preparation and evaluation of liposphere based topical drug delivery system containing nsaid drug

“PREPARATION AND EVALUATION OF LIPOSPHERE

BASED TOPICAL DRUG DELIVERY SYSTEM CONTAINING

A NSAID DRUG”

THESIS

Submitted in partial fulfillment for the degree of

MASTER OF PHARMACY

In

Industrial Pharmacy

In The Faculty of Medicine

Sant Gadge Baba Amravati University, Amravati.

RESEARCH SCHOLAR

Mr. LEELADHAR PRAJAPATI

B. PHARM

RESEARCH GUIDE

Dr. P. S. KAWTIKWAR

(M. PHARM, Ph.D.)

2012-2013

DEPARTMENT OF PHARMACEUTICS

SUDHAKARRAO NAIK INSTITUTE OF PHARMACY, PUSAD-445204

SANT GADGE BABA AMRAVATI UNIVERSITY, AMRAWATI

MAHARASHTRA. (INDIA)

Affectionately Dedicated To, God,

Who is always with me.

My family,

Whose affection and love are infinite with me in adversity and prosperity.

The Guide,

To whom, I shall remain indebted for giving new shape and path to my life.

(Research Guide) Dr. P.S. Kawtikwar M.Pharm. Ph.D.

Professor & H.O.D.

Department of Pharmaceutics,

S. N. Institute of Pharmacy,

Pusad- 445 204.MS (India)

CERTIFICATE

This is to certify that the investigations described in this thesis

entitled “Preparation and evaluation of liposphere based topical drug

delivery system containing a NSAID drug” was carried out by Mr.

Leeladhar Prajapati in the laboratories of Department of pharmaceutics,

S.N. Institute of pharmacy, Pusad under my supervision and guidance.

The thesis work was carried out and submitted in partial fulfillment

of the requirements for the Degree of Master of Pharmacy in

Industrial Pharmacy, in the faculty of Medicine of Sant Gadge Baba

Amravati University, Amravati.

This thesis is now ready for examination and evaluation.

Date: Dr. P. S. Kawtikwar

Place: [Research Guide]

Dr. D. M. Sakarkar M.pharm. Ph.D. Principal, S.N. Institute of Pharmacy, Pusad -445204, MS (India)

CERTIFICATE

This is to certify that the investigations described in this thesis


delivery system containing a NSAID drug” was carried out by Mr.

Leeladhar Prajapati in the laboratories of Department of pharmaceutics,

S.N. Institute of pharmacy, Pusad under my supervision and guidance.

The thesis work was carried out and submitted in partial fulfillment of the

requirements for the Degree of Master of Pharmacy in Industrial

Pharmacy, in the faculty of Medicine of Sant Gadge Baba Amravati

University, Amravati.

The thesis is now ready for examination and evaluation.

Date: Dr. D. M. Sakarkar

Place: Principal

Mr. Leeladhar Prajapati

B. Pharm.

Department of Pharmaceutics

S. N. Institute of Pharmacy

Pusad-445204, MS (India)

DECLARATION

It gives me great pleasure and satisfaction to declare that the thesis


delivery system containing a NSAID drug” was reviewed and submitted

in partial fulfillment of the requirements for the Degree of Master of

Pharmacy in Industrial Pharmacy, in the Faculty of Medicine of Sant

Gadge baba Amravati University, Amravati under guidance and supervision

of Dr. P.S. Kawtikwar and I hereby declare that

This thesis is now ready for examination and evaluation.

Date: Mr. Leeladhar Prajapati

Place:

INSTITUTIONAL ANIMAL ETHICS COMMITTEE

(Reg. No. CPCSEA/729/02/a/ CPCSEA)

Prof. S.V. Tembhurne

Member Secretary (IAEC)

Ref. No. SNIOP/IAEC/2012-13/CPCSEA/IAEC/IP-PL/12-2012

Mr. Leeladhar Prajapati

(M.pharm Student)

Department of Pharmaceutics

Sudhakarrao Naik Institute of Pharmacy, pusad

Dist-Yavatmal-445204

This is to certify that the proposal of Mr.Leeladhar Prajapati

for the Study entitled “Preparation and evaluation of liposphere based

topical drug delivery system containing a NSAID drug” was approved by

Institutional Animal Ethics Committee (IAEC) of S.N. Institute of Pharmacy

Pusad in the IAEC meeting held on dated 07/01/2013 and the proposal

number CPCSEA/IAEC/IP-PL/12-2012

Hence the certificate is issued.

Place: Pusad [Prof. S.V. Tembhurne]

SSUUDDHHAAKKAARRRRAAOO NNAAIIKK IINNSSTTIITTUUTTEE OOFF PPHHAARRMMAACCYY

Nagpur road, Pusad Dist: Yavatmal (M.S) 445204

Phone (07233)-247308; Fax (07233)-247308

Web site: www.sniop.ac.in

http://www.sniop.ac.in/

LIST OF TABLES

Table

No.

Title Page

No.

1. Composition and active ingredients for formulations of lipospheres 19

2. Factors influencing morphology of Lipospheres 25

3. Factors influencing entrapment efficiency 26

4. Factors influencing drug release 27

5. List of instrument used 45

6. List of chemical and reagent used 46

7. Formulations codes of lipospheres different batchs (F1-F12) 51

8. Topical gel formulation of optimized batch 55

9. Physical characters of Aceclofenac drug 60

10. Interpretation of Aceclofenac IR 62

11. UV calibration curve reading of Aceclofenac in phosphate buffer

saline pH 7.4 63

12. Evaluations of lipospehers 64

13. Mean particle size (mps) of different formulation of liposphers 66

14. Cumulative percentage drug release from various formulations of

lipospheres (F1-F4) 72





17. Drug release kinetics for the various formulations of lipospheres 75

18. Result of pH, Viscosity, Drug content and Spreadability 77

19. Cumulative percent (%) drug release of optimize batch (F8, LF8) 79

20. Percentage inhibition for anti-inflammatory activity 81

21. Stability studies for the formulation LF7 and LF8 82

LIST OF FIGURES

Fig.

No.

Title Page

No.

1 Interactions of phospholipids in aqueous media. 6

2 Schematic liposphere 7

3 A cross sectional view of human skin 8

4 Epidermis with Stratum corneum including a corneocytes cluster 9

5 Skin appendages 13

6 Plasma drug concentration profile for controlled release 17

7 Schematic representation of the methods of production of LS: melt

dispersion and solvent evaporation 21

8 UV spectrum of aceclofenac 61

9 FTIR Spectra of aceclofenac drug 61

10 Calibration Curve of Aceclofenac 63

11. Entrapment efficiency (F1-F12) 64

12 Photograph of optimized liposphers batch F7 65

13 Photograph of optimized liposphers batch F8 65

14 FT-IR spectra of Aceclofenac 67

15 FT-IR Spectra of physical mixture 68

16 DSC Thermogram of Aceclofenac 69

17 DSC Thermogram of optimized formulation F8 and F6 70

18 SEM images of drug loaded lipospheres 70,71

19 In vitro drug release of batch F1,F2,F3,F4 72



22 First order drug release plot of optimized batch F7 76

23 First order drug release plot of optimized batch F8 76

24 Higuchi’s drug release plot of optimized batch F7 76

25 Higuchi’s drug release plot of optimized batch F8 76

26 Peppas korsmeyer’s drug release plot of optimized batch F7 76

27 Peppas korsmeyer’s drug release plot of optimized batch F8 76

28 Lipospheres based gel of optimized batch 78

29 In vitro release profile of optimized formulation F8 and LF8 80

30 Skin-irritation testing (Draize patch test) 80

LIST OF ABBREVATIONS

Base unit Symbol

Hour h

Second s

Minute min

Milliliter ml

Micrometer μm

Nanometer nm

Millimeter mm

Milligram mg

Gram g

Cubic meter m3

Revolutions per minute rpm

Centimeter cm

Potassium Bromide KBr

Centipoise cps

BP British Pharmacopoeia

LS Liposphere

ACKNOWLEDGEMENT

First and foremost I would like to express my deepest pray to ALL

MIGHTY, love and thanks to my father Mr. Teerath Prasad Prajapati, my mother

Mrs. Shivkali Prajapati, for their extreme patience and support. I know this took a

long time but your sacrifice and understanding has allowed me to persevere.

Let me start by expressing my sincere and special thanks to my esteem guide

Respected Dr. P. S. Kawtikwar Sir, H.O.D. Dept. of Pharmaceutics, Sudhakarrao

Naik Institute of pharmacy, Pusad. I am thankful to him for giving me freedom to

work, timely advice and valuable suggestions. Under his constant guidance,

encouragement, and positive attitude towards work has instilled more confidence in

me. “Thank you Sir” for all you has done.

I owe a great debt of gratitude to Dr. D. M. Sakarkar Sir, Principal,

Sudhakarrao Naik Institute of pharmacy, Pusad for providing the necessary

facilities. This thesis would not have become a reality without his constant quest for

knowledge and critical evaluation.

I’m grateful to Prof. R. B. Wakade Sir, Prof. A.H.harsulkar sir, Prof.

A.M.Mahale sir, & Prof. J.K. Jadhav, Prof. A.R. Tapas sir and prof. S. V.

Tembhurne sir for their proper orientation to my work to make it possible.

I express my deepest thanks to VAV Life Science Ms. Stuti Singh mam and

Mr. Swanand Malode sir for valuable suggestions and motivation.

I express my deepest and special thanks to my elder Brothers Anand and

Yogesh & my sister for their keen interest, love and motivation in my life.

I am thankful to all teaching and non-teaching staff of our college for valuable

suggestions and motivation, which they bestowed on me right from the inception to

the successful completion of the work.

I’m really very much grateful to Mr. M.D.Wandhare, for his tips, his

guidance and his cooperation during my dissertation work.

I express my deepest and special thanks to my colleagues,Prashant, Girish, Ketan,

Mayur, Lukesh, Anant, Pankaj, Ghanashyam, Dhanraj, Amol, Rahul, Sushil,

Nikita, Radhe, Anish, Akanksha, Nilakshi, for their keen interest, love and support

in my dissertation work.

There are many others whose names flashed across my mind when I enlist

those who have given grateful support to me. It would rather impracticable to

mention each of them separately but I am conscious my obligation and thanks them

collectively.

Leeladhar Prajapati

Table of Contents

SR. S.NO. TITLE PAGE NO.

1.

INTRODUCTION

1-28

2.

LITERATURE REVIEW

29-35

3.

AIM AND OBJECTIVES

36

4.

PLAN OF WORK

37

5.

DRUG PROFILE

38-44

6.

LIST OF MATERIAL AND EQUIPMENT

45-47

7.

EXPERIMENTAL WORK

48-59

8.

RESULTS AND DISCUSSION

60-82

9.

SUMMARY AND CONCLUSION

83-85

10.

FUTURE SCOPE

86

11.

REFERENCES

87-97

12.

