). This effectively implies

for reversion of bio-homing trend of the carrier, hence the process is referred to as inverse

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

RES by pre-injection of a large amount of blank colloidal carriers or macromolecules like



dextran sulphate. This approach leads to RES blockade and as a consequence impairment

of host defense system. Alternate strategies include modification of the size, surface

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

4. Dual Targeting:

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

their own intrinsic antiviral effect thus synergising 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. A major advantage is that the virus replication

process can be attacked at multiple points, excluding the possibilities of resistant viral

strain development.

5. Double Targeting:

For a new future trend, drug 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.

The temporal control of drug delivery has been developed in terms of control drug

release prior to the development of drug targeting. If spatial targeting is combined with

temporal, controlled release results in an improved therapeutic index by the following

two effects. First, if drug release or activation is occurred locally at therapeutic sites,

selectivity is increased by multiplication of the spatial selectivity with the local

release/activation. Second, the improvement in the therapeutic index by a combination of

a spatially selective delivery and a preferable release pattern for a drug, such as zero

order release for a longer time period of the drugs. When these two methodologies are

combined, it may be called “Double targeting”



6. Combination Targeting:

Petit and Gombtz, 1998 have suggested the term 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 favor targeting the specific compartments, organs or their

tissues within the vasculature.

Carriers Used In Targeted Drug Delivery Systems 4,7

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

transportation of the loaded drug(s). They are drug vectors, which sequester, transport

and retain drug en route, while elute or deliver it within or in vicinity of target. Following

is the categorical presentation of these potential targetable systems.





Problems Associated With Targeted Drug Delivery Systems 5

Several problems have been identified which require alterations in targeting

strategies particularly, in vivo. These include:

Rapid clearance of targeted systems especially antibody targeted carriers.

Drug- antibody inactivation during conjugation.

Immune reactions against intravenous administered carrier systems.

Target tissue heterogeneity.

Problems of insufficient localizations of targeted systems into tumor cells.

Down regulation and sloughing of surface epitopes.

Diffusion and redistribution of released drug leading to non-specific

accumulation.

Nanoparticles are difficult to manufacture in large quantities.

Nanoparticles has bioacceptibility restrictions.

Poor stability, rapid and quantitative interception of liposomes and their contents

by cells of RES.

Magnetic Microspheres

Splendid achievements have been made in management of diseases through

invention of drugs over the past decade, which are fulfilling the challenge of modern drug

therapy i.e. optimization of the pharmacological action of the drugs coupled with the

reduction of their toxic side effects in vivo. Recently a lot of interest has been shown in

targeted drug delivery system, magnetic microspheres being one of them.

Targeting by magnetic microspheres i.e. incorporation of magnetic particles into

drug carriers8, 9 (polymers) and using an externally applied magnetic field is one way to

physically direct this magnetic drug carriers to a desired site8, 10,11. Widder et al. first



reported on the use of magnetic albumin microspheres. Morimoto and widder and senyei

extensively reviewed their preparation and drug release properties12.

Magnetic microspheres are supramolecular particles that are small enough to

circulate through capillaries without producing embolic occlusion (< 4μ m) but are

sufficiently susceptible (ferromagnetic) to be captured in minor vessels and dragged into

the adjacent tissue by magnetic fields of 0.5-0.8 tesla (T) 13.

Evolution of Magnetic Microspheres

There are several techniques (like liposomes, resealed erythrocytes, platelets,

monoclonal antibody and non magnetic microspheres) by which drugs can be delivered to

targeted areas2.

Although above mentioned techniques are quite efficient but drug carrier in case of

liposomes, erythrocytes and platelets suffer major stability problem, hence shelf life of

such preparation is tremendously reduced or they need special storage conditions which

is not economically viable. While in monoclonal antibody preparation, selection and

isolation of an appropriate antigen for developing monoclonal antibody is again a very

brain-taxing problem. However nonmagnetic microspheres do not show any serious

stability problem but they show poor site specificity and are rapidly cleared off by RES

(reticuloendothelial system) under normal circumstances13.

Magnetic fields are believed to be harmless to biological systems and adaptable to

any part of the body. Up to 60% of an injected dose can be deposited and released in a

controlled manner in selected non-reticuloendothelial organs.

So magnetic targeting of microspheres was developed to overcome two major

problems encountered in the drug targeting namely RES clearance (RES readily takes up



a variety of microparticles including liposomes, microspheres as well as other colloidal

particles) and target site specificity. Figure 1 shows methods of microsphere targeting.

Fig.1: Methods of microsphere targeting

Principle Of Magnetic Drug Targeting 4,14

Magnetic drug delivery by particulate carriers is a very efficient method of

delivering a drug to localized disease site. Very high concentrations of chemotherapeutic

or radiological agents can be achieved near the target site, such as tumour, without any

toxic effects to normal surrounding tissue or to whole body. Fig.2 highlights the concept

of magnetic targeting by comparing systemic drug delivery with magnetic targeting. In

magnetic targeting, a drug or therapeutic radioisotope is bound to a magnetic compound,

injected into patient’s blood stream, and then stopped with a powerful magnetic field in

the target area. Depending on the type of drug, it is then slowly released from the

magnetic carriers (e.g. release of chemotherapeutic drugs from magnetic microspheres) or

confers a local effect. It is thus possible to replace large amounts of drug targeted



magnetically to localized disease sites, reaching effective and up to several –fold

increased localized drug levels (wider et al., 1979; Gupta and Hung, 1989; Hafeli et

al.,1997).Figure 3 shows the principle of magnetic drug targeting.

Systemic Drug Delivery Magnetic Targeting

Fig.2: Concept of magnetic drug targeting

Fig 3: Principle of magnetic drug targeting



Benefits Offered By Magnetically Responsive Microspheres 4,15

Magnetically responsive microspheres (MRM) are site specific and by the

localization of these microspheres in the target area, the problem of their rapid clearance

by RES is also surmounted.

Linear blood velocity in capillaries is 300 times less i.e. 0.05 cm/sec as compared

to arteries, so much smaller magnetic field, 6-8 Koe, is sufficient to retain them in the

capillary network of the targeted area. Moreover, restricting microspheres to capillary

bed of targeted area offers more benefits.

a) Diffusion occurs maximally in capillary network so efficient delivery of drug to

diseased tissue is achieved.

b) Microspheres can transit in to extravascular space thereby creating an

extravascular drug depot for sustained release of drug within the targeted area.

c) Therapeutic responses in targeted organs at only one tenth of the free drug dose.

d) Controlled release with in target tissue for intervals of 30 minutes to30 hrs. as

desired.

e) Avoidance of acute drug toxicity directed against endothelium and normal

parenchyma.

f) Adaptable to any part of the body.

g) This drug delivery system reduces circulating concentration of free drug by a

factor of 100 or more.

h) Magnetic carrier technology appears to be a significant alternative for the

biomolecule malformations (i.e. composition, inactivation or deformation).

In case of tumor targeting, microspheres can be internalized by tumor cells due to

its much increased phagocytic activity as compared to normal cells. So the problem of

drug resistance due to inability of drugs to be transported across the cell membrane can

be surmounted.



Limitations 4

However, this novel approach suffers from certain disadvantages also as given

below:

• Drug(s) can’t be targeted to deep-seated organs in the body. So this approach is

confined to the targeting of drugs in superficial tissues only like skin, superficial

tumors or to joints etc.

• Magnetic targeting is an expensive, technical approach and requires specialized

manufacture and quality control system.

• It needs specialized magnet for targeting, advanced techniques for monitoring,

and trained personnel to perform procedure.

• Magnets must have relatively constant gradients, in order to avoid local over-

dosing with toxic drugs.

• A large fraction (40-60%) of the magnetite, which is entrapped in carriers, is

deposited permanently in target tissue.

Magnet Design 13,16

The force exerted by a gradient magnetic field is an important parameter that governs

magnetic targeting of micro carriers. The relationship of magnetic force to field gradient

and magnetic moment of particles is expressed by following equation : -

F=M∇H

Where,

F= Force on particles

M=Magnetic moment of particles after saturation magnetization

∇H= Magnetic field gradient



This equation explains that spheres with increased magnetic moments will

experience force sufficient for extra vascular migration of proportionately lower field

gradients. The magnetic moments of microspheres can be increased in three ways: -

a) By clustering magnetite at the center of each sphere to produce large macro

domains.

b) By magnetizing the spheres to saturation levels prior to vascular targeting.

c) By substituting one of the newer ferromagnetic materials that has high

susceptibility than Fe3O4.

Techniques Of Preparation2

There are mainly two techniques, which are commonly employed for

microspheres preparation: -

a) Phase separation emulsion polymerization (PSEP)

b) Continuous solvent evaporation (CSE).

Phase separation emulsion polymerization:

Schematic diagram of preparation of magnetically responsive microspheres by

PSEP technique



Continuous solvent evaporation:

Schematic diagram of preparation of magnetically responsive microspheres by CSE

Factors Affecting Rate Of Drug Delivery 2

The amount and rate of drug delivery via magnetically responsive microspheres

can be regulated by varying size of microspheres, drug content, magnetite content, their

hydration state and drug release characteristic of carrier. Actually all these factors are

interconnected. The size of microspheres is related to their drug content by a direct

proportionality. However, drug content is also governed by the solubility characteristic of

the drug and method of preparation of microspheres. Hydration step of microspheres

affect their body distribution and drug release rate from the microspheres. The magnetic

content and magnitude of applied field governs the retention of microspheres at targeted

sites. In case of microspheres with higher magnetic content, smaller magnetic field are

sufficient for efficient retention of microspheres in the targeted area. But by

incorporating excessive magnetite into the microspheres, the effective space available for

the drug in microspheres is reduced appreciably. So amount of drug and magnetite



content of microspheres needs to be delicately balanced in order to design an efficient

therapeutic system.

Drugs Generally Used For Magnetized Targeting

Adriyamycin Doxorubicin

5-Fluro uracil Oxantrazole

Cisplatin Hydrocortisone

Dactinomycin Diclofenac sodium17

Dexamethasone18

Employed carriers

Carriers generally used for entrapping drug and magnetite are: -

Poly lactide Ova albumin

Casein Fibrinogen

Ethyl cellulose Chitosen

Calcium alginate Gelatin

Nitrocellulose Polyvinyl alcohol (PVA)

Starch19 Polyalkylcynoacrylates

Agarose Poly ethylene glycol (PEG) 20

Carnauba wax Polystyrene

Human serum albumin (HSA) Bovine serum albumin (BSA)

N-Isopropyl acrylamide and their copolymer



Applications

a) The most popular applications of magnetic carrier technology are bioaffinity

chromatography, wastewater treatment, immobilization of enzymes or other

biomolecules and preparation of immonological assay 15.

It is also used in the delivery of insulin, nitrates as well as in selective β

blockers, in general hormone replacement immunization and cancer

chemotherapy.

b) Magnetic delivery of chemotherapeutic drugs to liver tumors : -

The first clinical cancer therapy trials using magnetic microspheres were

preformed by Lubbe et.al. in Germany for the treatment of advanced solid

cancer21, 22.

While current preclinical research is investigating use of magnetic particles

loaded with different chemotherapeutic drugs such as mitoxantrone, paclitaxel 23.

c) Magnetic targeting of radioactivity: Magnetic targeting can also be used to deliver

the therapeutic radio isotopes (Hafeli, 2001) 24. The advantage of this method over

external beam therapy is that the dose can be increased, resulting in improved

tumor cell eradication, without harm to adjacent normal tissue.

Magnetic targeted carriers, which are more magnetically responsive

iron carbon particles, have been radio labeled in last couple of years with isotope

such as 188 Re (Hufli et al. 2001) 25, 90 Y, 111 In and 125I (Johnson et al. 2002) 23 and

are currently undergoing animal trials.

d) Treatment of tumors with magnetically induced hyperthermia: Developments by

Jordan and chan led to the current hyperthermia application of single domain

dextran- coated magnetite nanoparticles in tumors (Jordan et al. 1993; chan et

al.1993) 26. The first clinical trials are going on in Germany (Jordan et al. 2001) 27.