ERRATA

98

Abstract

SNIOP, PUSAD 2012-2013

Abstract

The purpose of this study was to prepare lipospheres containing aceclofenac

intended for topical delivery with the aim of exploiting the favorable properties

of this carrier system and developing a sustained release formula to overcome

the side effects resulting from aceclofenac oral administration. Lipospheres

were prepared using different lipid cores (carnauba wax, bees wax, steryl

alcohol) and phospholipids coats (egg phosphatidylchoine and soya

phosphatidylcholine) by melt dispersion technique. Characterization of the

prepared lipospheres formulation carried out through photomicroscopy,

scanning electron microscopy (SEM), particle size analysis, diffential scanning

caorimetry (DSC), and In vitro drug release and stability study. It was

uniformly dispersed after suitably gelled by Carbopol 940 preparation. The

characterization of the prepared lipospheres was based on topical gel

rheological study, pH, spreadability, drug content, skin irritation test. No

oedema and erythema were observed after administration of lipospheres based

aceclofenac gel on rabbit skin. The anti-inflammatory effect of liposphere

systems was assessed by the rat paw edema technique and was compared to the

marketed product. Results revealed that liposphere systems were able to entrap

aceclofenac at very high levels (101.2%). The particle size of liposphere

systems was well suited for topical drug delivery. DSC revealed the molecular

dispersion of aceclofenac when incorporated in lipospheres. Lipospheres were

substantially stable after 3 months storage at 2–8 °C. Liposphere topical gel was

found to possess superior anti-inflammatory activity compared to the marketed

product.

Key words: Aceclofenac, entrapment efficiency, formulation of topical gel,

animal experiment, stability, sustained release.

Chapter No.1 Introduction

SNIOP, PUSAD 2012-2013 Page 1

1. Introduction

One approach for increasing the beneficial action of drugs and

decreasing systemic adverse effects is to deliver the necessary amount of drugs

to the diseased sites, where they are most needed, for the appropriate period of

time1-3

. Although the drug delivery system concept is not new, great progress

has recently been made in the treatment of a variety of diseases. Particulate

carriers (e.g., polymeric nano- and Microparticles, fat emulsion, and liposomes)

possess specific advantages and disadvantages. For instance, in the case of

polymeric Microparticles, the degradation of the polymer might possibly cause

systemic toxic effects through the impairment of the reticuloendothelial system4

or by accumulation at the injection Site5, cytotoxic effects have indeed been

observed in vitro after phagocytosis of particles by human macrophages and

granulocytes6. In addition, organic solvent residues deriving from the

preparation procedures, such as the solvent evaporation technique often used

for liposome7 and polyester microparticles

8 can be present in the delivery

system and could result in severe acceptability and toxicity problems9. To solve

these adverse effects, lipid microspheres, often called lipospheres (LS), have

been proposed as a new type of fat-based encapsulation system for drug

delivery of bioactive compounds (especially lipophilic compounds). LS consist

of solid microparticles with a mean diameter usually between 0.2 and 500 μm,

composed of a solid hydrophobic fat matrix in which the bioactive compounds

are dissolved or dispersed10-12

. LS have some advantages over other delivery

systems, such as good physical stability, low cost of ingredients, ease of

preparation and scale-up, and high entrapment yields for hydrophobic drugs.

Because of their large range in particle size, LS can be administered by

different routes- such as orally, subcutaneously, intramuscularly, or topically-or

they can be used in cell encapsulation, thus allowing them to be proposed for

treatment of a number of diseases13 15

. For instance, the in vivo distribution of

LS demonstrated a high affinity to vascular wells (including capillaries),



inflamed tissues, and granulocytes16-17

. LS have been used for the controlled

delivery of various types of drugs, including vasodilator and antiplatelet drugs,

anti-inflammatory compounds, local anesthetics, antibiotics, and anticancer

agents; they have also been used successfully as carriers of vaccines and

adjuvants18

. For a biocompatible formulation suitable for human administration,

triglycerides and monoglycerides have been chosen as the biomaterials for LS

because of their high biocompatibility, high physicochemical stability, and drug

delivery release. LS were prepared by two alternative approaches, namely, the

melt dispersion and the solvent evaporation techniques. Liposphere formulation

is an aqueous micro dispersion of solid water insoluble spherical micro the

lipospheres are made of solid hydrophobic triglycerides with a monolayer of

phospholipids embedded on the surface of the particle. Liposphere formulation

is appropriate for oral, parenteral and topical drug delivery system. The solid

core containing a drug dissolved or dispersed in a solid fat matrix and used as

carrier for hydrophobic drugs. Several techniques, such as solvent

emulsification evaporation, hot and cold homogenization and high pressure

homogenization have been used for the production of liposphere. Benefits of

liposphere drug delivery system are;

a) Improving drug stability.

b) Possibility for controlled drug release.

c) Controlled particle size.

d) High drug loading.

In addition, use of lipospheres for oral administration, it can protect the drug

from hydrolysis, as well as improve drug bioavailability. Therefore, the present

review articles focused on achievements of lipospheres formulation to deliver

the drugs in the targeted sites.

Polymers as carriers19

for “difficult to deliver” drugs, “to be targeted

drugs” and to achieve a desired release pattern is a popularly known and widely

exploited concept in formulation Technology. With the emergence of polymer



era, delivery systems like microspheres20

nanoparticles21

found their way into

pharmaceutical market. Natural, semi synthetic and synthetic polymers are now

being widely explored in the formulation of multiparticulate systems apart from

several other applications. Multi particulate systems comprise of several small

discrete units containing drug substance entrapped or encapsulated in polymer

and offer several advantages over single unit dosage forms. They show

predictable gastric emptying resulting in less inter and intra-subject variability,

gastric emptying independent of the state of nutrition, high degree of dispersion

in the digestive tract, lower risk of dose dumping and reduced local irritation,

increased bioavailability, reduced risk of systemic toxicity. Despite these

advantages, use of polymers in drug delivery poses several safety concerns.

Polymers used in the preparation of micro/nanospheres can produce toxic

degradation products causing systemic toxic effects through the impairment of

reticuloendothelial system (RES).

Polymers precipitate toxic effects due to accumulation of products at injection

site. In vitro studies have shown cytotoxic effects after phagocytosis of polymer

particles by human macrophages and granulocytes. Eg: Pre degraded poly (L-

lactic acid) (P-PLLA) and non treated PLLA (N-PLLA) particles, both having

diameters not exceeding 38 μm, were injected intraperitoneally in mice.

Nondegradable polytetrafluoroethylene (PTFE) particles and the carrier

solution were used as control. Cells of the abdominal cavity were harvested after

1, 2, 3, 4, 5, and 7 days to study the effect on the morphology of cells and

viability due to degradation products. TEM (Transmission Electron Microscopy)

studies indicated that, upon injection of particles in the peritoneal cavity,

macrophages demonstrated signs of cell damage, cell death, and cell lysis due to

phagocytosis of a large amount of PPLLA particles.

Methods like solvent evaporation used in the preparation of liposomes and

polyester Microparticles 22

leave organic solvent residues in the product which

can result in severe acceptability and toxicity problems.



Due to several limitations with polymeric delivery systems, extensive

attempts are being made to develop alternate carriers. Lipids23

especially, are

now being studied widely due to their attractive properties namely

physicochemical diversity, biocompatibility, biodegradability, ability to increase

the oral bioavailability of poorly water soluble drug moieties, thus making them

ideal candidates as carriers for problematic drugs.

1.1.1 Advantages of lipid based delivery systems:

Lipid based delivery systems disperse, solubilize and maintain solubility of

drug in GI fluids.

Bioavailability of most of the lipophilic drugs is altered in the presence of

lipid content in food.

Lipid carriers mimic such lipid food and thus reduce the food effect on

bioavailability of drugs and render flexibility to dosage regimen.

Transfer drug into bile-salt mixed micelle and promote lymphatic uptake of carrier-

drug particles.

Influence gut wall permeability.

Normalize and/or modify pharmacokinetic parameters. However few concerns

related to using lipids as carriers can be overcome by well characterizing

physicochemical and testing methodologies for lipid drug delivery systems and

are as follows:

Limiting solubility of drug in lipid core which determines entrapment

efficiency.

Quantity of excipient required.

Stability of drug.

Chemical stability issues like drug and carrier compatibility.

Physical stability of lipid dosage forms like polymorphic phase transitions of

drug and lipid during preparation and storage and stability of semisolids.



Also research should be focused towards developing reliable in vitro and in vivo

testing methodologies for lipid based delivery systems and understanding the in

vivo fate of drug carried by such delivery systems and influence of co

administered drugs/lipids on the bioavailability of drugs.

Lipid based drug delivery systems like solid lipid nanoparticles (a

technology owned by Skye Pharma) and lipospheres are now being studied

widely. Solid lipid nanoparticles24

are nanosized lipid carriers in which lipidic

core contain the drug in dissolved or dispersed state. These systems were

designed to substitute polymeric carriers due to the inherent toxicity.

Lipospheres are lipid based dispersion systems in which drug is dissolved or

dispersed in lipidic core, the surface of which is embedded with emulsifier layer.

1.1.2 Advantages of lipospheres carriers:

1. Easily available, low cost, GRAS (Generally Recognized As Safe) listed raw

materials.

2. Feasible simple production techniques that do not employ high energy

process like homogenization which will otherwise compromise the stability of

labile active pharmaceutical ingredient.

3. High entrapment for lipophilic drugs.

4. Extended release of entrapped drug after a single injection from few hours to

several days

5. Good physical stability

6. Administration by several routes.

1.2. Colloidal drug delivery systems

Many of the drug substances are characterized by poor aqueous

solubility, which causes many formulation problems. Besides the use of co-

solvents, drug24

complexation and solubilization in surfactant micelles,

incorporation in colloidal carrier systems represents an alternative way to



render poorly water soluble drugs applicable for effective therapy. Furthermore

incorporation of drugs in particulate carriers provides a possibility to

manipulate the drug release. In last few years the colloidal carriers have been

used for site specific targeting especially in cancer chemotherapy. Based on the

carrier material the conventional vehicles used as drug carriers can be divided

into 2 groups

1. Polymeric carriers.

2. Lipidic carriers.

a. Liposomes.

b. Lipoproteins.

c. Lipid O/W emulsions.

d. Lipospheres.

The lipidic carriers are more preferred than polymeric carriers to avoid

potential toxicological problems. The vehicles of all the above lipidic carriers

are composed of physiological lipids such as phospholipids, cholesterol,

cholesterol esters and triglycerides.

Phospholipid molecule



Micelle Lipid Bilayer Lipospheres

Figure 1: Interactions of Phospholipids in Aqueous Media.

Lipospheres

Figure 2: Structure of Liposphere Figure 3: Schematic Liposphere

1.3. The structure of skin

Skin is anatomically divided into three principal and distinct layers, from

the outside of skin inward, including stratum corneum (10–20 μm thick), viable



epidermis (50–100 μm thick), and dermis (1000-2000 μm thick). A fatty

subcutaneous layer resides beneath the dermis. It should be pointed out that all

the thickness specified here are representative only, since the actual thickness of

each layer varies several fold from place to place on the body. Adnexal

appendages, including hair follicles, associated sebaceous glands and pili

muscles, apocrine and eccrine sweat glands, can be found dispersing throughout

of the skin, varying in number and size depending on body site. The cross

section of skin structure is shown is (Figure 4)

Fig 4: A cross sectional view of human skin, Source: From Ref. (Lu and Flynn, 2009)

1.3.1 Stratum corneum (SC)

Stratum corneum is the outmost superficial layer of the skin and also the

principal barrier element of the skin. SC consists of several layers of completely

keratinized flattened dead cells, corneocytes, each of which is about 30 μm in

diameter with a hexagonal shape and 0.5-0.8 μm in thickness. These acutely

flattened corneocytes are highly organized and stacked vertically 15 to 25 cell

layers, which are embedded into a specialized and well structured intercellular

lipid matrix.



The most simplistic organizational description of SC is advocated by which is

the classic “brick-and-mortar” assembly (Figure 5). The intracellular space of

corneocytes is literally packed with structural protein, semi crystal line α

keratin intermixed with more amorphous β keratin. The intracellular space is

dense, offering little freedom of movement to drug molecules. Thus, the

corneocytes work as “brick” being thermodynamically impenetrable. While the

intercellular space of corneocytes is filled with a lipid “mortar” formed of

cholesterol, free fatty acids, and ceramides, which seals horny structure.

Fig.5: Brick-and-mortar model of Stratum corneum and penetration routes

through it, Source from Ref. (Elias, 1981)

However, the brick-and-mortar skin model is not enough to describe the

panorama of the SC. In fact, the cells from basal layer of epidermis, which we

describe further in the text, to the SC are built up in clusters, which represent

the basic skin permeation resistance unit.It is these clusters that are separated by

surface corrugations (wrinkle line), which often reach several micrometers into

the basal layer of the epidermis (Figure 6).

Fig.6: Epidermis with Stratum corneum including a corneocytes cluster,

Source from Ref. (Cevc and Vierl, 2010)



In addition, the basolateral side of stratum corneum is in direct contact with the

living epidermal mass, where the corneocytes contain water at high

thermodynamic activity of the physiological milieu. On the other hand, its

external surface interfaces the environment, where air tends to have a far lower

water activity. Consequently a water gradient is established and water diffuses

out through the stratum corneum. Under such a normal hydration situation, the

stratum corneum takes up moisture to the extent of 15% to 20% of its dry

weight. It should be pointed out here that the hydration condition of SC plays

an important role on the drug molecules skin penetration, which would be

discussed further in the text.

1.3.2 Viable epidermis

The viable epidermis is underlying the stratum corneum. It is

multilayered when viewed under microscope, including, from bottom to top,

basal layer (stratum germinativum), spinous layer (stratum spinosum), granular

layer (stratum granulosum) and lucid layer (stratum lucidum). Each layer is

defined by position, shape, and morphology and also reflects the progressive

differentiation of keratinocytes which eventuates into their death and placement

as chemically and physically resistant “brick” in stratum corneum. However,

when physicochemically considered, the viable epidermis is just a group of

tightly massed live cells, which results in a singular diffusion area or resistance

in percutaneous absorption process. Water found in this live epidermis has an

activity equivalent to that of 0.9 % NaCl.

While the interface between stratum corneum and epidermis is flat, the one

between epidermis and dermis is papillose, which increases their contact

surface area and then allows for the diffusion of nutrients or other biological or

medicated molecules between dermis and epidermis.



The epidermis itself is avascular. Besides keratinocytes, Langerhans cells also

can be found in viable epidermis. They are antigen presenting cells in the skin’s

immunological responses. Moreover, another kind of cells, melanocytes, are

strategically placed in the epidermis just above the epidermis and dermis

junction. When influenced by melanocyte-stimulating hormone or ultraviolet

radiation, melanocytes synthesize and deposit the pigment granules into skin,

which gives rise to the skin coloration.

1.3.3 Dermis

Dermis is directly adjacent to the epidermis and extends from the

epidermal-dermal junction to the subcutaneous tissue (Figure 4). Dermis

consists of a net-work of irregular connective tissue, which provides the

mechanical support for the skin.

The matrix of this connective tissue consists of structure fibers, such as

collagen, reticulum, and elastin. These fibers are embedded in an amorphous

mucopolysaccharidic gel called the ground substance.

The dermis can be arbitrarily divided in into a superficial papillary layer

and a deep reticular layer. The upper papillary layer is thin, one fifth of

thickness of the dermis, and protrudes in to the epidermis giving rise to the

dermal papilla, and also provides the support of the delicate capillary plexus

which nurtures the epidermis.

The deepest layer of the skin is a far coarser fibrous matrix, the reticular

dermis, which is the main structural element of the skin. Equally importantly,

the microcirculation which subserves the skin is entirely housed in the dermis.

Blood flow through skin can vary by a factor of 100 fold depending on

environmental conditions. The dermis is also penetrated by sensory nerve

endings and an extensive lymphatic network. Moreover, skin appendages such

as sweat gland, sebaceous glands, hair follicles, and arrector pili muscles are

anchored within the dermis.



The main cell inhabitants of the dermis are fibroblasts, mast cells and

macrophages. Fibroblasts synthesize the structural fibers, while mast cells are

thought to synthesize the ground substance. Macrophages work as immune

response. In addition, plasma cells, chromatophores, fat cells, nerve cells and

endings can also be found along with blood vessels, nerves and lymphatics.

1.3.4 Skin appendages

Skin appendages include hair follicles and their associated sebaceous

glands, eccrine glands, apocrine glands, and arrector pili muscles. The hair

follicle unit is composed of the hair, hair follicle, associated sebaceous gland,

and pili muscles. Hair is a compact of keratinized structures, which consist of

three layers, including an outermost cuticle, a cortex of densely packed

keratinized cells, and a medulla of loose flattened cells. Hairs can be found

mostly everywhere on the body except for the soles of the feet, the palms of the

hand, and mucocutaneous junctions. There are100 follicles per square

centimeter, representing one thousandth of the skin’s surface. A hair emerges

from a follicle, which is set within the dermis at a slight angle. The hair follicle

consists of three major components, including internal root sheath, external root

sheath, and dermal papilla (Figure 7). This arrangement results in a solid

implantation of the hair root in the hair follicle. In addition, each follicle is

anchored to the surrounding connective tissue by an individual strand of

arrector pili muscle.

Furthermore, each follicle is associated with one or more flask like

sebaceous glands, which secret an oily secretion, sebum. Then the sebum is

forced upward around the hair shaft and onto the skin surface. Sebum mainly

consists of squalene, cholesterol esters, wax esters, and triglycerides. It has

several biological functions including the regulation of steroidogenesis and

androgen synthesis, and providing antibacterial and water resistance to skin.



Fig.7 Skin appendages: (a) Structure of the skin (b) Structure of the hair follicle

(c) Cross-section of the hair, source from Ref. (Wosicka and Cal, 2010)

Eccrine glands (sweat glands) are distributed over the entire body except the

genitalia and lips. These simple tubular glands open directly on the skin

surfaces and extend to the footings of the dermis. There are between 150 and

600 glands per square centimeter of body surface depending on body site

.However, the estimated number of actual sweating glands is much less than

that value, since many of these glands remains dormant. Thus, these glandular

openings occupy approximately one ten thousandth of the skin surface. Eccrine

sweat is slightly acidic (pH=5) due to traces of lactic acid, which is moderately

bacteriostatic.

1.3.5 Skin penetration routes

When a skin drug delivery system is applied topically, drug-containing

carriers or free drug in the system could interact with either the stratum

corneum or the sebum filled ducts of the pilosebaceous glands.

Thus, two principle absorption routes are involved, including the

transepidermal route, where the drug delivery system interacts with or diffuses



through stratum corneum, and transfollicular route, where they interact with or

diffuse through the follicles.

In the case of the transepidermal route, since the impermeable character of the

corneocytes, the intercellular space of corneocytes provides the only continuous

phase, which is also the predominant penetration pathway (intercellular route or

intercorneocyte pathway) from the skin surface to the viable epidermis.

However, the tortuous zigzag bestowed by staggered corneocytes arrangement

(typically 18–21), corneocyte layers as well as the highly organized crystalline

lamellae structures of the mortar lead to an outstanding barrier property of the

labyrinthine intercellular route.

The transportation of molecules across this layer is primarily passive diffusion,

in accordance with Fick’s law, and no active transport processes have been

identified to date.Thus the permeability of stratum corneum as a penetration

resistor is proportional to the diffusive mobility of drug molecules within it

(diffusion coefficient, Dsc, also proportional to the capacity of the SC to

solubilize the drug molecules relative to vehicle (partition coefficient, Ksc) but

inversely proportional to the thickness of stratum corneum (hsc). Consequently,

at the steady state and sink condition, drug permeation can be described as

following:

where Jsc (μg cm-2

h-1

) is the steady state flux through stratum corneum. C is

the concentration of drug in the topical drug delivery system. When considering

transfollicular route, initially it was not considered to be a significant skin

penetration route, as evidence suggested that they accounted for only

approximately 0.1% of the skin surface area .Recently, it has been demonstrated

that hair follicles may act as a significant penetration pathway and/or potential



reservoirs for topically applied compound. As mentioned before, owing to the

presence of sebum in follicles, the permeation through follicular route can be

described as following:

Where J sebum (μg cm-2

h-1

) is the steady state flux through sebum/hair follicle.

C is the concentration of drug in the topical drug delivery system. Ksebum and

Dsebum are diffusion coefficient through sebum and drug partition coefficient

in sebum/water, respectively.

In short, either or both routes can be important depending on the

physicochemical properties of a drug as well as the condition of the skin, since

the percutaneous absorption is a spontaneous passive diffusion process which

takes the path of least resistance.

1.4. Sustained drug delivery system

Modified release delivery systems may be divided conveniently into the

following categories 25

Delayed release

Sustained release

Site specific targeting

Receptor targeting

Sustained release, sustained action, prolonged action, controlled release,

extended action, timed release, depot and repository dosage forms are terms

used to identify drug delivery systems that are designed to achieve a prolonged

therapeutic effect by continuously releasing medication over an extended period

of time after an administration of single dose.



The term “sustained release” is used to describe a dosage form

formulated to retard the release of a therapeutic agent such that its appearance

into systemic circulation is delayed and/or prolonged and its plasma profile is

sustained in duration. The onset of pharmacological action is often delayed and

duration of its therapeutic effect is often sustained. Controlled release dosage

forms are designed to release drug in vivo according to predictable rates that

can be verified by in vitro measurements. Controlled release technology implies

a quantitative understanding of the physicochemical mechanism of drug

availability to the extent that the dosage form release rate can be specified.26

Various designations such as smart, targeted, intelligent, novel and therapeutic

have been given to sustained release systems.

The sustained release dosage forms continue to lure both the market and

the researchers by virtue of improved patient compliance and reduced

incidences of adverse drug reactions. The field of sustained release technology

is vastly growing and as a consequence has witnessed a remarkable

sophistication. Many new technologies and devices are continuously being tried

for providing a more reliable control and precision over the release of the

actives.27

1.4.1 Rationale of sustained drug delivery systems:

In general, the goal of sustained release dosage form is to maintain

therapeutic blood or tissue level of the drug for extended period of time. This is

generally accomplished by attempting to obtain “zero order” release from the

dosage form. Zero order release constitutes drug release from the dosage form

which is independent of the amount of drug in the delivery system. Sustained

release system generally do not attain this type of release and usually try to

mimic zero order release by providing drug in slow “first order” fashion (i.e.



concentration dependent). Thus sustained release dosage form consists of two

parts:

An immediately available dose to establish the blood level quickly in an amount

sufficient to produce the desired pharmacological response (i.e. Loading dose).