Magnetic hyperthermia is also possible with larger magnetic particles, as shown

by the group of moroz et al. (2002) 28.

e) On going investigations in magnetic hyperthermia are focused on the

development of magnetic particles that are able to self-regulate the temperature

they reach. The ideal temperature for hypothermia is 43˚C - 45˚C, and particles

with a curie temperature in this range have been described by kuznetsov et al.

(2002) 29.

f) Other magnetic targeting application: It can be used for encapsulation of peptide

octreotide and the protein tumor necrosis factor alpha (TNF-α) (Johnson et al.

2002) 23. Advantages of such an approach are target gene transfection at rapid

speed and high efficiencies.

It is also possible to use only the mechanical- physical properties of magnetic

particles or ferrofulids for therapy. One example is the embolization (clogging) of

capillaries under the influence of a magnetic field (Flores and Liu, 2002) 30. In

this way, tumors could specifically starved of their blood supply. Another elegant

example is the use of magnetic fluids to prevent retinal detachment, thus

preventing the patients from going blind (Dailey et al. 1999) 31.

g) Magnetic control of pharmacokinetic parameters and drug release: Langer et al.

embedded magnetite or iron beads into a drug filled polymer matrix and then

showed that they could activate or increase the release of the 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) 32. The microenvironment with

in the polymer seemed to have shaken the matrix or produced “micro cracks ײ and

thus made the influx of liquid, dissolution and efflux of the drug possible. In this

way, it was possible to magnetically activate the release of insulin from a depot



underneath the skin (Kost et al., 1987) 33. Done repeatedly this would allow for

pulsative drug delivery.

h) Magnetic system for the diagnosis of disease: The most important diagnostic

application of magnetic particles is as contrast agent for magnetic resonance

imaging (MRI). Suini et al. tested 0.5-1μm sized ferrites in vivo for the first time

in 1987 (Suini et al., 1987) 34. Since then, smaller supramagnetic iron oxides

(SPIOs) have been developed into unimodular nanometers sizes and have since

1994 been approved and used for the imaging of liver metastasis (ferumoxide

based feridex I.V, or Endorem) or to distinguish loops of bowel from other

abdominal structures (GastroMark, or Lumirem in Europe).

i) Magnetic systems for magnetic cell separation: The era of using magnetic

particles with surface markers against cell receptors started in 1978 with a seminal

paper by Kronick et al. (1978) 35. Currently, many different kits for the sample

preparation, extraction, enrichment and analysis of entire cells based on surface

receptor, and subcellular/ molecular component such as protein, mRNA, DNA are

available (Bosnes et al., 1997) 36. Analytical procedures such as many different

immunoassays are often based on magnetic separation (Meza, 1997) 37.

CANCER

“The main problem of cancer therapy is not the lack of efficient drugs, but that

these drugs are very difficult to concentrate in the tumour tissue without leading to toxic

effects on neighbouring organs and tissues.”

Cancer is a Latin word meaning a crab. A malignant tumor, like the crab, has a fat

main body with extensions, like the crab’s feet, which invade the surrounding tissues.



Irrespective of the aetiology, cancer is basically a disease of cells characterized by

the loss of normal cellular growth, maturation and multiplication and thus homeostatis is

disturbed. 38

The main features of cancer are:

1. Excessive cell growth, usually in the form of tumour.

2. Invasiveness, i.e., ability to grow into surrounding tissue.

3. Undifferentiated cells or tissues, more similar to embryonic tissue.

4. The ability to metastasize or spread to new sites and establish new growths.

5. A type of acquired heredity in which the progeny of cancer cells also retain cancerous

properties.

6. A shift of cellular metabolism towards increased production of macromolecules from

nucleosides and amino acids, with an increased catabolism of carbohydrates for

cellular energy39.

Causes Of Cancer



Pathophysiology Of Cancer40

A healthy individual has trillions of cells that divide at an orderly rate with a

controlled pace. However, as a result of various carcinogens and exposure to ultraviolet

light which cause DNA mutations, three things happen which turn the once normal cell

into a cancer cell:

(1) The conversion of protooncogenes to oncogenes.

(2) The inhibition of tumor suppressor genes.

(3) The inhibition of DNA repair genes.

In the first step protooncogenes, which encode proteins for cell growth and which

are normally tightly regulated, become oncogenes whereby they never stop producing

growth related proteins. In the second step, genes that would normally suppress these

oncogenes get turned off. In the third step the genes which encode the proteins which fix

DNA mutations get turned off stunting the cells ability to regain control. Basically if any

of these three things do not occur then the cell would either remain normal or the cancer

cell would form but not survive. The immune system also contributes to destroying

cancer cells by recognizing abnormalities on the cancer cells membrane.

Cancer Pathogenesis And Cancer Chemotherapy

General principles:

The term cancer refers to a malignant neoplasm (new growth).

Cancer arises as a result of a series of genetic and epigenetic changes, the main

genetic lesions being:

- Inactivation of tumor suppressor genes.

- The activation of oncogenes (mutation of the normal genes controlling cell

division and other processes.)



Cancer cells have four characteristics that distinguish them from normal cells:

- Uncontrolled proliferation

- Loss of function because of lack of capacity to differentiate.

- Invasiveness

- The ability to metastasize.

Cancer cells have uncontrolled proliferation owing to changes in:

- Growth factors and/or their receptors

- Intracellular signaling pathways, particularly those controlling the cell

cycle and apoptosis.

- Telomerase expression

- Tumor-related angiogenesis.

Simplified Outline Of The Genesis Of Cancer

Chemical, viruses, irradiation, etc Acquired mutations Inherited mutations Altered gene expression Proto-oncogenes Oncogenes Decreased expression of tumor Sis, erbB, ras, myc, gene for cyclin D, etc suppressor genes: p53, Rb1, etc Other factors Uncontrolled cell proliferation, Decreased apoptosis, Dedifferentiation alterations in telomerase Development of primary tumor Production of metalloproteinases etc. Invasion of nearby tissue by tumor cells. Angiogenesis Metastasis Development of secondary tumors



Various Cancer Therapies And Their Limitations

Conventional cancer therapies include; surgery, chemotherapy and radiation

therapy. Each has its own limitations in providing a complete cure. Surgical resection is

limited by the ability to expose and remove the tumor and can only remove those tumors

detectable by current imaging techniques. Any cells that are not removed by the surgeon

have the ability to proliferate causing a recurrence. Surgery is also not effective against

micrometastases that may have migrated from the site of primary tumor.

Chemotherapy, whether given systemically or by regional perfusion of a

particular organ, is impeded by the lack of specificity of the drugs for cancer cells.

Therefore, therapy is often limited due to systemic toxicity before truly therapeutic drug

levels in the tumor can be achieved. Drug concentrations in the tumor must also be

sustained for prolonged periods of time for maximum efficacy, so as to catch all the

cancer cells during cell cycle. Chemotherapeutic drugs, usually act on rapidly dividing

cells, so cells of the intestinal lining and bone marrow can be extensively damaged during

treatment.

Radiation therapy can be specifically directed to the site of tumor, but is also

limited by the potential for damage to non-cancerous cells. Radiation therapy like surgery

is a local modality used in the treatment of cancer. Its success depends on the inherent

difference in radio sensitivity between the tumor and the adjacent normal tissues.

Radiation therapy for most solid tumors involves the administration of radiation in the

form of X-rays or gamma rays to a tumor site. Radiation therapy is associated with both

acute toxicity and long-term sequel. Acute reactions occur during or immediately after

therapy. Common manifestations include systemic symptoms such as fatigue, local skin

reaction, gastrointestinal toxicity with nausea, vomiting and dysphagia or diarrhea.



Chemoembolization is an extension of traditional percutaneous embolization

techniques. With Chemoembolization, investigators embolize tumors with Gelfoam or

Ivalon particles soaked with chemotherapeutic agents, providing vascular occlusion, with

sustained therapeutic level of chemotherapy in the tumor areas. Generalize ischemia

would reduce the ability of the cell to relive itself from the toxicity of chemotherapy.

Such therapy is standard therapy for non-resectable primary hepatocellular carcinoma

(HCC). For Metastatic cancer, however, the benefits are less clear except for Metastatic

neuroendocrine tumors. Chemoembolization, especially in patients with liver metastases

should presently only be performed in the setting of clinical trials41.

None of these therapies alone or in combination have achieved complete cure for

all cancer types in all patients. Therefore many researchers have been exploring

controlled release or targeted delivery options for the treatment of this disease.42, 43

Anti Neoplastics44

Antineoplastic drugs (also known as cytotoxic drugs) are used in the treatment of

malignant neoplasms when surgery or radiotherapy is not possible or has proved

ineffective as an adjunct to surgery or radiotherapy, or as in leukemia, as the initial

treatment. Therapy with Antineoplastics is notably successful in a few malignant

conditions & may be used to palliate symptoms and prolong life in others.

The two main groups of drugs used in the treatment of malignant disease are the

alkylating agents and the antimetabolites Nitrogen Mustards, ethyleneimine compounds

and alkyl sulphonates are the main alkylating agents. Other compounds with an

alkylating action are the various nitrosoureas. Cisplatin and dacarbazine appear to act

similarly.



Classification

A. DRUGS ACTING DIRECTLY ON CELLS (Cytotoxic drugs) 1. Alkylating agents

Nitrogen Mustards Mechlorethamine (Mustine HCl) Cyclophosphamideε, & Ifosfamide Chlorambucil, Melphalan. Ethylenimine Thio-TEPA Alkyl sulfonate BusulfanεNitrosoureas Carmustine (BCNU), Lomustine

(CCNU) Triazine Dacarbazine (DTIC)

2. Antimetabolites Folate antagonist Methotrexateε (Mtx) Purine 6-Mercaptopurineε (6-MP), 6-Thioguanine

(6-TG), Azathioprineε

Pyrimidine 5-Fluorouracilε (5-FU) Antagonist Cytarabine (cytosine arabinoside)

3. Vinca alkaloids vincristineε (oncovin), vinblastineε 4. Taxanes Paclitaxel, Docetaxel. 5. Epipodophyllotoxin Etoposideε 6. Camptothecin analogues Topotecan, Irinotecan 7. Antibiotics Actinomycin Dε, doxorubicin,

Daunorubcin, Mitoxantrone, Bleomycinsε, Mitomycin, Mithramycinε.

8. Miscellaneous Hydroxyurea, Procarbazineε, L-Asparaginaseε, Cisplatinε, Carboplatin.

B. DRUGS ACTING IN HOROMONAL MILIEU 1. Glucocorticoids Prednisolone & Other

2. Estrogens Fosfestrol, Ethinylestradiol 3. Antiestrogen Tamoxifenε4. Antiandrogen Flutamide 5. 5-ά reductase inhibitor Finasteride 6. GnRH analogues Naferelin, Goserelin.



Summary Of The Main Sites Of Action Of Cytotoxic Agents That Act On

Dividing Cells 45



Platinum Coordination Complexes 45

Rosenberg and co-workers first identified the platinum coordination complexes as

cytotoxic agent in 1965. Cis-Diammine-dichloroplatinum (II) (Cisplatin) was the most

active of these substances in experimental tumor systems & has proven to be of great

clinical value.

Cisplatin has broad activity as an antineoplatic agent, & the drug is especially

useful in the treatment of epithelial malignancies. It has become the foundation for

curative regimens for advanced testicular cancer & cancers of the head & neck, bladder,

esophagus & lung.

Mechanism Of Cellular Uptake Of Cisplatin

Pt2+(NH3)2Cl2 + H20 ---> [Pt2+(NH3)2Cl(H20)]+ + Cl-

[Pt2+(NH3)2Cl(H20)]+ + H20 ---> [Pt2+(NH3)2(H20)2]2+



Mechanism Of Action

Cisplatin appears to enter cells by diffusion. The chloride atoms may be displaced

directly by reaction with nucleophiles such as thiols; replacement of chloride by water

yields a positively charged molecule & is probably responsible for formation of the

activated species of the drug, which then reacts with nucleic acids & proteins. The

platinum complexes can react with DNA, forming intrastrand & interstrand cross-links.