The remaining amount of total dose (maintenance dose) is then gradually

released to maintain constant blood level of the drug. 28

Figure 8: Plasma drug concentration profiles for conventional tablet or capsule

formulation, a sustained release formulation and a zero order controlled release

formulation.

1.4.2. Factors influencing the design and performance of sustained

release products:

To establish criteria for the design of controlled release products a

number of variables must be considered such as:

T

I

M

E



1.4.2.1. Drug properties:

Physicochemical properties of drug including stability, solubility,

partitioning characteristics, charge and protein drug binding play a dominant

role in the design and performance of controlled release systems.

1.4.2.2. Route of drug delivery:

Physiological constraints imposed by particular route, such as, first pass

metabolism, GI motility, blood supply and sequestration of small foreign

particles by the liver and spleen.

1. Target sites.

2. Acute or chronic therapy.

3. The disease.

4. The patient29

.

1.4.3.3. Advantages of sustained drug delivery systems over conventional

dosage forms

Improved patient compliance and convenience due to less frequent drug

administration.

Reduction in fluctuations in steady state levels and therefore better control

of disease condition and reduced intensity of local or systemic side effects.

Increased safety margin of high potency drugs due to better control of

plasma levels.

Maximum utilization of drug enabling reduction in total amount of dose

administered.



Reduction in health care costs through improved therapy, shorter treatment

period, less frequency of dosing, reduction in personal time to dispense,

administer and monitor patients.

Improved bioavailability of some drugs. 30

1.5. Formulation:

Lipospheres are generally composed of:

1.5.1. Lipid core which is a combination of different lipids (fats, oils):

Table 1: Composition and Active ingredients for formulations of lipospheres

Triglycerides

Witepsol W35, Witepsol H35; Compritol 888 ATO

(Glyceryl behenate); Dynasan 112; Precirol ( Glyceryl

palmito stearate); tricaprin, trilaurin, tripalmitin,

tristearin, trimyristin.

Monounsaturated fatty

acid

Cis forms of monounsaturated fatty acids have lower

melting point than triglycerides hence used as a mixture

with higher saturated fatty esters

Partially hydrogenated

vegetable oils

Soybean oil, coconut oil, cotton seed oil.

Oils Olive oil, wheat germ oil, evenin primrose oil, arachis

oil, safflower oil, corn oil, rice bran oil.

Waxes Bees wax, spermaceti, cetyl palmitate, arachidyl oleate,

carnauba wax, cetyl alcohol, cholesteryl butyrate

1.5.2. Active pharmaceutical ingredient

1.5.3. Emulsifiers:

Phospholipids pure-egg phosphatidyglycerol, phosphatidylethanolamine,

dimyristoyl phosphatidylglycerol, soybean phosphatidylcholine

Surfactants: Tween-80, butyl alcohol



1.5.4. Stabilizers:

Gelatin, pectin, carrageenan, polyvinyl alcohol, polyoxyethylene sorbitan

trioleate, Pluronic PE 8100, lauryl sarcosine.

1.6. Methods of preparation:

1.6.1 Melt dispersion technique 31

In this method, drug is dissolved or dispersed in the melted lipidic phase (figure

9). Aqueous phase is composed of water or suitable buffer which is heated to the

same temperature as lipid phase. The aqueous phase is kept under stirring during

which emulsifier is added. To the aqueous phase containing emulsifier, lipid

phase containing drug is added drop by drop while maintaining the temperature

and stirring speed. After this “hot emulsification phase”, the temperature of the

mixture is rapidly brought down to room temperature or below room

temperature by adding ice cold water or ice under continuous stirring. This cold

resolidification results in the formation of discrete lipospheres which can be

filtered. Several drugs like bupivacaine, glypizide , aceclofenac , retinyl acetate,

progesterone,sodium cromglycate, diclofenac , carbamazepine , C14-diazepam,

proteins like somatostatin , thymocartin , casein , bovine serum albumin ,

R32NS1 malaria antigen , tripalmitin based lipospheres for labon-chip

applications have been prepared by melt dispersion methods. Lipids carrying

antigens exert their adjuvant effect to immunogenicity of antigens and the effect

was found to decrease in the following order for the lipids studied: ethyl

stearate>olive oil>tristearin>tricaprin>corn oil>stearic acid. Also inclusion of

negatively charged lipids like dimyristoyl phosphotidyglycerol in the lipid core

was found to improve the antibody response to encapsulated malaria antigen.



1.6.2 Solvent evaporation method 31

In this method, lipid is dissolved in an organic solvent. Commonly used organic

solvents include ethyl acetate, ethanol, acetone or dichloromethane. This lipid

phase is emulsified into aqueous phase containing emulsifier. Organic solvent is

evaporated by stirring the oil in water emulsion for 6-8 h under ambient

conditions. Discrete lipospheres can be collected by filtration through paper

filter after the water rises to the surface. Examples of the drugs formulated as

lipospheres by this method include paclitaxel, thymocartin, bovine serum

albumin, triptorelin leuprolide .

Fig 9: Schematic representation of the methods of production of LS: melt

dispersion and solvent evaporation



1.6.3 Co-solvent solvent evaporation method 31

In this co-solvent - solvent evaporation method employing chloroform and N-

methyl pyrollidone to create a clear solution, although low yield and large

particle size is obtained, which is altered by variation in the solvent used.

Lipospheres made up of polar and non-polar lipids using synthetic stabilizers

instead of phospholipids which are the deviation from the definition of

liposphere reported by Domb in his patent. Although their work is not related to

protein delivery but they tried it with hydrophilic drug and reported around 50%

entrapment by double emulsification method

1.6.4 Sonication method 32

In this technique, the drug is mixed with lipid in a scintillation vial which is pre-

coated with phospholipids. The vial is heated until the lipid melts, and then

vortexed for 2min to ensure proper mixing of the ingredients. A 10 ml of hot

buffer solution is added into the above mixture and sonicated for 10min with

intermittent cooling until it reaches to the room temperature.

1.6.5 Rotoevaporation method 32

In this technique, lipid solution with drug is prepared in a round bottom flask

containing 100 grams of glass beads (3 mm in diameter) mixed thoroughly till a

clear solution is obtained. Then, the solvent is evaporated by using

rotoevaporizer under reduced pressure at room temperature and a thin film is

formed around the round bottom flask and the glass beads. Raise the temperature

upto 40 °C until complete evaporation of the organic solvent. Known amount of

0.9 % saline is added to the round bottom flask and the contents are mixed for 30

min at room temperature and then the temperature is lowered to 10 °C by



placing in ice bath and mixing is continued for another 30min until lipospheres

are formed.

1.6.6. Microfluidizer method 32

Lipospheres can also be prepared by using a microfluidizer which is equipped

with two separate entry ports. From one entry port, a homogenous melted

solution or suspension of drug and carrier is pumped and from second entry port,

an aqueous buffer is pumped. The liquids are mixed in the instrument at elevated

temperatures where the carrier is melted and rapidly cooled to form the

lipospheres. The temperature of the microfluidizer can also be changed at any

stage of the lipospheres processing to manipulate the particle size and

distribution.

1.6.7 Polymeric lipospheres 32

Polymeric biodegradable lipospheres can also be prepared by solvent or melt

processes. The difference between polymeric lipospheres and the standard

liposphere formulations is the composition of the internal core of the particles.

Standard lipospheres, as those previously described, consist of a solid

hydrophobic fat core that is composed of neutral fats like tristearin, while in the

polymeric lipospheres, biodegradable polymers such as polylactide (PLD) or

PCL substitute the triglycerides. Both types of lipospheres are thought to be

stabilized by one layer of phospholipid molecules embedded in their surface.

1.6.8 Microemulsion 32

In this method, drug is added to the melted lipid. Aqueous phase is prepared by

adding surfactant like Tween 80 into water maintained at same temperature as

lipid phase. This is followed by the addition of co-surfactant like butyl alcohol to

the aqueous phase. The aqueous phase containing surfactant and co-surfactant is



added to lipid phase kept under stirring. Rapid cooling of the above mixture

results on formation of discrete lipid particles. Flurbiprofen lipospheres

prepared by this method. Presence of Tween80 at 2%, butyl alcohol at 2ml and

water at 50ml found to give discrete lipospheres of superior quality.

1.6.9 Multiple Emulsions 32

In this method, drug solution (aqueous phase) is added to melted lipid. The

primary emulsion formed as a result is added into aqueous solution containing

emulsifier kept at the same temperature as primary emulsion. The multiple

emulsions formed as a result is subjected to rapid cooling to form lipospheres.

Morel et al reported a 90 % entrapment efficiency of D-Trp-6- LHRH peptide

from stearic acid-egg lecithin based lipospheres prepared by this technique.

Drugs like thymopentin, cyclosporine and peptides like papain were investigated

for liposphere formulations by this method.



1.7. Factors influencing quality attributes of lipospheres

1.7.1. Factors influencing morphology of lipospheres

Table 2: Factors influencing morphology of lipospheres

S.No. Factors Influence

1.

Drug

loading

Proportion of larger particles formed was high on

increasing the drug amount. At maximum drug: lipid

(1:1)33

insufficient coating of drug by lipid leads to the

formation of aggregates during cooling phase resulting in

irregular, fluffy and fragile particles.

2.

Type of

lipid

Combination of apolar (tristearin, tripalmitin or tribehenin)

with polar lipids (glycery monostearate, glyceryl

monooleate) gave lipospheres satisfactory in terms of size,

shape and recovery.

3.

Type of

impeller

Lipospheres were produced using different impeller types

33 and particle characteristics of formed lipospheres were

studied. Impellers used were of rotor (2-blade, 3-blade)

type, helicoidal rotor (4-blade) type, double truncated cone

rotor. Lipospheres could not be produced using 2-blade

rotor and resulted in the formation of elliptical particles.



1.7.2. Factors influencing entrapment efficiency

Table 3: Factors influencing entrapment efficiency

S.No. Factors Influence

1.

Type of

lipid

Hydrophobicity of lipids promotes entrapment of

drugs. Long chain triglycerides (tristearin and

triarachidin) are generally more hydrophobic than short

chain triglycerides like tricaprin and trilaurin.

Accordingly the free drug contents of formulations

containing the long chain triglycerides were found to

be lower than short chain triglycerides34

. Also long

chain triglycerides were found to increase the

bioavailability of drug as they increase in

gastrointestinal residence time of drug compared to

medium chain and short chain fatty acids35

Lipid

excipients reduce the activity of P-glycoprotein and

MDR (multi drug resistant) associated protein 2 by

down regulating the protein expression and increase in

cell membrane permeability in addition to lymphatic

uptake.

2.

Amount of

Phospholipid

As the phospholipid (coat) amount increases, formation

of alternative systems like liposomes was observed

which will compromise drug entrapment. Experiments

with triglyceride, phospholipid at a 1:0.5 to 1: 0.25

w/w 36

revealed that 70-90% of phospholipid polar

heads were accessible on liposphere surface thus

enhancing the loadability of drug.



3.

Effect of

method of

preparation

Melt dispersion method was found to be superior over

solvent evaporation method in terms of entrapment

efficiency as melt method promotes drug incorporation

core where as solvent evaporation promotes drug

incorporation in coat.

1.7.3. Factors influencing drug release

Table 4: Factors influencing drug release

S.No. Factor Influences

1.

Release

pattern

The release mechanism of drugs namely tetracaine,

etomidate an prednisolone37

entrapped in lipid particles.

Dynasan 112 (glycerol trilaurate), Compritol 888 ATO

(glycerol behenate) were used as lipid carriers and Pluronic

F 68 (Poloxamer 188), Lipoid S 75 (soy lecithin), Lipoid KG

were used as emulsifiers. Tetracaine and etomidate

lipospheres have shown burst release and prednisolone

lipospheres gave prolonged release.

2.

Effect of

particle

size

Smaller particles have larger surface area exposed to

dissolution medium and higher diffusion coefficient. If the

drug resides in the outer shell diffusion distance becomes

shorter resulting in fast (burst) release.

3.

Type of

lipid

Highest T8h value was obtained with stearyl alcohol

lipospheres compared to fatty acids like stearic acid. Stearyl

alcohol possesses hydroxyl groups promoting matrix

hydration by providing a hydrophilic pathway for water

molecules to solubilize the drug and increase in dissolution

rate. Lowest T 8h value was obtained from stearic acid

lipospheres because of interaction of stearic acid with metal



ions in medium forming sodium soaps which are crystals

that contain fatty acid and metal carboxylate ion pairs

retarding the release.

4

Effect of

stabilizer

Lipospheres formulated with gelatin as stabilizer released

80% of total drug in 8hrs resulting in sigmoid mode of

release whereas formulations with Poloxamer 40738

resulted

in a biphasic pattern (burst release followed by slow release)

1.8. Applications of liposphers

1.8.1. Parenteral route

Lipospheres have been exploited for the delivery of anesthetics like lidocaine

bupivacaine for the parenteral delivery of antibiotics like ofloxacin, norfloxacin,

chloramphenicol palmitate and oxytetracycline , and antifungal agents, such as

nystatin and amphotericin B for the parenteral delivery of vaccines and

adjuvants.

1.8.2. Transdermal route 39

Properties of lipospheres like film forming ability, occlusive

properties;controlled release from solid lipid matrix resulting in prolonged

release of drug and retarded systemic absorption of drugs; increasing the

stability of drugs which are susceptible to extensive hepatic metabolism, make

them attractive candidates for topical delivery.

1.8.3. Oral delivery 40, 41

Several categories of drugs like antibiotics, anti-inflammatory compounds,

vasodilators, anticancer agents, proteins and peptides are being formulated as

oral lipospheres.

Chapter No.2 Literature Review


2. Literature Review

Sandipan dasgupta et al (2012)42

Nanostructured Lipid carriers (NLC)

based gel for Topical Delivery of aceclofenac preparation

,characterization ,and in vivo evaluation.stearic acid as the solid lipid and

oleic acid as the liquid lipid ,pluronic F68 as the sur factant and

phospholipon 90G as the co-surfactant were used NLC prepared by melt

–emulsification and high speed homogenization methods.the anti-

inflammatory effect of NLC gel was assesed by rat paw edema

technique and compared to marketed aceclofenac gel.

Esimone et al (2012) 43

Formulation and evaluation of goat fat and shea

butter based lipospheres of benzyl penicillin lipospheres of benzyl

penicillin were formulated using the conventional thin film hydration

technique five different combinations of shea butter, surfactant (span 80)

and goat fat were the key variables employed in the formulations the

presence of goat fat however seems to impact negatively on the in vivo

stability of liposphere.

Abraham J. Domb et al (2012)44

Preparation and characterization of an

oral pro-dispersion liposphere formulation for cyclosporin, a water

insoluble drug with limited bioavailability. Pro-dispersion formulation

consisted of a solid fat, dispersing agents and amphiphilic solvents as the

major components besides cyclosporin A (CsA) were prepared in the

present work. For preparation of this formulation, phospholipid was

dissolved in pharmaceutically acceptable water soluble organic solvent,

thereafter CsA along with other components was added and formulation

optimization was carried out. After formulation preparation, particle size

determination and in vitro release study was carried out. Additionally,



ultracentrifugation, TEM, Cryo-TEM and DSC techniques were used for

in vitro characterization of formulation. The prepared system was also

compared with marketed Neoral® microemulsion formulation.

Vignesh Muruganandham et al (2012)45

Formulation, Development &

Characterization of Ofloxacin Microspheres Ofloxacin is anti bacterial

agent that has a wide range of activity against gram (-ve) and gram (+ve)

microorganisms. Multiple doses of Ofloxacin are required to attain

steady state concentration. The main objective of this study was to

formulate, develop and characterize Ofloxacin microspheres to prolong

the release rate so as to decrease the necessity of multiple dosings

especially in patients with renal impairment. The Ofloxacin

microspheres were prepared using natural polymers by non-ionic

crosslinking technique. Five different formulations were prepared with

respective quantities of the polymer (Chitosan) with copolymer (Gelatin

and sodium alginate) with drug in different drug-polymer ratio of 1:0.5,

1:0.75 and 1:1. The prepared microspheres were evaluated for

percentage drug loading, entrapment efficiency, surface morphology,

and in-vitro release characteristics to identify the effect of addition of

these polymers.Cumulative release data were fitted into kinetic models.

The Scanning Electron Microscope analysis revealed a smooth and

spherical surface morphology with mean particle size of the

microspheres ranging from 7 to 14 μm. Drug loading was found to

increase with the increase in the concentration of encapsulating polymer,

chitosan, sodium alginate and gelatin concentration. Drug release obeyed

the first order kinetics45

.

Sanming Li et al (2012)46

Nanostructured lipid carriers (NLC)-based gel

was developed as potential topical system for flurbiprofen (FP) topical



delivery. The characterizations of the prepared semisolid formulation for

topical application on skin were assessed by means of particle size

distribution, zeta potential analysis, X-ray analysis, in vitro percutaneous

penetration, rheological study, skin irritation test, in vivo

pharmacodynamic evaluation and in vivo pharmacokinetic study. The

NLC remained within the colloidal range and it was uniformly dispersed

after suitably gelled by carbopol preparation. It was indicated in vitro

permeation studies through rat skin that FP-NLC-gel had a more

pronounced permeation profile compared with that of FP loaded

mcommon gel. Pseudoplastic flows with thixotropy were obtained for all

NLC-gels after storage at three different temperatures. No oedema and

erythema were observed after administration of FPNLC- gel on the

rabbit skin, and the ovalbumin induced rat paw edema could be inhibited

by the gel.

Satheesh Babu et al (2011)47

Manufacturing techniques of lipospheres

Lipid microspheres, often called lipospheres (LS), have been proposed as

new type of lipid-based encapsulation system for drug delivery of

bioactive compounds especially lipophilic compounds. LS consist of

solid microparticles with a mean diameter usually the size range between

0.2 to 500μm, composed of a solid hydrophobic fat matrix, where the

bioactive compound(s) is dissolved or dispersed. The lipospheres have

several advantages over other colloidal delivery systems (including nano

& micro emulsions, nanaoparticles, hydrogels and liposomes).

Loganathan Veerappan et al (2010)48

formulation development and

evaluation of flurbiprofen liposphere microencapsulation is a rapidly

expanding technology in the production of controlled release dosage



forms. Most of the non-steroidal antiinflammatory drugs (Nsaid) have

been widely used for the treatment of acute and chronic arthritic

conditions. flurbiprofen appears to be more active as an anti-

inflammatory agent than other nsaid products and is usually well

tolerated. flurbiprofen is frequently prescribed for the treatment of

rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. By

formulating sustained release dosage form of this drug leads to

minimization of damage to the gastro intestinal mucosa. Development of

formulation was made with different formulation variables and suitable

formulation was selected for further evaluations.

Kamal Dua et al (2010)49

Aceclofenac is a new generation non-steroidal

anti-inflammatory drug showing effective anti-inflammatory and

analgesic properties. It is available in the form of tablets of 100 mg.

Importance of aceclofenac as a NSAID has inspired development of

topical dosage forms. This mode of administration may help avoid

typical side effects associated with oral administration of NSAIDs,

which have led to its withdrawal. Furthermore, aceclofenac topical

dosage forms can be used as a supplement to oral therapy for better

treatment of conditions such as arthritis. Ointments, creams, and gels

containing 1 % (m/m) aceclofenac have been prepared. They were tested

for physical appearance, pH, spreadability, extrudability, drug content

uniformity, in vitro diffusion and in vitro permeation. Gels prepared

using Carbopol 940 (AF2, AF3) and macrogol bases (AF7) were

selected after the analysis of the results. They were evaluated for acute

skin irritancy, anti-inflammatory and analgesic effects using the

carrageenan-induced thermal hyperalgesia and paw edema method.



Maha Nasr et al (2008)50

liposphere as a carrier for topical Delivery of

Aceclofenac preparation characterization and in vivo. The aim of

exploiting the favorable properties of this carrier system and developing

a sustained release formula to overcome the side effects resulting from

aceclofenac oral administration. Lipospheres were prepared using

different lipid cores and phospholipid coats adopting melt and solvent

techniques. liposphere systems were found to possess superior anti-

inflammatory activity compared to the marketed product in both lotion

and paste consistencies. Liposphere systems proved to be a promising

topical system for the delivery of aceclofenac.

Jia-You Fang et al (2007)51

acoustically active lipospheres (AALs)

were prepared using perfluorocarbons and coconut oil as the cores of

inner phase. These AALs were stabilized using coconut oil and

phospholipid coatings. A lipophilic antioxidant, resveratrol, was the

model drug loaded into the AALs. AALs with various percentages of

perfluorocarbons and oil were prepared to examine their

physicochemical and drug release properties. Co-emulsifiers such as Brij

98 and Pluronic F68 (PF68) were also incorporated into AALs for

evaluation. AALs with high resveratrol encapsulation rates (_90%) were

prepared, with a mean droplet size of 250–350 nm. The AALs produced

with perfluorohexane as the core material had larger particle sizes than

those with perfluoropentane. Resveratrol in these systems exhibited

retarded drug release in both the presence and absence of plasma in

vitro; the formulations with high oil and perfluorocarbon percentages

showed the lowest drug release rates.

Hagalavadi Nanjappa Shivakumar et al (2007)52

Design and statistical

optimization of glipizide loaded A 32 factorial design was employed to



produce glipizide lipospheres by the emulsification phase separation

technique using paraffin wax and stearic acid as retardants. The effect of

critical formulation variables, namely levels of paraffin wax (X1) and

proportion of stearic acid in the wax (X2) on geometric mean diameter

(dg), percent encapsulation efficiency (% EE), release at the end of 12 h

(rel12) and time taken for 50% of drug release (t50), were evaluated

using the F-test. Mathematical models containing only the significant

terms were generated for each response parameter using the multiple

linear regression analysis (MLRA) and analysis of variance (ANOVA).