The N7 of guanine is very reactive & platinum cross-links between adjacent guanines on

the same DNA strand; guanine-adenine cross-links also readily form. The formation of

interstrand cross-links is a slower process & occurs to a lesser extent. DNA adducts

formed by Cisplatin inhibit DNA replication & transcription & lead to breaks &

miscoding. Although no conclusive association between platinum-DNA adduct formation

& efficacy has been documented, the ability of patients to form & sustain platinum

adducts appears to be an important predictor of clinical response.


Chapter 2 Objectives

OBJECTIVES

The purpose of this study was to consider how externally applied magnetic fields

can be used to guide materials inside the body. Methods of guiding magnetic particles in

a controlled fashion through the arterial system in vivo using external magnetic fields are

explored. This type of magnetic guidance system is needed for an effective drug delivery

system.

This work is fueled by the general nanotechnology initiative and the general

desire for less invasive surgery using electromagnetic field-directed microspheres. The

current nanotechnology initiatives are motivated by the added functionality derived from

reducing the overall size of working systems.

Magnetically responsive microspheres (MRM) are site specific and by

localization of these spheres in the targeted area, the problem of their rapid clearance by

RES is also surmounted.

Magnetic microspheres have potential use as magnetic seeds for drug delivery.

Such microspheres are paramagnetic and have been made to range in size from

approximately one micron to greater then 600 microns. Magnetic fields are believed to be

harmless to biological systems and adaptable to any part of body. MRM ensures that

maximum amount of injected dose can be deposited and released in controlled manner in

selected non-RES organs.

There have been many attempts in the past to create platform technologies that

can guide and deliver drugs, make repairs, and essentially give one’s hands the dexterity

to seamlessly manipulate nature to macro and micro particles. This range of

maneuverability and control over matter allows noninvasive surgery and the ability to



pass through tissue and even cell walls instead of cutting or lysing them to obtain internal

access to a material or body.

These above advantages makes magnetic microspheres as an ideal candidate for

the targeted and controlled drug delivery system.

Cisplatin (Cis-Diammine-dichloroplantinum (II)) is relatively simple molecule.

Cisplatin forms stable complexes with cancer cell DNA. Distortion of the DNA structure

leads to the disruption of two key processes that enable these abnormal cells to multiply:

replication and transcription. It also disrupts the cell's natural ability to repair itself by

either blocking and slowing down repair proteins, or negatively altering the function of

nucleotide excision repair (NER) proteins.

Cisplatin is the drug of choice in management of many solid malignancies. It is

used in treatment of non-Hodgkin’s, lymphomas, tumors of brain, cervix, ovary, breast,

prostrate, trophoblastic tumours, neuroblastoma, melanoma and AIDS associated kaposis

sarcoma.

I.V administration of Cisplatin produces adverse effects like ototoxicity, renal

failure, loss of hearing, optic neuritis, cerebral blindness, papilloedema, tubular-necrosis,

focal encephalopathy and cardiac abnormalities. All these adverse effects limits the

amount of drug to be given to the patient.

Hence, to overcome these inherent drawbacks associated with parenteral drug

delivery of Cisplatin an attempt is being made to provide an alternative drug delivery

system of Cisplatin in the form of magnetically responsive microspheres to reduce the

adverse effects and to enhance therapeutic efficacy of this drug and have following

advantages:



i. Reduction in dose.

ii. Controlled drug release.

iii. Avoidance of acute drug toxicity.

iv. Site-specific delivery.

v. Adaptable to any part of the body.

In the present attempt Cisplatin loaded magnetic microspheres were formulated

by phase separation emulsion polymerization technique and evaluated for the following

parameters.

• Percent practical yield.

• Particle size analysis of magnetic microspheres.

• Drug entrapment efficiency.

• X-ray diffractrometry.

• Determination of magnetite content.

• In vitro magnetic responsiveness.

• In vitro drug release studies.

• In vivo drug targeting studies.

• Stability studies.


Chapter 3 Review of Literature

REVIEW OF LITERATURE

DRUG REVIEW OF CISPLATIN46, 47

Structure: Cl NH3

Pt

Cl NH3

N2Cl2PtH6, [Pt (NH3)2Cl2]º Mol.Wt : 300.09

Category:

Cytotoxic

Doses:

By intravenous infusion, 20 mg per sq.m of body surface daily for 5 days in

Metastatic testicular tumors and 50-70 mg per sq.m of body surface every 3-4 weeks in

advanced bladder cancer.

Description:

Yellow powder or orange yellow crystals.

Solubility:

Soluble in N, N-dimethylacetamide (DMA) and dimethylsulphoxide (DMSO),

sparingly soluble in dimethylformamide, slightly soluble in water; practically insoluble in

ethanol (95%).

Standards:

Cisplatin contains not less than 97.0 percent and not more than 102.0 percent of

H6Cl2N2Pt.



Absorption, Fate And Metabolism

After rapid intravenous administration of usual doses, the drug has an initial

elimination half-life in plasma of 25 to 50 minutes; concentrations of total drug, bound

and unbound, fall thereafter, with a half-life of 24 hours or longer. More than 90% of the

platinum in the blood is covalently bound to plasma proteins. High concentrations of

Cisplatin are found in the kidney, liver, intestine, & testes, but there is poor penetration

into the CNS. Only the kidney excretes a small portion of the drug during the first 6

hours; by 24 hours up to 25% is excreted, & by 5 days up to 43% of the administered

dose is recovered in the urine. When given by infusion instead of rapid injection, the

plasma half-life is shorter & the amount of drug excreted is greater. Biliary or intestinal

excretion of Cisplatin appears to be minimal.

Routes Of Administration

1) Cisplatin is administered intravenously, not more frequently than every 3 to 4

weeks. It is usually given as a single dose of 50 to 120 mg per meter square body

surface.

2) Cisplatin has also been administered by the intra-arterial & intraperitoneal routes

& by instillation into the bladder.

3) Cisplatin has also been investigated as a less toxic but apparently less effective

Cisplatin albumin complex, as a liposomal formulation & as an implant

containing Cisplatin in a protein matrix, MPI-5010.

Adverse Effects And Clinical Toxicities

1) Severe nausea and vomiting.

2) Nephrotoxicity, which has largely been abrogated by the routine use of hydration

& diuresis.

3) Ototoxicity, tinnitis & hearing loss.



4) Peripheral neuropathy at higher doses or after multiple cycles of treatment, which

may worsen after discontinuation.

5) Mild to moderate myelosuppression with transient leukopenia, thrombocytopenia

& anemia.

6) Electrolyte disturbances, including hypomagnesemia, hypokalemia &

hypophosphatemia.

7) Hyperuricemia, seizures, hemolytic anemia & cardiac abnormalities.

8) Anaphylactic-like reactions, characterized by facial edema, bronchoconstriction,

tachycardia & hypotension.

9) Optic neuritis, cerebral blindness.

10) Cisplatin is potentially mutagenic and teratogenic.

Incompatibility

Cisplatin is rapidly degraded in the presence of bisulphate or metabisulphite, and

admixture with preparations containing these as preservatives may result in loss of

activity. Sodium bicarbonate may also increase the loss of Cisplatin from solution, and in

some cases may cause precipitation. The stability of Cisplatin when mixed with

fluorouracil is reported to be limited with 10% loss of Cisplatin in 1.2 to 1.5 hrs.

Mixtures with Etoposide in 0.9% sodium chloride injection formed a precipitate if

mannitol and potassium chloride were present as additives, but not when the diluent was

5% glucose with 0.45% sodium chloride. Cisplatin has also been reported to be

incompatible with aluminum in dispensing equipment.

Stability

Decomposition of Cisplatin in aqueous solution is primarily due to reversible

substitution of water for chloride, and its stability is enhanced in sodium chloride

solutions because of the excess of chloride ions available. A solution in 0.9% sodium



chloride injection has been reported to lose 3% of the drug in less than one hour and to

remain stable at the equilibrium value for 24 hrs at room temperature. Stability is

decreased if exposed to intense light, but the effect of normal lighting conditions is

apparently smaller. It has been recommended that admixtures of Cisplatin with mannitol

and magnesium sulphate (in glucose 5% with NaCl 0.45%) stored at room temperature in

polyvinyl chloride bags should be used with in 48 hrs, but may be stored for 4 days at 4ºC

or frozen and stored at -15ºC for up to 30 days. However, solutions containing 600ug per

mL or more of Cisplatin precipitates out when refrigerated and are slow to redissolve.

Storage

Store in a tightly closed, light resistant container.

Preparations

CISPLAT, PLATINEX – 10mg/ml, 50mg/50ml vial.

Proprietary Preparations

Australia: Abiplatin, Platiblastin.

Canada, Belgium, and USA: Platinol

Switzerland: Platiblastine-S; Platinol

Italy: Citoplatino; Platamine; Platinex; Pronto Platamine.



Review Of Past Work Done On Cisplatin

The pharmacological activity and pharmacokinetics of Cisplatin (CDDP)-loaded

polymeric micelles were examined by Nishiyama N, Kato Y, Sugiyama Y, Kataoka K.

to reveal their usefulness as a novel tumor-directed drug carrier system of CDDP. In

biodistribution assay, free CDDP or CDDP-loaded micelles were administered

intravenously to Lewis lung carcinoma-bearing mice. Antitumor activity and

nephrotoxicity were respectively evaluated by the measurement of tumor size and plasma

blood urea nitrogen (BUN) after single bolus i.v. administration of each drug. Results

showed that the time profile of the plasma Pt level after the injection of the micelles

exhibited a time-modulated disappearance as observed in saline in vitro. The micelles

exhibited 5.2- and 4.6-fold higher AUC of Pt in the plasma and tumor, respectively, with

minimal change in the kidney, in comparison with free CDDP, suggesting that prolonged

circulation of Pt in circulation and specific accumulation in the tumor were achieved

utilizing the micellar drug carrier system. Administration of the micelles at the dose

exhibiting antitumor activity similar to free CDDP did not increase the plasma BUN,

whereas free CDDP induced its remarkable increase.48

Chen FA, Kuriakose MA, Zhou MX, DeLacure MD, Dunn RL. reported the

use of an injectable biodegradable polymer to deliver Cisplatin for intratumoral treatment

of human head and neck squamous cell carcinoma (HNSCC) in a chimeric mouse model.

The objectives of this research project were (1) to determine the release kinetics of

Cisplatin from the polymer delivery system, (2) to identify the MTD of polymer-

delivered Cisplatin, and (3) to evaluate its therapeutic efficacy. The results revealed that

the polymer delivery system released 80% of the loaded Cisplatin in vivo over a 7-day

period. The polymer-delivered Cisplatin exhibited higher MTD (36 mg/kg) than free

Cisplatin (18 mg/kg) and had a statistically significant tumor suppression effect

compared with free Cisplatin when used at their respective MTD.49



Thomas Chandy, Robert F Wilson, Gundu H R Rao, Gladwin S Das

demonstrated the possibility of entrapping an antiproliferative agent, Cisplatin, in a series

of surface coated biodegradable microspheres composed of poly(lactic acid)--

poly(caprolactone) blends, with a mean diameter of 2--10 um. The microspheres were

surface coated with poly ethylene glycol (PEG), chitosan (Chit), or alginate (Alg).