Both formulation variables studied exerted a significant influence (p <

0.05) on the response parameters. Numerical optimization using the

desirability approach was employed to develop an optimized formulation

by setting constraints on the dependent and independent variables. The

drug release from lipospheres followed first-order kinetics and was

characterized by the Higuchi diffusion model. The optimized liposphere

formulation developed was found to produce sustained anti-diabetic

activity following oral administration in rats.

Vandana B. Patravale et al (2007)53

to develop solid lipid nanoparticles

(SLN) of tretinoin (TRE) with the help of facile and simple

emulsification-solvent diffusion (ESD) technique and to evaluate the

viability of an SLN based gel in improving topical delivery of TRE. The

feasibility of fabricating SLN of TRE by the ESD method was

successfully demonstrated in this investigation. The developed SLN

were characterized for particle size, polydispersity index, entrapment

efficiency of TRE and morphology. Studies were carried out to evaluate

the ability of SLN in improving the photostability of TRE as compared

to TRE in methanol. Encapsulation of TRE in SLN resulted in a

significant improvement in its photostability in comparison to



methanolic TRE solution and also prevented its isomerization.

Furthermore, the skin irritation studies carried out on rabbits showed that

SLN based TRE gel is significantly less irritating to skin as compared to

marketed TRE cream and clearly indicated its potential in improving the

skin tolerability of TRE. In vitro permeation studies through rat skin

indicated that an SLN based TRE gel has permeation profile comparable

to that of the marketed TRE cream.

ChapterNo.4 Plan of Work


4. Plan of Work

Present proposed research work was planned as follows-

A) Literature survey.

B) Selection of drug, lipid core material and coat material.

C) Procurement of drug, core material and coat material.

D) Preliminary study of drug, core material and coat material.

E) Formulation of Liposphere.

F) Evaluation of Liposphere.

1. Photo microscopic analysis.

2. Scanning Electron microscopy.

3. Particle size analysis.

4. Differential scanning Calorimetry.

5. Fourier transforms infrared spectroscopy (FTIR).

6. In vitro dissolution test.

H) Formulation of lipospheres based gel.

I) Evaluation of Lipospheres based gel.

1. pH.

2. Drug content.

3. Viscosity.

4. Spreadability.

5. In vitro permeation of liposphere based gel.

6. Skin-irritation testing (Draize patch test).

7. In vivo Anti-inflammatory Study of Liposphere.

8. Stability study.

Chapter No. 3 Aim and Objective


33.. Aim and Objectives

Aim

Preparation and evaluation of liposphere based topical drug delivery system

containing a NSAID drug.

Objectives

1. To study formulation and evaluation of liposphere as a carrier for

topical delivery of NSAID drug.

2. To optimize the formulation using suitable experimental technique,

regarding particle size, stability, release property, surface morphology,

hydrophobicity, drug entrapment efficiency etc.

3. To study in vitro release of NSAID from liposphere

4. In vivo Anti-inflammatory study of liposphere.

Chapter No.6 List of Material and Equipment


6. List of material and equipment

Table 5: List of Materials Used

Sr. No. Instrument Used (Model No.) Make

1. FTIR Spectrophotometer Aligent Cary 630 ATR

2. UV–1700 Spectrophotometer,

double beam

Shimadzu, 1700, Japan

3. Electronic balance Citizen scale (CY 104),

Germany

4. Brookfield viscometer (Dial type) Middleboro, MA-02346, USA

5. Differential Scanning

Calorimetry (DSC 60)

Mettler Toledo, Zaventem

(U.S.)

6. Mechanical Stirrer Remi lab stirrer, Mumbai

7. Magnetic Stirrer Remi lab stirrer, Mumbai

8. Intel play Qx3 microscope

(200X magnification)

Edmund Scientific (U.S.)

9. Dissolution test apparatus Electro lab , Mumbai

10. Scanning Electron Microscope

(JSM 6380A)

JOEL, Japan



11 Centrifuge machine Remi, Mumbai

12. Digital pH Meter Chemi line (CL-110)

13. Plethysmometer (UGO-Basile, 7140,Comerio,

Italy

14. Stability chamber Skylab,Mumbai

List of chemicals and reagents used

Aceclofenac was procured as a gift sample from Concept

pharmaceutical, india. Soy phosphatidylcholine-35% was kindly gifted by

perfect Biotech industries Pvt Ltd, Nagpur. LIPOVA-E120 (Egg

phosphatidylcholine) gifted by VAV Life sciences Pvt Ltd. Mumbai. All other

chemicals were procured from Research lab fine Chem industries, Mumbai.

They are-

Table 6: List of chemical used

Chemicals Grade

Absolute Ethanol AR grade

Methanol LR grade

Potassium chloride LR grade

Sodium chloride AR grade



AR grade-Analytical reagent, LR grade-Laboratory reagent

Potassium dihydrogen phosphate AR grade

Acetone LR grade

disodium hydrogen phosphate AR grade

Acetic anhydride AR grade

Disodium hydrogen phosphate AR grade

Carbopol 940P AR grade

Triethanolamine (TEA) AR grade

Chapter No.5 Drug Profile


5. Drug Profile

5.1. Drug: Aceclofenac 54,55,56,57

5.1.1. Structure:

Chemical Name [(2, 6-dichlorophenyl)amino]

phenylacetoxyacetic acid.

Molecular formula C16H13Cl2NO4

Molecular weight 354.18

Melting point 149-153 0C

Description A white or almost white, crystalline

powder. Insoluble in water, soluble in

ethanol and acetone

Mode of action Highly selective β2 agonist

Dose 100 mg

Plasma half-life 2 to 3 h

Plasma protein binding 40-50%

Category Anti inflammatory

Analgesic



Adverse reactions

Gastrointestinal System – Duodenal ulcer, gastrointestinal perforation

Urinary System – Interstitial nephritis

Central and Peripheral Nervous System – Optic neuritis

Psychiatric – Hallucination, Drowsiness, Confusion

Skin and Appendages – Epidermal necrolysis, Erythema multiforme,

dermatitis

Respiratory – Aggravated asthma

Haematological – Aplastic anaemia

Contraindications

1. Active peptic ulceration

2. Recurrent indigestion (relative contraindication)

3. Care should be taken in patients on anticoagulants

4. Care should be taken in patients with hypertension or heart failure

5. Pregnancy and lactation

6. History of sensitivity to aspirin or other NSAISD drugs.

Drug Interactions

1. Anticoagulants

2. Alcohol and smoking

3. Lithium

4. Diuretics

5. Antihypertensive drugs

6. Diflunisal

7. Anti-platelet agents and selective serotonin reuptake inhibitors



5.2. Lipid profile

5.2.1. Lecithin58

5.2.1.1. Structure:

CH2

CH

OCH

2

P

O

OCH2CH

2N(CH

3)3

O-CO-R1

O-CO-R2

O

R1 and R

2 are fatty acids

+

_

Non proprietary names Lecithin

Synonyms Soya lecithin, soyabean lecithin,

phosphatidylcholine.

Chemical name and CAs

registry no

Lecithin [8002-43-5]

Empirical formula CH2OCOR’- - - CHOCOR’’- - - CH2OPOO-

OCH2CH2N (CH3)3

Functional category Emollient; Emulsifying agent; Solubilizing

agent

Description They may vary in color from brown to light

yellow, depending upon whether they are

bleached or not or on the degree of purity.



when they are exposed to air, rapid oxidation

occurs, also resulting in a dark yellow or

brown color

Solubility Insoluble but swells up in water and in Nacl

solution forming a colloidal suspension,

soluble in about 12 parts cold, absolute

alcohol; soluble in chloroform, ether,

petroleum ether, in mineral oil and sparingly

soluble in benzene

Incompatibility Incompatible with esterase owing to

hydrolysis

Regulatory status Gras listed included in the FDA inactive

ingredient guide. included in nonparenteral

and parenteral medicine licensed in UK

Safety It is highly biocompatible and oral doses of

up to 80 g daily have been given

therapeutically in the treatment of Tardive

dyskinesia.



5.2.2. Stearyl alcohol

5.2.2.1. Structure

Chemical Name octadecyl alcohol or 1-octadecanol)

Molecular formula CH3(CH2)16CH2OH

Molecular weight 270.49

Melting point 55-58°C

Description Freely soluble in chloroform and in

ether; soluble in ethanol (95 per cent);

practically insoluble in water

5.2.3. Bees wax

Structure

CAS No 8012-89-3

Chemical Name Bees wax

Molecular formula C15H31COOC30H61

http://en.wikipedia.org/wiki/File:1-Octadecanol.png



Molecular weight 415

Melting point 62 to 64 °C

Description white,solid Insoluble in water, soluble

in alcohol

Category Cosmetic Ingredients & Chemicals

5.2.4. Carnauba wax

A coumpound is a pure substance and Carnuba is a mixture. Carnauba wax

contains mainly esters of fatty acids (80-85 %), fatty alcohols (10-15 %), acids

(3-6 %) and hydrocarbons (1-3 %). Specific for carnauba wax is the content of

esterified fatty diols (about 20%), hydroxylated fatty acids (about 6 %) and

cinnamic acid (about 10 %). Cinnamic acid, an antioxidant, may be

hydroxylated or methoxylated. The major components of carnauba wax are

aliphatic and aromatic esters of long-chain alcohols and acids, with smaller

amounts of free fatty acids and alcohols, and resins. Another site claim common

components as C30 alcohols and C26 acids.

CAS No 8015-86-9

Synonyms Carnuba;carnauba, brazil wax, carnubawax, carnaba

wax, wax, carnaubawachs, carnauba wax yellow,

carnauba wax flakes.

Molecular formula -------

Molecular weight ----

http://www.chemicalbook.com/ProdClassDetail/CosmeticIngredientsChemicals_0_EN.htm



Melting point 81-86 °C

Description Soluble on warming in chloroform, in ethyl acetate and

in xylene; practically insoluble in water and in ethanol

Category Cosmetic Ingredients & Chemicals

http://www.chemicalbook.com/ProdClassDetail/CosmeticIngredientsChemicals_0_EN.htm

Chapter No.7 Experimental Work


7. Experimental work

7.1. Preformulation study

Identification test

7.1.1. Aceclofenac

Organoleptic properties

Aceclofenac was tested for organoleptic properties such as

appearance, color, odor, taste, etc.

7.1.2. Melting point

The melting point of the aceclofenac was determined by capillary

method.

7.1.3. Solubility determination59

The approximate solubility of Pharmacopeial substance is indicated

by descriptive terms in accompanying table.

7.1.4. Ultra-violet scanning

The scanning of Aceclofenac was performed in 7.4 pH phosphate

buffer saline and λmax was found to be 276 nm which was complies with the

λmax reported in BP.

7.1.5. Fourier transforms infrared (FTIR) spectroscopy

Procedure: Small quantity of aceclofenac 1 mg was placed on diamond

ATR crystal then it was scanned between 4000-500 cm-1

range.

7.1.6. Lipids

7.1.6.1. Soyalecithin

Saponification value60

About 35 g Potassium hydroxide was weighed, dissolved in 20 ml of

water; sufficient ethanol was added (96 %) to produce 1000 ml and allowed

to stand overnight. Clear liquid was poured off. About 2 gm of soya lecithin



was accurately weighed and taken into a 200 ml conical flask, 25 ml of

ethanolic solution of KOH was added, boiled under a reflux condenser for 1

h with rotating the contents frequently. When the solution was still hot, the

excess of alkali was titrated with 0.5 M Hcl using 1 ml of phenolphthalein

solution as indicator.

Blank determination was carried out excluding the substance being

examined. The saponification value was calculated from expression

Saponification value = 28.5 V/W

Where, V- difference in ml between titration

W- Weight in g of substance taken.

7.2. Preparation of standard calibration curve of aceclofenac

7.2.1. Preparation of phosphate buffer saline pH 7.4

About 2.38 g of disodium hydrogen phosphate (Na2Po4), 0.19 g of

potassium dihydrogen phosphate (KH2PO4) and 8.0 g of sodium chloride

(Nacl) was taken in volumetric flask and volume was made with water to

produce 1000 ml. This solution had a pH of about 7.461

.

7.2.2. Determination of λmax for aceclofenac

Stock solutions of Aceclofenac was prepared by dissolving in 100 ml

of phosphate buffer saline solution pH (7.4), solutions were further diluted

and analyzed spectrophotometrically between 220 to 370 nm to determine

λmax.

7.2.3. Preparation of standard calibration curve of aceclofenac

The calibration curve was plotted within the concentration range of 2-10

µg/ml. Appropriate dilutions were prepared and absorbance was measured

for each solution at 276 nm since maximum absorbance was observed at this

wavelength. Graph was plotted for absorbance vs. concentration.



y = 0.0109x R² = 0.994.

Correlation co-efficient value indicated the linear correlation between

concentration and absorbance.62

Where

Y is absorbance

X is concentration

R2 is coefficient of regression.

7.3. Formulation of liposphere by melt dispersion technique

Lipospheres (LS) were prepared by melt dispersion method. In this

method different lipid materials (Carnauba wax, Bees wax and stearyl

alcohol) along with the drug aceclofenac were used to form core material in

the ratio 2:1 and 3:1, egg phosphatidylcholine and soy phosphatidylcholine

were used as the coat material so as to give the core to coat ratio (cr/ct) of

2:1 and 3:1. The lipid core material was melted at 75 ºC using water bath

and then added with the required amount of aceclofenac by dispersing it in

the molten lipid. Separately 1000 mg of phospholipid was added in 100 ml

of phosphate buffer saline, which was preheated at 75 ºC and the given

mixture was homogenized using mechanical stirrer until a uniform

dispersion was obtained. To this dispersion at 75 ºC the previously prepared

drug was added to molten lipid at 75 ºC and dispersed with the help of

mechanical stirrer at 4000 rpm speed. The homogenized milky solution was

then rapidly cooled down to 10-20 ºC with continued stirring for another 5

min for formation of uniform dispersion of lipospehers63

.



Table 7: Formulation codes of lipospheres (F1-F12)

Egg pc –Egg phosphatidylcholine, soy pc-soya phosphatidylcholine

Cr-core material, Ct-coat material

7.4. Evaluations of Lipospehers

7.4.1. Separation of Unentrapped Aceclofenac from the Prepared

Lipospheres

Aceclofenac lipospheres were separated from free unentrapped

aceclofenac by centrifugation at 20,000 rpm for 30 min. The pellets formed

were washed with 10 ml phosphate buffered saline and recentrifuged again

for 30 min64-66

. The lipospheres were decanted and kept in the refrigerator

for further investigations.

7.4.2. Determination of entrapment efficiency

The entrapped drug concentration was determined by lysis of the

lipospheres with absolute alcohol65-66

. Accurately weighed amount of loaded

lipospheres (50 mg) was dissolved in 10 ml absolute alcohol and covered

well with aluminum foil to prevent evaporation. The solution was sonicated

for 15 min to obtain a clear solution. An aliquot of 1 ml of this solution was

added to 9 ml of absolute alcohol. The solution was sonicated for another

15 min. The concentration of aceclofenac in absolute alcohol was

Code Lipid core

Material

Lipid

coat

material

Quantity

of lipid

core (mg)

Quantity

of lipid

coat(mg)

Drug

(mg)

Ratio

(Cr/Ct)

F1 Stearyl alcohol Egg pc 2000 1000 500 2:1

F2 Carnauba wax Egg pc 2000 1000 500 2:1

F3 Bees wax Egg pc 2000 1000 500 2:1

F4 Stearyl alcohol Soya pc 2000 1000 500 2:1

F5 Carnauba wax Soya pc 2000 1000 500 2:1

F6 Bees wax Soya pc 2000 1000 500 2:1

F7 Stearyl alcohol Egg pc 3000 1000 1000 3:1

F8 Carnauba wax Egg pc 3000 1000 1000 3:1

F9 Bees wax Egg pc 3000 1000 1000 3:1

F10 Stearyl alcohol Soya pc 3000 1000 1000 3:1

F11 Carnauba wax Soya pc 3000 1000 1000 3:1

F12 Bees wax Soya pc 3000 1000 1000 3:1



determined spectrophotometrically at wavelength 276 nm after appropriate

dilution. Each sample was analyzed in triplicate. The entrapment efficiency

was calculated through the following relationship:

Entrapment efficiency percentage = Entrapped drug/ Total drug×100

7.4.3. Photo microscopic analysis

A drop of lipospheres preparation was placed on a slide for morphological

examination under optical stereo microscope and photographed at a

magnification of ×200 by means of a fitted camera.

7.4.4. Particle size analysis

The size of the prepared Lipospheres was measured by the optical

microscopy method using a calibrated stage micrometer. It is carried out by

using a compound microscope at 10 x lances. Dried lipospheres were first

re-dispersed in distilled water and placed in a glass slide and the number of

division of calibrated eye piece was counted by a micrometer using a stage

micrometer 67

.

7.4.5. Fourier transforms infrared (FTIR) spectroscopy

FTIR spectra of pure Aceclofenac and the optimized liposphere formulation

were recorded with a FTIR spectrophotometer.

FTIR Spectroscopy: There is always a possibility of drug Lipid interaction

in any formulation due to their intimate contact. The technique employed in

the present study for this purpose is IR spectroscopy.IR spectroscopy is one

of the most powerful analytical techniques, which offers the possibility of

chemical identification.



7.4.6. Differential scanning calorimetry (DSC)

Samples of aceclofenac, and drug loaded lipospheres of the selected

formulation were submitted to DSC analysis using differential scanning

calorimeter calibrated with indium. The analysis was carried out on 1 mg

samples sealed in standard aluminum pans. Thermograms were obtained at a

scanning rate of 10 °C/min using dry nitrogen flow of (25 ml/min). Each

sample was scanned between zero and 400 °C.

7.4.7. Scanning electron microscopy (SEM)

The detailed surface characteristics of the selected aceclofenac

lipospheres formulation were observed using a scanning electron

microscope. The lipospheres sample was attached to the specimen holder

using a double coated adhesive tape and gold coated (~20 nm thickness)

under vacuum using a sputter coater for 5–10 min at 40 mA and then

investigated at 30 kV 68

.

7.4.8. In vitro release of aceclofenac from lipospheres

Prepared Lipospheres equivalent to 100 mg of aceclofenac lipospheres

were accurately weighed and filled into gelatin capsules or tight in the

Muslin cloth. Degassed 7.4 Phosphate buffer saline (PBS) medium (900 ml)

was placed into the dissolution tester jars and the temperature was

maintained at 37 ±0.5 °C. A USP II (paddle) dissolution apparatus

(Electrolab) at 100 rpm was used. Samples of 5 ml were drawn at time

points of 30, 60, 120, 180, 240, 300, 360, 420 and 480 min and an equal

amount of fresh dissolution medium was replaced each time. After suitable

dilution, the samples withdrawn were analyzed spectrophotometrically at a

wavelength 276 nm 69

. Results were the mean of three runs. The amounts of

drug present in the samples were calculated with the help of appropriate

calibration curve constructed from reference standard. Percentage drug

release was calculated.



7.5. Formulations of lipospheres based gel

Lipospheres based gel was prepared according to the formula (Table 9).

The suitable lipospheres formulation for the topical delivery of aceclofenac

was selected based on the evaluation of characteristics like: particle size,

entrapment efficiency, and in vitro release. It was found that the formulation

F7 and F8 were more suitable among the other formulations. Different

gelling agents such as: carbopol 940 P, xanthan gum, chitosan, and

hydroxypropyl methyl cellulose (HPMC) were used for the conversion of

the lipospheres dispersion into the gel formulation. Based on the

compatibility with the lipospheres dispersion and the ease of spreadability,

carbopol was selected as the gelling agent. Appropriate quantity of carbopol

940P was soaked in water for a period of 2 h. Carbopol was then neutralized

with triethanolamine (TEA) with stirring. Then specified amount of

liposphere, glycerin and permeation enhancer (Mineral oil) was mixed.

Solvent blend was transferred to carbopol container and agitated for

additional 20 min. The dispersion was then allowed to hydrate and swell for

60 min, finally adjusted the pH with 98 % TEA until the desired pH value

was approximately reached (6.8-7). During pH adjustment, the mixture was

stirred gently with a spatula until homogeneous gel was formed. All the

samples were allowed to equilibrate for at least 24 hours at room

temperature prior to performing rheological measurements70-76

.



Table 8: Formulation of gel optimized Batch

Ingredients

(1.5%w/w)

Formulations Gels 50g

F7 F8 LF7 LF8 Blank Lipospheres

gel

Carbopol 940P 0.5 0.5 0.5 0.5 0.5

Lipospheres 0.75 0.75 0.75 0.75 --

Glycerin 5 5 5 5 5

Triethanolamine

(TEA)

q.s q.s. q.s. q.s. q.s.

Permeation

enhancer

--- --- 0.5 0.5 ---

Distilled water 43.7

5

43.75 43.18 43.18 44.5

7.6. Characterization of lipospheres based topical gel

7.6.1. pH

pH of gel was determined using digital pH meter. About 1 g of gel was

stirred in distilled water till a uniform suspension effected. The volume was

made up to 40 ml and pH of the solution was measured 77

.

7.6.2. Drug content78

Take 1g gel in a 100 ml of volumetric flask and dissolve with little amount

of methanol and mixture was shaken till solution was affected. The volume

was made up to 100 ml with methanol. The solution was filtered through

Whatman filter paper (No. 41). Further dilute 5 ml to 50 ml with methanol.

The absorbance of the solution was measured at 276 nm against reagent

blank.

7.6.3. Viscosity79

Viscosity of the gel was determined by using Brookfield viscometer (Dial

type). As the system is non-Newtonian spindle no. 4 is used. Viscosity is

measured for the fixed time 2 min for 100 rpm.



7.6.4. Spreadability

80-82

The spreadability of the gel was determined using the following

technique: 0.5 g gel was placed within a circle of 1 cm diameter premarked

on a glass plate over which a second glass plate was placed. A weight of 500

g was allowed to rest on the upper glass plate for 5 min. The increase in the

diameter due to spreading of the gels was noted.