Cisplatin recovery in microspheres ranged from 25--45% depending on the emulsification

system used for the preparations. Scanning electron microscopy revealed that the PLA--

PCL microspheres were spherical in shape and had a smooth surface texture. The amount

of drug release was much higher initially (20--30%), this was followed by a constant

slow-release profile for a 30-day period of study. It was found that drug release depends

on the amount of entrapped drug, on the presence of extra Cisplatin in the dispensing

phase, and on the polymer coatings. This PEG or Alg-coated PLA/PCL microsphere

formulation may have potential for the targeted delivery of antiproliferative agents to

treat restenosis.50

Barroug A, Kuhn LT, Gerstenfeld LC, Glimcher MJ devloped a new system

for the local delivery of chemotherapy to malignant solid tumors based on calcium

phosphate (CaP) nanoparticles. The adsorption of the anti-neoplastic drug Cisplatin was

characterized on three types of apatitic CaP (poorly and well crystallized hydroxyapatite,

and carbonated apatite). Adsorption isotherms obtained in chloride-free phosphate

solutions at pH = 7.4 (24 and 37 degrees C) indicated that Cisplatin adsorption increases

with temperature and increases with decreasing crystallinity. Release studies in phosphate

buffer saline (containing the chloride ion essential for release) showed that while the

cumulative amount of released drug was same for all the apatites at 20 days

(approximately 70% of the total bound), the least crystalline material released the drug

more slowly. The drug release rate increased slightly with temperature. Cytotoxicity



testing was conducted in a K8 clonal murine osteosarcoma cell line to verify that drug

activity was retained after adsorption onto the apatite crystals. K8 cells were plated onto

dried films of the apatite/Cisplatin conjugates and after 24 h, viability was measured with

tritiated uridine. The apatite/Cisplatin formulations exhibited cytotoxic effects with a

dose dependent diminishment of cell viability.51

Xiao CJ, Qi XR, Aini W, Wei SL prepared Cisplatin multivesicular liposomes

with high encapsulation efficiency and sustained-release character, and compared the

release characteristics with conventional liposomes prepared by reverse-phase

evaporation method. Results showed that the mean diameter of Cisplatin multivesicular

liposomes was (16.6 +/- 1.0) micron. The encapsulation efficiency of Cisplatin was more

than 80%. The release profile in vitro fitted with a first-order equation. Co-membrane

stabilizer had remarkable stabilizing effect on the multivesicular.52

Sugitachi A, Kashiwaba M, Takagane A, Asahi H, Saitoh K, Takahashi M

et.al. devised a novel sustained release system for aqueous cis-platinum (CDDP) to study

the in vitro degradability of the carrier materials and the release profile of the CDDP.

They first prepared fibrin hydrogels with clinically used biomedical materials, and

irradiated the gels with ultraviolet (UV)-rays to form gradually degradable drug carriers.

They loaded aqueous CDDP into the carriers under negative pressure. These prepared

materials were incubated in fibrinolytic test medium at 37 degrees C for the in vitro

studies. The UV-irradiated materials slowly degraded and dissolved within 10-15 days,

while non-UV-treated carriers disintegrated in 4-5 days. Each carrier showed a sustained

release of CDDP. Most of the CDDP delivered was revealed to be the protein-binding

(larger than 10 kDa) form. Free-CDDP was almost nil. The antineoplastic efficacies of

this new drug delivery system developed using an original technique are now being

investigated.53



REVIEW OF MACROMOLECULE

Macromolecule:

Bovine Serum Albumin (BSA), Fraction V.

Molecular Weight:

66,210

Structure and Shape:

Bovine serum albumin is a globular protein consisting of 581 amino acids

stabilized by 17 disulphide bridges. The molecule is cigar shaped is estimated to be 141

A long and 41 A in diameter.

Solubility:

It is soluble in water, insoluble in organic solvents like acetone, acetonitrile,

methanol and ethanol.

Properties:

Isoelectric point is 4.9.

It is precipitated by strong acids like trichloroacetic acid, sulphuric acid.

It is reported to have several hydrophobic binding sites and functions in blood as

an 'Organic Carrier'. Many fatty acids are also known to bind to BSA.

It is biocompatible, biodegradable, non-toxic and non-antigenic.

Storage:

It is stored at 0-80C protected from light.

Applications:

In pharmaceuticals, Bovine serum albumin is used to prepare drug loaded

microspheres and nanoparticles. Albumin microspheres and nanoparticles have received



wide attention because of their stability, shelf life, controllable drug-release property,

high loading capacity, biodegradability, biocompatibility, non-toxicity, non-antigenicity,

drug binding properties of native albumin and organ targeting properties.

In chromatography, bovine serum albumin is reported to be useful in

chromatographic resolution of pharmaceutically active enantiomeric compounds.

Enantiomers like benzoin, ibuprofen, ketoprofen, oxazepam, tamazepam, and warfarin

are separated on bovine serum albumin chiral stationary phases (CSPs) 54.



REVIEW OF MAGNETICALLY MODULATED DRUG DELIVERY SYSTEMS

A great deal of work has been done by scientists and researchers on magnetically

modulated drug delivery systems. The references cited below have been taken from

various national and international journals and published articles in various books.

Emir Baki Denkbas , Ebru Kilic¸ay , Cengiz Birlikseven , Eylem Ozturk

prepared magnetic chitosan microspheres with a size range of 100 to 250 mm (size

distribution ±15 to ±40 mm, respectively) by the suspension cross-linking technique for

use in the application of magnetic carrier technology. The magnetic material (i.e. Fe2O3 )

used in the preparation of the magnetic chitosan microspheres was prepared by

precipitation from FeSO4 and Fe2(SO4 )3 solutions in basic medium and then ground to

the desired size (i.e. 1–5 mm). The morphological and magnetic properties of the

microspheres were characterized by different techniques (i.e. SEM, optical microscopy,

magnetometry). The results demonstrated that the stirring rate of the suspension medium

and the Fe3O4 / chitosan ratio were the most effective parameters for the size / size

distribution and the magnetic quality of the microspheres, while the chitosan molecular

weight (MW) had no significant effect on these properties for the given MW range (i.e.

150 to 650 kDa). The best magnetic quality of the magnetic chitosan microspheres was

around 9.1 emu/g microsphere at 10 kG magnetic field intensity.55

Yue Chang, Zhixing Su devloped a new method for the preparation of magnetic

particles by linking γ-methylacryloyl oxypropyl trimethoxysilane (KH-570) with

magnetic powder after it was hydroxylated and then graft copolymerizing with N-

isopropylacrylamide. Thus, thermosensitive magnetic microsphere was produced.

Modified magnetic particles were characterized by the method of IR, X-ray photoelectron

spectroscopy and transmission electron microscopy. The studies of adsorption and



desorption of emulsifiers on modified magnetic particles showed the modified magnetic

particles have good thermosensitivity.56

Jain S, Mishra V, Singh P, Dubey PK, Saraf DK, Vyas SP Prepared negatively

charged magnetic liposomes using soya lecithin (Soya PC), cholesterol and phosphatidyl

serine (PS) for their preferential presentation to circulating blood phagocytes (monocytes

and neutrophils). In vivo cellular sorting study under magnetic guard indicated an

increase in relative count of neutrophils and monocytes. This study suggested the

potential of negatively charged and RGD-coated magnetic liposomes for

monocytes/neutrophils-mediated active delivery of drugs to relatively inaccessible

inflammatory sites, i.e. brain. The study opens a new perspective of active delivery of

drugs for a possible treatment of cerebrovascular diseases.57

Wada S, Tazawa K, Furuta I, Nagae H. performed study to clarify the

usefulness of Dextran magnetite (DM) for the oral cancer hyperthermia. Tumors were

induced in golden hamster tongue by 9,10-dimethyl 1-1,2-benzanthracene (DMBA)

application. DM suspension was locally injected into the tumor-bearing tongue and

tongues were heated up to 43.0-45.0 degrees C, by AC magnetic field of 500 kHz.

Histological examination revealed a brown uniform DM accumulation at the stroma in

the margin of the tumors. Many of tumor cells disappeared at the site adjacent to this

accumulation. These results strongly suggested the usefulness of this local hyperthermic

system in the oral region was accessible to this treatment.58

Shi K, Li C, He B. Prepared a novel magnetic drug carrier-carboxymethyl

dextran magnetic nanoparticles(CMD MNPs) . Adriamycin (ADR) was coupled with two

types of carrier-neutral dextran MNPs and anionic CMD MNPs, by periodate oxidation.

The physico-chemical characteristics and the magnetic guidance effects in vitro and in



vivo of ADR-CMD MNPs were studied. The distribution profiles of liver and spleen in

mice were studied for both ADR-dextran conjugate MNPs and ADR-CMD conjugate

MNPs. The results showed ADR-CMD conjugate MNPs possessed superparamagnetism,

the mean diameter was 56 nm, the mass magnetic susceptibility was 1.06 x 10(-4) emu.

g-1, and the drug loading was 12.4%. The distribution profiles in liver and spleen revealed

that conjugation with neutral dextran MNPs, excessive accumulation of loaded ADR was

found in liver and spleen after intravenous administration, while conjugation with CMD

MNPs gave a markedly lower concentration in these organs, which indicated less uptake

of ADR-CMD conjugate MNPs by reticuloendothelial system(RES) and the advantage of

delivering loaded drug to sites other than the RES; thus it opened a new perspective for

the active delivery of drug.59

Christopher S. Brazel designed magnetic and Magnetothermal Responsive

Systems for Drug Delivery and concluded that environmentally responsive polymers,

such as the pH-sensitive enteric coatings used in pharmaceuticals, have allowed greater

control over diffusive properties in membranes and controlled release carriers. Many

significant achievements have made magnetic-sensitive release more feasible since the

early experiments at MIT with 1 mm magnetic beads in ethylene vinyl acetate. The

ability to create magnetic nanoparticles with desired compositions has led to the

investigation of hydrogels with aqueous-dispersed cobalt-modified hematite (Co-γFe2O3)

and FePt for magnetically controlled release of model agents. In their early work, the

magnetic particles moved in response to an oscillating magnetic field, causing an increase

in diffusivity for imbedded solutes. Their current work focuses on using the heating of

these particles controlled by magnetic field to change polymer phase behavior and

modulate the release of active agents. The magnetic particles were designed to have a low

Curie temperature, so that they become paramagnetic around 45 oC and do not heat any



further. With this heating, the polymer system, poly (2-hydroxyethyl methacrylate) with

grafter N-isopropylacrylamide oligomers, opened up to deliver the agent until the

magnetic field was removed and the heat dissipates.60


Chapter 4 Methodology

METHODOLOGY MATERIALS USED

Sr.No Materials Grade Manufacturer

1. Cisplatin I.P Sun Pharma, Mumbai, and Cipla.Banglore

2. Bovine serum albumin Fraction V

Himedia, Mumbai

3. Di-Sodium Hydrogen Phosphate L.R E.Merck (India) Limited, Mumbai.

4. Potassium Dihydrogen Phosphate

L.R S.D. Fine Chem. Limited, Mumbai

5. Cotton seed oil L.R Acros organics, New jersey, USA

6. Diethyl Ether A.R LOBA chemie Pvt. Ltd, Mumbai

7. Dimethyl Sulphoxide A.R S.D. Fine Chem. Limited, Mumbai

8. Tween 80 L.R Himedia, Mumbai

9. Silicone oil L.R LOBA chemie Pvt. Ltd, Mumbai.

10. Diethyl Dithio carbamate L.R Thomas Baker, Mumbai.

11. Ferrous sulphate L.R S.D. Fine Chem. Limited, Mumbai

12 Sodium thiosulphate L.R S.D. Fine Chem. Limited, Mumbai



EQUIPMENTS

Sr. No Equipment Company

1 UV- Vis Spectrophotometer (UV-1201)

Shimadzu Corporation, Japan

2 IR 200 Spectrometer Thermo Nicolet Corporation,

Madison, WI.

3 Refrigerated Centrifuge

Super spin R-V/FA

Plasto Crafts

4 0.22 Micron Filters PALL Pharmalab filtration Pvt Ltd. Mumbai

5 Magnetic Stirrer 2 MLH Remi Equipments, Mumbai

6 Scanning Electron Microscope,

JSM-T330A

JEOL, Japan

7 Millex-HIV 0.45 μm filter units Millipore Molsheim, France

8 Metabolic Shaker Ravi Scientific Instruments, Delhi

9 High speed tissue Homogenizer Remi Motors Ltd

10 Humidity control oven Lab control, Mumbai

11 X-Ray diffractrometer Philips, PUU 1700,Holland.

METHODS:

Preformulation Studies:61

Preformulation is defined as the phase of research and development process

where physical, chemical and mechanical properties of a new drug substance are

characterized alone and when combined with excipients, in order to develop stable, safe

and effective dosage form.



A thorough understanding of physicochemical properties may ultimately provide

a rationale for formulation design, or support the need for molecular modification or

merely confirm that there are no significant barriers to the compounds development.

Hence, preformulation studies on the obtained sample of drug were performed for

identification and compatibility studies.

1) Identification of drug:

The obtained sample was examined by Infrared spectral analysis and was compared

with the reference spectrum of Cisplatin.

2) Solubility Analysis:

Preformulation solubility analysis was done, which included the selection of

suitable solvent, to dissolve the drug as well as various excipients used for the

fabrication of magnetic microspheres.

3) Melting Point Determination:

Melting point determination of the obtained sample was done as it is a first point

indication of purity of sample, since the presence of relatively small amount of impurity

can be detected by lowering as well as widening in the melting point range. Melting point

of obtained sample was determined by “Theils tube apparatus”.

4) Compatibility Studies:

Compatibility of the drug (Cisplatin) with excipients such as bovine serum

albumin (BSA) and magnetite (Fe2O3) used to formulate magnetically responsive

microspheres (MRM) was established by Infrared spectral analysis. I.R. Spectral analysis

of Cisplatin, BSA, magnetite and combination of the drug with BSA and Magnetite was

carried out to investigate any changes in chemical composition of the drug after

combining it with the excipients.



Standard Calibration Curve:46

10 mg of Cisplatin was dissolved in 5 ml 0.1 N HCl and volume made up to 100

ml using phosphate buffer saline (PBS) pH 7.4. From this stock solution aliquots of

1,2,3,4,5,6 & 7 were pipetted out and volume made up to 10 ml with PBS pH 7.4 to

obtain concentrations in the range of 10 to 70 μg/ml. The absorbance of the solutions

were measured at 210 nm using UV-Vis Spectrophotometer. A graph of Concentration

Vs Absorbance was plotted.

Preparation Of Coated Magnetite Particles:17

The magnetite was prepared by reacting 10% w/v ferrous sulphate (containing 5%

tween 80) with 20% w/v sodium hydroxide solution, followed by washing off the

precipitate with dilute ammonia in order to get magnetite free of sulphate ions. This was

then dried at 100 °C and passed through # 300 sieve. The magnetite particles thus formed

tend to agglomerate due to their surface energy and under the influence of induced

magnetic field (when magnetic field is applied). Therefore the magnetite particles were

coated with a nonmagnetic material, silicon oil to reduce the mutual attractive forces

between the particles. The coating was done by packing the magnetite particles in a

funnel and percolating 1% w/v solution of silicon oil in ether through the magnetite. The

oil coating also imparts hydrophobicity to the particles and facilitates their uniform

dispersion in water.

Tests For Identity:62

The above-prepared magnetite was tested for its identity:

• 1 ml solution of digested magnetic microspheres when allowed to react with 1 ml

(5 %w/v) solution of potassium ferrocyanide an intense blue precipitate formed,

which was insoluble in dilute hydrochloric acid and decomposed on addition of

sodium hydroxide solution.



• 2 ml strongly acidic solution of digested magnetic microspheres was allowed to

react with 2 ml (0.2 % w/v) solution of 8-hydroxy-7-biodoquinoline-5-sulphonic

acid a stable green colour was produced.

Preparation Of Magnetically Responsive Microsphere: 17,63

Magnetic microspheres of Cisplatin were prepared by phase separation emulsion

polymerization (PSEP) technique. 1 ml solution of BSA (in freshly prepared phosphate

buffer saline pH 7.4) containing dispersed magnetite (30% w/w of albumin) and 0.5 ml of

DMSO containing 10 mg of dissolved Cisplatin) was added into 10 ml of cottonseed oil

(4 °C) containing 4% w/v. of surfactant span 20 and the above mixture was stirred for 10

min. The resultant emulsion was then added dropwise (50±10 drops/min) using glass

syringe with 23-gauge needle into 150 ml of cottonseed oil (preheated to 130 ± 5 °C)

along with stirring at 1400 rpm. Heating and stirring of the oil were continued for 10 min

after the addition of emulsion. The resulting suspension was then allowed to cool to room

temperature with continuous stirring and washed three times with 60 ml anhydrous

diethyl ether, each time centrifuging at 3000 g for 15 min. The washed microspheres

were suspended in 10 ml anhydrous ether and unincorporated Fe2O3 was removed by

transferring the suspension into a tared tube, in presence of a 300 Gauss bar magnet

placed at rim of the decanting tube. The magnetic microspheres thus obtained were dried

in a dessiccator and stored in airtight amber coloured bottles at low temperature. Four

batches of magnetic microsphere viz. F-1, F-2, F-3 & F-4 were prepared by employing

the above method.



TABLE 1: FORMULATION PLAN OF CISPLATIN MAGNETIC

MICROSPHERES

Formulations Ratio (Drug : Polymer) Drug (mg) Polymer

(mg)

Fe203 (30% w/w of

polymer)

F-1 1:1.5 10 15 4.5

F-2 1:2 10 20 6.0

F-3 1: 2.5 10 25 7.5

F-4 1:3 10 30 9.0

Evaluation Of Magnetic Microspheres:

1) Percentage Practical Yield : 64

Percentage practical yield is calculated to know about percent yield or efficiency

of any method, thus it helps in selection of appropriate method of production. Practical

yield was calculated as the weight of the dried magnetic microspheres recovered from

each batch in relation to the sum of the starting material.

% PY = (Practical Yield/ Theoretical Yield) X 100

2) Particle Size Analysis: 65

Particle size analysis was done by scanning electron microscopy (SEM). SEM is

the most commonly used method for characterizing drug delivery systems, due to

simplicity in sample preparation and ease of operation. The three dimensional

information about macro- (0.1-10mm) meso (1-100 μm) & microstructure (10-1,000nm),

is often found with in the same micrograph. SEM has been used to determine particle size

distribution, surface topography, texture and to examine the morphology of fractured or

sectioned surface.



Particle size analysis was done by SEM using JEOL JSM-T330A scanning

microscope. Small volume of suspension of magnetic microspheres was placed on an

electron microscope brass stub. The stubs were placed briefly in a drier and then coated

with gold in an ion sputter. Picture of magnetic microspheres were taken by random

scanning of the stub. The diameter of about 30 magnetic microspheres were measured

from the photomicrographs of each batch. Finally, average mean diameters were

obtained.

3) Drug Entrapment Efficiency :66

Unentrapped drug: Ten milligrams of drug-loaded microspheres were weighed

and suspended in 10 ml solution (0.5 ml 0.1 N HCl + 9.5 ml PBS) for 5 minutes. The

suspension was then filtered through 0.45μ filter and supernatant was analysed by UV-

Vis. Spectrophotometer for free drug content.

Entrapped drug: The residue obtained after filtration was then digested in 5 ml

solution containing 2.5 ml of 50 % v/v trichloroacetic acid and 2.5 ml 0.1N HC1 and kept

for 24 hrs to precipitate the protein. The digested homogenate was centrifuged for 5 min.

and the supernatant was analyzed for drug content by measuring the absorbance at 210

nm by UV-Vis spectrophotometer after appropriate dilutions with PBS.

Entrapment Efficiency = Experimental Drug Content x 100 Theoretical Drug Content

*All the following evaluation parameters were performed after removal of unentrapped

drug and unincorporated magnetite

4) X-ray diffractrometry:67

X-ray diffraction is currently of prime importance in elucidating the structure of

natural product, physical properties of metals, polymeric materials and other solids. X-ray



diffraction also provides a convenient and practical means for the qualitative

identification of crystalline compounds.

An x-ray diffraction pattern is unique for each crystalline substance. Thus, if an

exact match can be found between the pattern of an unknown and an authentic sample,

chemical identity can be assumed.

The best formulation (F-3) was subjected to X-ray diffractrometry. X-ray

diffractrometer Philips, Holland, PUU 1700 was used to ensure the presence of Fe2O3 in

prepared formulation.

5) Determination Of Percent Magnetite Content: 68,69

Determination of Fe2o3 content in prepared MRM was conducted by employing a

conventional method using thiosulfate and potassium iodide for quantitative analysis.

This was done by destroying the microspheres by gently heating while mixing them with

the help of magnetic stirrer for few minutes, leading to a yellow homogeneous solution.

An accurately weighed amount of magnetic microspheres (after destruction by

gentle heating) was dissolved in mixture of water (200ml) and con. HCl (200) by heating

it to the boiling point. The solution was boiled for 15 seconds and cooled rapidly. Then

potassium iodide (3 gm) was added and kept in dark for 15 minutes, the liberated iodine

was then titrated with 0.1N sodium thiosulphate (Na2S2O4) using starch as indicator. A

blank titration was carried out. The difference between titrations gave the amount of

iodine liberated by ferric ion.

Each ml of 0.1N sodium thiosulphate ≅ 0.005585g of ferric ion.



6) In Vitro Magnetic Responsiveness:17

An apparatus was designed to study the magnetic responsiveness (Figure 5). 250

ml separating funnel used as reservoir (A) was fitted with stopcock (B) to regulate the

flow of liquid from the reservoir. A rubber tubing (C) of length 10 cm formed the link

between the reservoir and an “L” shaped glass tubing (D) (30 cm long vertically and 40

cm long horizontally). This tube had its end bent to a length of 6.5 cm in the upward

direction and it was attached to another rubber tubing of 2.5 cm. A screw cock (E) was

fitted to this rubber tube to adjust flow rate of the fluid. A trap (F) was made in the

middle of the horizontal portion of the glass tube with an exit tube (making an angle)

with it. To this trap, a rubber tubing of 2.5 cm length was joined and closed with the help

of a pinchcock (G). An electromagnet (H) of strength 7000 Oe was placed

perpendicularly over the trap such that the horizontal portion and trap lie in the center of

the applied magnetic field.

Figure 4: Apparatus for in vitro magnetic responsiveness study

Magnetic microspheres equivalent to 20 milligrams of Cisplatin were weighed

and suspended in isotonic saline solution containing 1 drop of tween 80. The flow rate

was adjusted to 3 ml/min with the help of stopcock (B). The magnetic field was applied



and the prepared suspension of magnetic microspheres was injected slowly into the upper

end of rubber tubing (C). Ten minutes after injection, the flow of fluid from (A) was

stopped with the help of stopcock. The microspheres retained by the magnet, were

collected from the trap and analyzed for drug content.

7) In Vitro Drug Release Studies: 70

Magnetic microspheres equivalent to 10 milligrams of Cisplatin were weighed

and transferred into a conical flask containing 50 ml of PBS pH 7.4. Then the flask was

kept in a metabolic shaker and the shaker was adjusted to 50 horizontal shakes per minute

at 370C ± 0.5 °C. One ml aliquot of release medium were withdrawn at time intervals of

15 min, 30 min, 1, 2, 4, 8, 16, 24 hrs and replaced by the same volume of PBS. These

samples were filtered through 0.45 μm membrane filter. The Filtrate was diluted suitably

and estimated by UV-Vis. spectrophotometer at 210 nm.

Plate No.1

METABOLIC SHAKER USED FOR IN VITRO RELEASE STUDY



8) In Vivo Drug Targeting Studies: 17,63,71

This study was carried out to compare the targeting efficiency of drug loaded

magnetic microspheres in presence and absence of magnetic field in terms of percentage

increase in targeting to various organs of reticuloendothelial system like liver, kidney,

lungs and spleen.

Dose Calculation:

Dose of Cisplatin to be administered in rats was calculated according to body

surface ratio of rat to human beings.

Dose (mg/200 gm of rat) = Human dose (mg) x BSA ratio

Dose = 30 x 0.018

= 0.54 mg/200 gm of rat.

For 200 gm rat, Dose is = 0.54 mg

There fore, for 225 gm rat Dose = (0.54 x 225)/ 200 = 0.6075 mg or 607.5 mcg

Eight healthy adult Sprague Dawley rats weighing 225-235 gms were divided into

2 groups, each containing 4 rats. The tail of each rat was demarcated into 3 parts with

picric acid solution. Section-l, the dose administration site, measuring 3 cm from the

base; section-2, the target-site, measuring 4 cm from section-l; and section-3, the

remaining tail length. The ventral caudal artery at section-l was cannulated, and section-2

was placed between two poles of an electromagnet.

The rats of first group were anaesthetized and a dose of drug loaded magnetic

microspheres equivalent to 607 mcg of Cisplatin in sterile phosphate buffer saline

solution was introduced through polyethylene tubing, cannulated through caudal artery, at

the exposed section 1 of the tail. The magnetic field was applied on section 2, the

preselected target site, with the help of electromagnet having field strength 7000 Oe.The



magnet was removed from the site (section 2) after 30 min of administration of the

magnetic microspheres. Two rats were sacrificed after 1 hour and the other two after 3

hours. The organs such as tail, lung, liver, spleen and kidney were isolated. The

individual organs of each rat were homogenized separately and digested with 2 ml

solution containing 1 ml of 50 % v/v trichloroacetic acid and 1 ml 0.1N HC1 and kept for

24 hrs in refrigerator to precipitate the protein. Then the drug was extracted after multiple

washings and centrifuged at 15,000 rpm to obtain the supernatant. The supernatant was

filtered through an ultra filter membrane of pore size 0.22 microns and subjected to

extraction procedure. The rats of second group, taken as control were administered with

the same dose of magnetic microspheres of Cisplatin in the absence of magnetic field and

the same procedure was followed.

Extraction procedure:

Ultra filtrate samples were mixed with a known amount of 10% solution of

diethyldithiocarbamic acid (sodium salt DDTC) in sodium hydroxide and digested at

37°C for 1 hr and then chilled. The drug chelate was then extracted with chloroform and

evaporated to dryness and redissolved in PBS pH 7.4. The drug content was estimated

using UV-Vis spectrophotometer at 210 nm.

9) Stability Studies73

Information on the stability of drug substance is an integral part of the systematic

approach to stability evaluation. The purpose of stability testing is to provide evidence on

how the quality of a drug substance or drug product varies with time under influence of

variety of environmental factors such as temperature, humidity and light, and to establish

a re-test period for drug substance or a shelf life for the drug product and recommended

storage conditions.



All 4 batches of Cisplatin magnetic microspheres were tested for stability. All the

preparations were divided into 3 sets and were stored at:

• 4°C in refrigerator

• 30º C ± 2º C/65% RH ± 5% RH in humidity control oven (GINKYA IM 3500

series).

• Ambient temperature and humidity.

After 15, 30 and 60 days drug content of all the formulations was determined by

the method discussed previously in entrapment efficiency section.

In vitro release study of a selected formulation was also carried out after storage

for one month.


Chapter 5 Results and Discussion

RESULTS AND DISCUSSION

Magnetic responsive microspheres of Cisplatin were prepared and evaluated for

their use as targeted and controlled drug delivery system.

In the present work total four formulations were prepared and their complete

composition is shown in Table 1.

Preformulation studies:

1) Identification: The IR spectrum of the obtained sample complied with the

reference standard IR spectrum of Cisplatin. This indicates that the obtained

sample is Cisplatin.

2) Solubility Analysis: The obtained sample was tested for solubility in N, N-

dimethylformamide (DMF), N, N-dimethylacetamide (DMA), and

dimethylsulphoxide (DMSO). The obtained sample was found to be soluble in all

above-mentioned solvents, which complied with solubility profile of reference

Cisplatin.

3) Melting Point Determination: The melting point of the obtained sample was

found to be 269 0C-271 0C, which complied with the reported melting point of

standard Cisplatin thus indicates the purity of obtained sample.

4) Compatibility Studies: Preformulation studies were carried out to study the

compatibility of Cisplatin with BSA and prepared magnetite prior to the

preparation of magnetic responsive microspheres of Cisplatin.

I.R spectra of pure Cisplatin, BSA and prepared magnetite and their combination

were obtained, which are shown in spectra 1, 2,3, & 4 respectively.



All the characteristic peaks of Cisplatin were present in spectrum 4. Thus

indicating compatibility between drug and excipients. It shows that there was no

significant change in the chemical integrity of the drug. Further, drug was identified as

Cisplatin by carrying out its monograph analysis.

Standard calibration curve of Cislpatin by UV-Vis spectroscopy:

Table 2 shows the absorbance of Cisplatin standard solutions containing 10-70

mcg/ml of drug in pH 7.4 phosphate buffer saline. Figure 5 shows the standard

calibration curve. The curve was found to be linear in the range of 10-70 mcg/ml at λmax

210 nm. The regression value was found to be 0.9998.

The calculation of drug content, in vitro magnetic responsiveness, in vitro release,

in vivo drug targeting studies, and stability studies are based on this calibration curve.

The prepared formulations were subjected to various evaluation parameters, which are

discussed with their results as follows: -

1. Percentage Practical Yield:

The result of % practical yield studies are shown in Table 3 Percent practical yield

increased as the amount of polymer added to each formulation increased, although it may

not be dependent upon drug concentration in the formulation. Maximum yield was found

to be 61.25 % for F-4.

2) Particle size analysis:

Scanning electron photomicrographs of all the four formulations are shown in

plates 2, 3, 4 and 5. Different magnifications were used while taking these

photomicrographs. Average particle size of magnetic microspheres of Cisplatin was

found to be 3 to 12 μm for F-1 to F-4. Particles of all formulations except F-3 were

smooth, oval and discrete whereas particles of F-3 were slightly rough surfaced but

discrete.



3) Drug Entrapment Efficiency:

Drug entrapment efficiency was calculated from the drug content. The drug

content in four batches of Cisplatin magnetic microspheres was studied. The amount of

drug bound per 10 mg of magnetic microspheres was determined in each batch. Table 4

and Figure 6 show the results of the drug entrapment efficiency in each of these

formulations. It was observed that the entrapment efficiency increased with the increase

in concentration of polymer in the formulations.

The maximum entrapment was found in F-4 (56.37%) and lowest entrapment in

F-1 (39.82%).

4) X-Ray Diffractrometry:

X-ray diffractrometry was performed to ensure the presence or incorporation of

magnetite in prepared magnetic microspheres. X-ray diffractogram showed almost

similar peaks (at 4.18,2.85 and 1.998) for formulation (F-3) as that of prepared magnetite

(at 4.18,2.49 and 2.21). Thus the result confirms the presence of magnetite in prepared

Cisplatin magnetic microspheres. Spectrum 5 and 6 shows X-ray diffractogram of

prepared magnetite and formulation F-3 respectively.

5) Percent Magnetite Content:

Determination of magnetite content in prepared MRM was conducted by

employing a conventional method using thiosulfate and potassium iodide for quantitative

analysis.

The amount of magnetite content per 10 mg of microspheres was determined in

all the prepared four batches. Table 5 and Figure 7 show the result of the magnetite

content in each formulation. It was observed that entrapment of magnetite increased with

increase in concentration of polymer added in consecutive formulations. The maximum

magnetite content was found in F-4 (55.48%).



6) In Vitro Magnetic Responsiveness:

Table 6 and Figure 8 show the result of magnetic responsiveness. The magnetic

responsiveness, after 10 min was found to be highest 72.68% for formulation F-4 and

least 51.36% for formulation F-1 under the influence of magnetic felid. While

8.44,11.38,16.26 and 24.19% microspheres were retained in absence of magnetic field for

formulation F-1–F-4 respectively. It was observed that magnetic responsiveness

increased with increase in entrapped magnetite content irrespective of increase in

concentration of polymer.

The percentage of microspheres failed to retain in the glass tube in absence and

presence of magnetic field was compared. In the absence of magnetic field, unretained

microspheres were significantly more than those in the presence of magnetic field.

Therefore, it is predicted that the microspheres prepared by this method can accumulate

in the capillaries following in vivo administration.

7) In Vitro Release Studies:

Pure Cisplatin and all the four formulations of Cisplatin magnetic microspheres

were subjected to in vitro release studies. These studies were carried out using metabolic

shaker (plate 1) in phosphate buffer saline pH 7.4. The cumulative percent drug release of

pure drug was found to be 93.60% at 3 hours. Cumulative percent drug release after 24

hours was 89.60%, 82.22%, 78.41%, and 76.35% for F-1-F-4 respectively by UV

spectroscopy.

The results obtained in in vitro release studies were plotted in five models of data

treatments as follows: -

• Cumulative percent drug release Vs. Time (Zero order rate kinetics).

• Log Cumulative percent drug retained Vs. Time (First order rate kinetics).



• Higuchi's classical diffusion equation (Higuchi matrix) in which cumulative

percent release was plotted against √T (root time).

• Log of cumulative percent drug released Vs. log Time (Peppas exponential

equation).

• According to Hixson Crowell's erosion equation, (% Retained)1/3 Vs. Time or

(1-Mt/M∝)1/3 Vs. Time.

The release data obtained for pure drug and formulations F-1, F-2, F-3, F-4 are

tabulated in Table 7-11. Figure 9 and 10 show plots of cumulative percent drug released

as a function of time for pure drug and for different formulations.

It was observed that the drug release from the formulations decreased with

increase in ratio of polymer added in each formulation.

When compared with pure drug, the in vitro release of microspheres is prolonged

over a period of 24 hrs.

The in vitro release of all the four batches of microspheres showed a bi-phasic

release with an initial burst effect even after washing. In the first hour, drug release was

38.64%, 36.03%, 35.81% and 33.81% for F-1, F-2, F-3 and F-4 respectively. Afterwards

the drug release followed a steady pattern approximating zero order release. The burst

release in the first hour even after washing indicates need of increase in washing period

in order to overcome burst effect. The mechanism for the burst release in the first hour

can be attributed to the drug loaded on the microspheres surface or imperfect entrapment

of the drug by the albumin.

The kinetic values obtained for different formulations are indicated in Table 12

and 13. The values of in vitro release were attempted to fit into various mathematical



models. Plots of zero order, first order, Higuchi matrix, Peppas and Hixson Crowell are

depicted in figures 10, 11, 12, 13 and 14 respectively.

The regression co-efficient for Zero order plot were found to be 0.9022, 0.9670,

0.9764 & 0.9894 for formulation F-1, F-2, F-3 and F-4 respectively.

The regression co-efficients for First order plot were found to be -0.9783, -

0.9901, -0.9875 & -0.9834 for formulation F-1, F-2, F-3 and F-4 respectively. Based on

the highest regression values, (r) the best fit model for F-1, F-2 and F-3 was first order,

and F-4 followed zero order release.

Hixson Crowell plot of the formulations is indicated in Figure 14. The

regressions co-efficient of formulation F-1 to F-4 were found to be -0.9627, -0.9877,

-0.9877 and -0.9874 respectively. These results indicate that F-3 and F-4 appear to fit in

Hixson Crowell model. Here it can be assumed that the release rate was limited by the

drug particles dissolution rate and erosion of the polymer matrix.

The regression co-efficient for formulation F-1 to F-4 of Higuchi matrix plot was

found to be 0.9677, 0.9885, 0.9851 & 0.9699 respectively. It was observed that F-2

followed Higuchi matrix suggesting drug release by diffusion.

The Peppas model is widely used, when the release mechanism is not well known

or when more than one type of release phenomenon could be involved. The 'n' value

could be used to characterize different release mechanisms.

Peppas – Korsmeyer Equation is given as: -

% R = K tn

or, Log %R = log K + n log t

Where, R = drug release, k = constant, n = slope, t = time.



The 'n' values for F-1 to F-4 were 0.3163, 0.2569, 0.2327, and 0.2019

respectively, which is less than 0.45. This indicates that the release approximates Fickian

diffusion mechanism.

8) In vivo drug targeting studies

Table 14 shows the result of in vivo targeting. In vivo drug targeting studies

showed that after 1 hour of administration of magnetic microspheres, 57.61% of drug was

recovered from the rat tail section-2 under the influence of magnetic field. The drug

concentration in other organ was found to be 10.69% in liver, 3.29% in kidney, 2.79% in

spleen and 1.81 % in lungs. Though the magnetic field was removed at 30 mins. after the

administration of magnetic microspheres, 41.97% of the drug was recovered from the rats

tail section-2 after 3 hrs. of the administration (Figure15). This localization may have

appeared due to the penetration of the microspheres by endocytosis in the rats tail section,

lack of phagocytosis and slow rate of blood flow in the tail.

These results were compared with those obtained form control rats (without

magnetic field). In control group of rats only 3.62% drug was recovered after one hour

and 2.31% was recovered after 3 hours from the rats tail section-2 (Figure15). However

the highest concentration was found in liver (46.1%) and spleen (18.11%).

9) Stability Studies:

Stability studies of the prepared microspheres were carried out, by storing all the

formulations F-1 to F-4 at 4ºC in refrigerator, Ambient temperature and humidity and 30º

C ± 2ºC/65% RH ± 5% RH in humidity control oven for sixty days. Two parameters

namely residual percent drug content and in-vitro release studies were carried out.

The results of drug content after 15,30 and 60 days are shown in Table 15. Figure

16 shows the plots of % residual drug Vs. time for different formulations after 60 days

storage.



These studies reveal that there is a reduction in drug content after storage for sixty

days at 4º C, ambient temperature and humidity and 30º C ± 2ºC/65% RH ± 5% RH. It

was also revealed that out of the four formulated batches, the one stored at 4º C showed

maximum residual drug followed by that stored at ambient temperature and humidity and

30º C ± 2ºC/65% RH ± 5% RH.

Table 16 shows the data for in vitro release studies, which were carried out after

storing a selected formulation (F-3) at 4º C, ambient temperature and humidity and 30º C

± 2ºC/65% RH ± 5% RH for sixty days. Figure 17 shows the plots.

In vitro release studies revealed that the formulation stored at 4º C showed

80.56 % release, the one which stored at ambient temperature and humidity showed 82.74

% and formulation stored at 30º C ± 2ºC/65% RH ± 5% RH showed 84.48 % release after

24 hours.

These results indicate that the drug release from the formulation stored at 30º C ±

2ºC/65% RH ± 5% RH was highest followed by formulation stored at ambient

temperature and humidity and 4º C.

On comparing this data with the previous release data of F-3, it was observed that

there was an overall increase in the drug release.

These results may be attributed to erosion of particles to some extent during

storage.



Plate No.2

Scanning Electron Photomicrograph of Formulation F-1

Plate No.3




Plate No.4


Plate No.5


































































SPECTRUM 5: X-RAY DIFFRACTROGRAM OF MAGNETITE

SPECTRUM 6: X-RAY DIFFRACTROGRAM OF FORMULATION (F-3)




Chapter 6 Conclusion

CONCLUSION

From the experimental results it can be concluded that: -

• Bovine serum albumin is a suitable and compatible biodegradable polymer for the

preparation of magnetically responsive microspheres of Cisplatin.

• Percent practical yield increases as the ratio of polymer to the drug added increased.

• Prepared coated magnetite particles were highly magnetically responsive and also

capable of forming uniform dispersion in water.

• Particle size analysis revealed that, the microspheres were in the range (3 to 12 µm)

and all the formulations showed ideal surface morphology. Particle size for

formulation F-3 was the smallest. Whereas particles of F-1and F-4 were slightly

rough surfaced but discrete.

• Increase in the amount of polymer added to the formulations increases the entrapment

efficiency of both drug as well as magnetite.

• Though formulation F-4 showed maximum entrapment of drug as well as that of

magnetite, the optimum drug to polymer ratio was found in formulation F-3 (1:2.5),

after which no significant increase in entrapment efficiency was observed with

increase in drug to Polymer ratio.

• The X-ray diffractrogram confirms the presence of magnetite in prepared Cisplatin

magnetic microspheres.

• The prepared magnetic microspheres of Cisplatin were found magnetically

responsive. It was also observed that magnetic responsiveness increased with increase

in entrapped magnetite content.



• In vitro release studies showed biphasic release pattern for all formulations, with an

initial burst effect even after washing, which may be attributed to the high drug

loaded onto the surface of the particles as well as insufficient washing.

• Formulation F-1 showed maximum cumulative percent drug release which can be

attributed to insufficient binding of drug, may be because of improper drug to

polymer ratio.

• Overall the curve fitting into various mathematical models was found to be average. It

was found that formulations F-1, F-2 and F-3 followed first order kinetics where as

F-4 followed zero order kinetics.

• It can be assumed from the data of Hixson Crowell plot for invitro release, that the

release rate was limited by the drug particles dissolution rate and erosion of polymer

matrix for formulation F-3 and F-4, as they appear to fit in this model.

• It was also observed that F-2 followed Higuchi matrix suggesting drug release by

diffusion.

• The ‘n’ values obtained from Peppas plot indicated that the microspheres followed

Fickian controlled release mechanism.

• On the basis of drug content, magnetic responsiveness, particle size morphology,

in vitro release and release kinetics, formulation F-3 was selected as an optimum

formulation for in vivo and stability studies.

• From the results of in vivo targeting studies it was observed that the percentage of

microspheres retained was significantly more in the presence of magnetic field than in

the absence of magnetic field. Hence magnetically responsiveness Cisplatin

microspheres can be successfully used for site specific targeting.



• Stability studies showed that maximum drug content and closest in-vitro release to

previous data was found at 4°C storage. Thus, it can be concluded that 4°C is the

most suitable temperature for storage of Cisplatin magnetic microspheres.

• The results of investigation demonstrated that magnetically responsive albumin

microspheres offer an alternative approach in achieving drug targeting. The results

obtained from this study clearly suggest that magnetically responsive microspheres

containing Cisplatin are retained at the target site, in the presence of a 7000 Oe

magnetic field, and are capable of releasing their drug for an extended period of time.

Hence, it is predicted that these microspheres could be retained on the target tissue in

vivo and release their drug for prolonged period of time.

• The present study was a satisfactory preliminary attempt in development of

magnetically modulated novel parenteral carrier for Cisplatin. However magnetic

field strength and gradient, distribution of the magnetite in the microspheres,

application time of the magnetic field and in vitro- in vivo co- relationship under

diseased state are the most likely parameters which need to be optimized for the

successful use of this delivery system in localized drug therapy.


Chapter 7 Summary

SUMMARY

In any drug delivery system therapeutic amount of drug is desired at the specific

site. 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 desired

differential distribution of drug by its targeted delivery would spare the rest of the body

and thus significantly reduce the overall toxicity while maintaining its therapeutic

benefits. The drug’s therapeutic index (TI) as measured by its pharmacological response

& safety, relies in the access and specific interaction of the drug with its candidate

receptor, whilst minimizing its interaction with non-target tissue. In addition, many

clinical scenarios require delivery of agents that are therapeutic at the desired delivery

point, but otherwise systemically toxic.

So this project proposed a method for targeted drug delivery by applying high

magnetic field gradients within the body to an injected super paramagnetic colloidal fluid

carrying a drug, with the aid of modest uniform magnetic field. Cisplatin, an anticancer

drug was used as the model drug. It is a potent cytotoxic drug with severe side effects.

In the present study an attempt was made to formulate Cisplatin magnetic

microspheres in order to study targeting efficiency, enhance bioavailability, reduce dose,

there by improving patient compliance. Cisplatin magnetic microspheres was formulated

using bovine serum albumin (biodegradable natural polymer) as carrier by phase

separation emulsion polymerization (PSEP) technique. Prior to formulation,

preformulation studies were carried out in order to establish compatibility between drug

polymer and magnetite by IR spectroscopy. Four formulations (F-1, F-2, F-3 and F-4)

were prepared by varying the ratio of polymer to drug.


Chapter 7 Summary

Preformulation studies revealed that the drug Cisplatin, magnetite and bovine

serum albumin were satisfactorily compatible, without any significant changes in the

chemical nature of the drug.

These formulations were subjected to various evaluation parameters like percent

practical yield, particle size analysis, drug entrapment efficiency, X-ray diffractrometry,

determination of magnetite content, in vitro magnetic responsiveness, in vitro drug

release studies, in vivo drug targeting studies and stability studies. The results of all the

parameters are tabulated and depicted graphically wherever necessary in the result and

discussion section.

Percentage practical yield was found to be maximum in formulation F-4. Particle

size of the drug-loaded magnetic microspheres revealed that the particles were in micron

range. Drug entrapment efficiency was found to be maximum in F-4. It was observed that

the entrapment efficiency increased with the increase in concentration of polymer added

in the consecutive formulations.

X-ray diffractrometry results ensured the presence or incorporation of magnetite

in prepared Cisplatin magnetic microspheres. The maximum magnetite content was

found in F-4. It was observed that entrapment of magnetite increased with increase in

concentration of polymer added in consecutive formulations.

The magnetic responsiveness was found to be highest for formulation F-4 and

least for formulation F-1 under the influence of magnetic felid. It was observed that

magnetic responsiveness increased with increase in entrapped magnetite content

irrespective of increase in concentration of polymer.

In vitro release study was analyzed using various mathematical models.

Cumulative percent drug release with respect to time was found to be highest for


Chapter 7 Summary

formulation F-1 showed maximum cumulative percent drug release. Based on the

regression coefficient values, the best-fit model for F-1, F-2 and F-3 was first order and

F-4 followed zero order release. Results of Hixson Crowell plot indicated that F-3 and

F-4 appear to fit this model. It was also observed that F-2 followed Higuchi matrix

suggesting drug release by diffusion.

The ‘n’ values obtained from Peppas plot indicated that the microspheres

followed Fickian controlled release mechanism. Hixson Crowell regression data shows

that the release rate was limited by the drug particles dissolution rate and erosion of

polymer matrix.

The in vivo drug targeting studies revealed that the percentage of microspheres

retained was significantly more in the presence of magnetic field than in the absence of

magnetic field. Percentage of microspheres retained in vivo under influence of magnetic

field was in the order tail section -2>liver> kidney >spleen> lung.

Drug content data of stability studies revealed that all the four formulated batches

stored at 4º C showed maximum residual drug followed by that stored at ambient

temperature and humidity and 30ºC ± 2ºC/65% RH ± 5% RH. In vitro release data of

stability studies for formulation F-3 indicate that very less variation in release was found

at 4ºC followed by ambient temperature and humidity and 30°C/65% RH.

From the above studies it can be concluded that 4°C is the most suitable

temperature for storage of Cisplatin magnetic microspheres.


Annexure

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72. The European Agency for the evaluation of medicinal products. Stability Testing

Guidelines: Stability testing of new drug substances and products. London:ICH –

Technical Coordination, EMEA. [online]. 2003 [Cited 2003 Feb 20];[21 Screens].

Available from: URL:http://www.emea.eu.int.


http://www.emea.eu.int/

Annexure


Classification of parenterally administered colloidal carrier systems

I R SPECTRUM 3: MAGNETITE

MAGNATITE

603.

85804.

64

889.

8110

19.7

1

1097

.60

1258

.25

1367

.14

1559

.82

1663

.58

2627

.02

2759

.67

2858

.44

2911

.71

3139

.75

3490

.19

76

78

80

82

84

86

88

90

92

94

96

98

100

102

104

106

108

110

%T

500 1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1)

I R SPECTRUM 1: CISPLATIN

CISPLATIN

424

04

738.

7980

0.36

1304

.75

1367

.02

1537

.79

1656

.9021

09.1

3

2564

.59

2755

.39

2902

.86

3209

.00

3285

.94

3411

.16

3504

.16

3573

.4936

53.4

5

3762

.63

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

%T

500 1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1)

I R SPECTRUM 4: CISPLATIN + BSA + MAGNETITE

CISPLATIN + BSA + Fe2O3

407

30

805.

91

901.

471023

.78

1096

.2711

66.4

812

11.3

3

1304

.10

1366

.06

1543

.60

1662

.97

2617

.51

2757

.85

2907

.87

3293

.26

3484

.39

92

94

96

98

100

102

104

106

108

110

112

114

116

118

120

122

124

%T

500 1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1)

I R SPECTRUM 2: BOVINE SERUM ALBUMIN (BSA)

BOVINE SERUM ALBUMINE

489.

6956

7.16

686.

72

740.

7183

3.2593

8.17

986.

0210

31.9

411

01.2

511

70.0

412

15.7

312

85.3

1

1369

.31

1448

.16

1511

.09

1553

.79

1607

.15

1662

.59

2628

.68

2760

.87

2907

.36

2960

.36

3068

.29

3225

.37

3299

.91

3392

.53

3536

.29

3898

.07

52

54

56

58

60

62

64

66

68

70

72

74

76

78

%T

500 1000 1500 2000 2500 3000 3500 Wavenumbers (cm-1)

TABLE 2: ABSORBANCE VALUES OF CISPLATIN STANDARD SOLUTIONS AT 210 nm.

Sr. No. Concentration (mcg/ml)

Absorbance

1 10 0.168

2 20 0.325

3 30 0.487

4 40 0.638

5 50 0.798

6 60 0.955

7 70 1.135

TABLE 3

PERCENTAGE PRACTICAL YIELD OF BOVINE SERUM ALBUMIN MAGNETIC MICROSPHERES OF CISPLATIN

Formulations Total amount of ingredients (mg)

Practical yield (mg) Percentage yield

F1 29.5 15.09 51.13%

F2 36.0 19.19 53.29%

F3 42.5 24.13 56.78%

F4 49.0 30.01 61.25%

TABLE 4: DRUG ENTRAPMENT EFFICIENCY OF MAGNETIC MICROSPHERES

Formulations Absorbance Concentration (μgm/ml)

Drug Content (mg/10mg)

% Drug Entrapment Efficiency

F-1 0.208 13.5 1.35 39.82%

F-2 0.206 13.0 1.30 46.76%

F-3 0.203 12.5 1.25 53.19%

F-4 0.181 11.5 1.15 56.37%

TABLE 5: PERCENT MAGNETITE CONTENT

Formulations Vol. of 0.1 N Na2S2O4

consumed (ml)

Amt. Of magnetite entrapped (mg/10mg)

% Magnetite entrapped

F-1 0.41 2.28 42.53%

F-2 0.50 2.79 47.81%

F-3 0.60 3.36 54.34%

F-4 0.64 3.57 55.48%

TABLE 6: IN VITRO MAGNETIC RESPONSIVENESS OF MAGNETIC MICROSPHERES

WITH MAGNETIC FIELD WITHOUT MAGNETIC FIELD

Formulations Conc. Of

drug

(μg/ml)

Amt. of drug in

microspheres retained by

magnets (mg)

% Drug retained

Conc. Of

drug

(μg/ml)

Amt. of drug in

microspheres retained by

magnets (mg)

% Drug retained

F-1 17.10 10.27 51.36 2.80 1.69 8.44

F-2 18.80 11.29 56.45 3.75 2.25 11.38

F-3 21.60 12.96 64.82 5.403 3.25 16.26

F-4 24.25 14.50 72.68 8.05 4.84 24.18

Magnetic microspheres equivalent to 20 mg/ml of Cisplatin were taken.

TABLE 7: IN VITRO RELEASE PROFILE FOR PURE CISPLATIN

Time (Mins) Absorbance Concentration

(mcg/ml)

Drug Release (mg/50

ml)

CLA (mg)

Cumulative Drug

Release (mg/50ml)

% Cumulative

Drug Release

30 0.375 22.0 1.10 - 1.100 36.60

60 0.412 25.5 1.26 0.110 1.370 45.60

90 0.495 30.5 1.52 0.236 1.761 58.70

120 0.562 35.0 1.75 0.388 2.138 71.26

150 0.625 39.0 1.95 0.563 2.513 83.76

180 0.657 41.0 2.05 0.758 2.808 93.60

TABLE 8: IN VITRO RELEASE PROFILE OF CISPLATIN FROM MAGNETIC MICROSPHERES FORMULATION-1

Time (T)

in hrs Root T Log T

Cum. Drug Released

(mg)

Cum % Drug

Released

Cum % Drug

Retained

Log Cum. %Drug

Released

Log Cum % Drug

Retained (% Retained)1/3

0.25 0.5 -0.602 2.021 20.21 79.79 1.305 1.901 4. 305

0.50 0.707 -0.301 2.464 24.64 75.36 1.391 1.877 4.223

1 1 0 3.803 38.03 61.97 1.580 1.792 3.957

2 1.414 0.301 4.983 49.83 50.17 1.697 1.700 3.688

4 2 0.602 5.934 59.34 40.66 1.773 1.609 3.438

8 2.828 0.903 6.526 65.26 34.74 1.814 1.540 3.262

12 3.464 1.079 7.215 72.15 27.85 1.858 1.444 3.031

16 4 1.240 7.696 76.96 23.04 1.886 1.362 2.845

24 4.898 1.380 8.960 89.60 10.4 1.952 1.017 2.182

Magnetic microspheres equivalent to 10 mg of Cisplatin were taken.

TABLE 9: IN VITRO RELEASE PROFILE OF CISPLATIN FROM MAGNETIC MICROSPHERES

FORMULATION-2

Time (T)

in hrs Root T Log T

Cum. Drug Released

(mg)

Cum % Drug

Released

Cum % Drug

Retained

Log Cum. %Drug

Released

Log Cum % Drug


0.25 0.5 -0.602 2.277 22.77 77.23 1.357 1.887 4.258

0.50 0.707 -0.301 2.861 28.61 71.39 1.456 1.853 4.148

1 1 0 3.603 36.03 63.97 1.556 1.805 3.999

2 1.414 0.301 4.168 41.68 58.32 1.619 1.765 3.878

4 2 0.602 4.577 45.77 54.23 1.660 1.734 3.785

8 2.828 0.903 5.127 51.27 48.73 1.709 1.687 3.652

12 3.464 1.079 6.061 60.61 39.39 1.782 1.594 3.402

16 4 1.240 7.132 71.32 28.68 1.853 1.457 3.061

24 4.898 1.380 8.222 82.22 17.78 1.915 1.249 2.160


TABLE 10: IN VITRO RELEASE PROFILE OF CISPLATIN FROM MAGNETIC MICROSPHERES FORMULATION-3

Time (T)

in hrs Root T Log T

Cum. Drug Released

(mg)

Cum % Drug

Released

Cum % Drug

Retained

Log Cum. %Drug

Released

Log Cum % Drug


0.25 0.5 -0.602 2.365 23.65 76.35 1.373 1.882 4.242

0.50 0.707 -0.301 2.967 29.67 70.33 1.472 1.847 4.127

1 1 0 3.581 35.81 64.19 1.554 1.807 4.004

2 1.414 0.301 3.873 38.73 61.27 1.588 1.787 3.942

4 2 0.602 4.275 42.75 57.27 1.630 1.757 3.854

8 2.828 0.903 4.885 48.85 51.15 1.688 1.708 3.712

12 3.464 1.079 5.473 54.73 45.27 1.738 1.655 3.563

16 4 1.240 6.729 67.29 32.71 1.828 1.514 3.198

24 4.898 1.380 7.841 78.41 21.89 1.894 1.334 2.784


TABLE 11: IN VITRO RELEASE PROFILE OF CISPLATIN FROM MAGNETIC MICROSPHERES

FORMULATION-4

Time (T)

in hrs Root T Log T

Cum. Drug Released

(mg)

Cum % Drug

Released

Cum % Drug

Retained

Log Cum. %Drug

Released

Log Cum % Drug


0.25 0.5 -0.602 2.817 28.17 71.83 1.449 1.856 4.156

0.50 0.707 -0.301 3.004 30.04 69.96 1.477 1.844 4.120

1 1 0 3.381 33.81 66.19 1.529 1.820 4.045

2 1.414 0.301 3.530 35.30 64.70 1.547 1.810 4.014

4 2 0.602 3.908 39.08 60.92 1.592 1.784 3.934

8 2.828 0.903 4.340 43.40 65.6 1.637 1.752 3.839

12 3.464 1.079 5.154 51.54 48.46 1.712 1.685 3.645

16 4 1.240 6.644 66.44 33.56 1.822 1.525 3.225

24 4.898 1.380 7.635 76.35 23.65 1.882 1.373 2.870


TABLE 12: KINETIC VALUES OBTAINED FROM IN VITRO RELEASE DATA OF DIFFERENT MAGNETIC

MICROSPHERE FORMULATIONS OF CISPLATIN

Plot of Log Cum. % Drug Retained V/s. Time (T)

(First Order Plot)

Plot Of Cum. % Drug Released V/s. Time (T)

(Zero order Plot) Formulations

Slope (n) First Order Rate Constant K = Slope x 2.303

Regression Co-efficient

(r)

Slope (n) Zero Order Rate Constant K= -Slope

Regression Co-efficient

(r)

F-1 -0.03324 -0.07655 -0.9783 2.59 -2.59 0.9022

F-2 -0.0246 -0.0566 -0.9901 2.281 -2.281 0.9670

F-3 -0.02093 -0.04820 -0.9875 2.087 -2.087 0.9764

F-4 -0.01949 -0.04489 -0.9834 1.998 -1.998 0.9894

TABLE 13: KINETIC VALUES OBTAINED FROM IN VITRO RELEASE DATA OF DIFFERENT MAGNETIC

MCROSPHERE FORMULATIONS OF CISPLATIN

Plot of Cum. % Drug Released V/s. Root Time (√T)

[Higuchi Matrix]

Plot Of Log Cum. % Drug Released V/s. Log Time (Log T)

[Peppas]

Plot Of [% Retained] 1/3 Vs. Time

[Hixson Crowell] Formulations

Slope (n) Regression Co-efficient (r)



F-1 14.73 0.9677 0.3163 0.9822 -0.0808 -0.9627

F-2 12.36 0.9885 0.2569 0.9884 -0.0638 -0.9877

F-3 11.16 0.9851 0.2327 0.9797 -0.0558 -0.9877

F-4 10.39 0.9699 0.2019 0.9381 -0.0525 -0.9874

TABLE 14

IN VIVO TARGETING STUDIES OF MAGNETIC MICROSPHERES OF CISPLATIN

Group I (With magnet) Group II Control (Without magnet)

After 1 hr. After 3 hr.

After 1 hr. After 3 hr.

Organs

Drug content

(μg)

% Drug content

Drug content

(μg)

% Drug content

Drug content

(μg)

% Drug content

Drug content

(μg)

% Drug content

Tail section-2 345 57.61 255 41.97 22 3.62 14 2.31

Lungs 11 1.81 21 3.46 50 8.23 90 14.81

Liver 65 10.69 95 15.63 195 32.1 28 46.1

Spleen 17 2.79 24 3.95 56 9.22 11 18.11

Kidney 20 3.29 34 5.59 32 5.27 57 9.38

Suspension of magnetic microspheres equivalent to 607.5 mcg Cisplatin was injected.

TABLE 15

STABILITY STUDIES FOR PERCENT DRUG CONTENT [AFTER STORAGE AT 4ºC,

AMBIENT TEMPERATURE AND HUMIDITY & AT 30ºC /65% RH]

Percent drug content at 4ºC Percent drug content at Ambient Temperature and Humidity

Percent drug content at 30ºC /65% RH

Formulations After

15 Days After

30 Days After

60 Days After

15 Days After

30 Days After

60 Days After

15 Days After

30 Days After

60 Days

F-1 39.74 39.62 38.88 39.36 39.10 38.54 39.30 38.95 38.30

F-2 46.64 46.38 45.64 46.29 45.89 45.12 46.25 45.76 44.62

F-3 53.02 52.88 52.26 52.96 52.32 51.96 52.88 52.15 51.58

F-4 65.18 56.04 55.38 56.14 55.94 54.78 56.08 55.70 54.42

TABLE 16: STABILITY STUDIES- IN VITRO RELEASE STUDY OF A SELECTED FORMULATION (F-3) AFTER

ONE MONTH STORAGE AT 4°C, AMBIENT TEMPERATURE AND HUMIDITY AND 30ºC /65% RH

4°C Ambient Temperature and Humidity At 30ºC /65% RH

Time (In hrs) Cum. Drug

Release (mg) % Cum.

Drug Release Cum. Drug

Release (mg) % Cum. Drug

Release Cum. Drug

Release (mg) % Cum.

Drug Release

0.25 2.282 22.82 2.242 22.42 2.168 21.68

0.50 2.886 28.86 2.814 28.14 2.826 28.26

1 3.552 35.52 3.564 35.64 3.588 35.88

2 3.964 39.64 3.988 39.88 4.036 40.36

4 4.388 43.88 4.426 44.26 4.494 44.94

8 5.066 50.66 5.212 52.12 5.308 53.08

12 5.628 56.28 5.796 57.96 5.948 59.48

24 8.056 80.56 8.274 82.74 8.448 84.48

FIGURE 17: PLOTS OF CUMULATIVE % DRUG RELEASED Vs. TIME OF F-3 FORMULATION AFTER 60 DAYS STORAGE

0

10

20

30

40

50

60

70

80

90

0 5 10 15 20 25 30

TIME (in hrs)

CU

M. %

DR

UG

REL

EASE

D

4°C R T 30ºC /65% RH

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