It was calculated using formula,

S = M. L / T

Where, S = spreadability

M = weight tied to upper slide

L = length of glass slide

T = time taken

Shorter time interval, to cover distance of 6.5 cm, indicates better

spreadability.

7.6.5. In vitro permeation of liposphere based gel83, 84

This study was carried out for the optimized batch selected, based on all

above evaluation parameters. One of the batches among them was

formulation prepared without permeation enhancer F8 and other was

formulation prepared with permeation enhancer LF8 was studied through

cellophane membrane using a fabricated dissolution testing apparatus. The

dissolution medium used was artificial tear fluid freshly prepared (pH 7.4).

Cellophane membrane, previously soaked overnight in the dissolution

medium, was tied to one end of a specifically designed glass cylinder (open

at both ends and of 5 cm diameter). A 5 ml volume of the formulation was

accurately pipetted into this assembly. The cylinder was suspended in 50 ml

of dissolution medium maintained at 37 ºC so that the membrane just

touched the receptor medium surface. The dissolution medium was stirred at

50 rpm using magnetic stirrer. Aliquots, each of 1 ml volume, were



withdrawn at regular intervals and replaced by an equal volume of the

receptor medium. The aliquots were suitablely diluted with the receptor

medium and analyzed by UV-Vis spectrophotometer at 276 nm.

7.6.6. Skin irritation testing (Draize patch test)85

The lipospheres gel in comparison to marketed aceclofenac gel was

evaluated by carrying out the Draize patch test on rabbits. Animal care and

handling throughout the experimental procedure were performed in

accordance to the CPCSEA guidelines. The experimental protocol was

approved by the institutional animal ethical committee. White rabbits

weighing 2.5–3 kg and were acclimatized before the beginning of the study.

Animals were divided into four groups as follows:

Group 1: No application (Control).

Group 2: Marketed formulation (Audigel 1.5% w/w gel)

Group 3: Lipospheres based gel containing LF7 (1.5% w/w)

Group 4: Lipospheres based gel containing LF8 (1.5%, w/w)

The back of the rabbits were clipped free of hair, 72 h prior to the

application of the formulations. Formulations, 0.5 g, were applied on the

hair free skin of rabbits by uniform spreading. Four rabbits will be use for

the skin irritancy study. All the test formulation will apply on single rabbit,

so total four surface areas will create on the skin of rabbit (3 cm×3 cm)

using hair depletion. To avoid biological variation, the study will perform on

four rabbits. The respective test sample will apply on specified area for

seven day and simultaneous observation for skin irritation such as redness,

edema and skin rash. The results will interpret in the form of grading scale:

A-no reaction; B-slight, patchy erythema; C-moderate but patchy erythema;

D-moderate erythema, and E-severe erythema with or without edema.



7.6.7. In vivo anti-inflammatory study of lipospheres47

The anti-inflammatory activity of the selected lipospheres formulation

(LF7 and LF8) applied and compared to the marketed product using the rat

paw edema test. The protocol of the present work was approved by the

experimental protocol was approved by the institutional animal ethical

committee. Male Wistar rats (130–150 g) were randomly divided into four

groups of 3 rats each. Group I control application, group II for marketed

formulation (AUDIGEL), group III for lipospheres based gel (LF7), group

IV for lipospheres based gel (LF8) formula the volume of paw edema

(milliliter) was measured in each animal using a plethysmometer to a

precision of two decimal places. The rats were marked on the left hind paw

just beyond the tibiotarsal junction, so that every time the paw was dipped in

the electrolyte fluid column up to a fixed mark to ensure constant paw

volume. The tested preparations were applied to the left hind paws of rats

using an amount equivalent to 1 mg of aceclofenac. After 1 h of topical

application, initial paw volume of the rats was measured by dipping the rat

paw into the electrolyte column just before carrageenan injection and the

increase in volume due to fluid displacement was noted from a digital

display, followed by the injection of 0.1 ml of 1% (w/v) carrageenan

solution in saline in the subplantar region of left hind paw of the rats.

Measurement of paw volume was done after 1, 2, 3, 4, 5, 6, 7 and 8 h. The

edema rate and inhibition rate of each group was calculated as follows: The

edema rate and percentage inhibition of each group were calculated as

follows:

.

The % inhibition of edema was calculated by formula:

% inhibition= 1-(a-x/b-y)*100



Where,

A= paw thickness of test animal after treatment

X= initial paw thickness of test animal

B=paw thickness of control animal after treatment

Y= initial paw thickness of control animal.

7.8. Stability studies86

Stability study was performed as per ICH guideline. The purpose of stability

testing is to provide evidence on how the quality of a drug substance or drug

product varies with time under the influence of a variety of environmental

factors such as temperature, humidity and light. Therefore, stability studies

provide data to justify the storage condition and shelf-life of the drug product.

For drug substance, such studies establish the retest date in addition to the

storage condition of raw material. Initial formulation are packed in the

aluminum lacquered tube and kept in the stability chamber, at different

stability condition.

All the selected formulations were subjected to a stability testing for

three months as per ICH norms at a temperature of 40 ºc ± 2 ºc /75 % ± 5

% RH. All selected formulations (LF7 and LF8) were analyzed for the

change in appearance, pH and drug content by procedure stated earlier.

Chapter No.8 Results and Discussion


8. Results and Discussion

8.1 Identification test

8.1.1. Aceclofenac

Physical characters of aceclofenac were found as

Table 9: Result of physical characters of aceclofenac drug

S.no. Characters Inference

1 Nature Amorphous powder

2 Color white

3 Odor Odorless

4 Taste Slightly Bitter

5

Solubility-

In methanol

In water

In ethanol

Soluble

Practically insoluble

Freely soluble

8.1.2. Melting point

The melting point of the aceclofenac was determined by capillary

method and found to 149-153 0C which compiled with melting point

reported in BP.

8.1.3. Ultra-violet scanning

The scanning of aceclofenac was performed in 7.4 pH phosphate

buffer saline and λmax was found to be 276 nm which was compiles with the

λmax reported in BP.



Fig.10: UV Spectrum of aceclofenac

8.1.4. Fourier Transforms Infrared (FTIR) spectroscopy-Scanning was

performed between 4000-500 cm-1

range.

Fig.11: FTIR of aceclofenac



Table 10:- Interpretation of aceclofenac FTIR

Sr. No. Functional Group Wave Number (cm-1

)

1 Amino group, OH, Aliphatic

and Aromatic CH 3600-2300

2 Carboxylic acid salt 1580

3 Aromatic ring 1580, 1515

4 Aromatic ether 1250, 1015

5 Isopropyl group 1180

6 Aliphatic ether, sec.alcohol 1100

7 1,4-disubstituted benzene 820

8.1.5. Lipids

8.1.5.1. Soyalecithin

Saponification value60

Saponification value = 28.5 V/W

Where, V- Difference in ml between titration

W- Weight in g of substance taken.

Result: Saponification value was found to be 190-195 which complies with

Merck Index76

.



8.2. Preparation of calibration curve of aceclofenac

Table 11: UV calibration curve reading of aceclofenac in phosphate buffer

saline pH 7.4

Sr. No. Concentration

(µg/ml)

Absorbance

1 0 0

2 2 0.0259

3 4 0.0452

4 6 0.0616

5 8 0.0896

6 10 0.1072

Result: The prepared dilutions obey’s Beers Lambert’s law in the entire

concentration range selected and the coefficient of correlation was found to

be 0.994.

Fig.12: UV Calibration curve of aceclofenac

From the scanning of drug in phosphate buffer pH 7.4, it was

concluded that the drug had λmax of 276 nm. From the standard calibration

curve of aceclofenac in pH 7.4 buffer (Figure), it was concluded that drug

y = 0.0109x R² = 0.9946

0

0.02

0.04

0.06

0.08

0.1

0.12

0 2 4 6 8 10 12

Ab

sorb

ance

Concentration μg/ml

Calibration curve



obeys Beer-Lamberts law in concentration range of 0-10 µg/ml. The linear

equation in pH7.4 Phosphate buffer was obtained as:

y = 0.0109x R² = 0.994.

Correlation co-efficient value indicated the linear correlation between

concentration and absorbance.

8.3. Evaluations of lipospehers

Table 12: Results of entrapment efficiency

DP=dispersed particle, AP= aggregated particle, PVA –Polyvinyl alcohol

Fig.13: Result of entrapment efficiency of liposphere (F1-F12)

96.3 70.42 76.35

101.65

32.86

88.84 78.07

96.92

55.48

120.2

50.2 49.38

1 2 3 4 5 6 7 8 9 10 11 12

Entrapment Efficiency

Code Lipid core

Material

Entrapment

±SD (n=3)

Stabilizer

(q.s.)

Appearance

F1 Stearyl alcohol 96.35±0.941 Pectin DP

F2 Carnauba wax 70.42.±1.38 Pectin DP

F3 Bees wax 76.35±2.92 Pectin AP

F4 Stearyl alcohol 101.65±0.99 ------ DP

F5 Carnauba wax 32.86±2.52 ----- DP

F6 Bees wax 88.84±1.19 Gelatin DP

F7 Stearyl alcohol 78.07±0.97 PVA DP

F8 Carnauba wax 96.92±2.51 PVA DP

F9 Bees wax 55.48±0.59 PVA AP

F10 Stearyl alcohol 120.2±2.40 PVA DP

F11 Carnauba wax 50.29±1.73 PVA DP

F12 Bees wax 49.38±1.81 Gelatin AP



8.3.1. Photomicroscopic analysis

The photomicrographs of aceclofenac lipospehers of formula F8 was

represented by Fig. 14 & 15. It reveals the uniform spherical shape of

lipospehers showing the solid lipid core and the phospholipid coat.

Fig.14 Fig. 15

Fig. Photomicrograph of optimized aceclofenac lipospehers prepared by melt

method at a magnification of 60 X (Fig.14) and 200X (Fig.15)

8.3.2. Particle size analysis

From Table 13 it was evident that the combination of carnauba wax (F8)

are stearyl alcohol (F7) with the highest amount the smallest particles. Use

of soy lecithin and egg phosphatidylcholine resulted in the smallest particle

size but the lipospehers were found to be aggregated with soya lecithin when

observed under microscope. Carnauba wax with egg phosphatidylcholine

gives the smallest particle size was decreased. As the core/coat ratio

increased, the particle size of lipospehers. This decrease in particle size was

probably due to the availability of higher amounts of carrier material and

emulsifier for the formation of discrete spherical particles.



Table 13: Mean particle size of different batchs (F1-F12)

8.4. Drug lipid compatibility

8.4.1. Fourier transform infrared (FTIR) spectroscopy

Drug-lipid compatibility in optimized lipospehers (F6, F7, and F8) was

evaluated by FTIR and DSC analysis. FTIR spectra are shown in Fig. 16, 17,

18, 19. Interactions between the ingredients used in topical formulations, such

as Aceclofenac, carnauba wax, bees wax, stearyl alcohol, Carbopol were studied

using FTIR spectroscopy. Aceclofenac was identified by the presence of

characteristic bands. The FTIR spectral analysis of aceclofenac alone (Fig. 9)

showed that principle peaks were observed at wave numbers 3316 (N-H

stretching vibration for amine), 2935 (aromatic C-H stretching), 1769 (carbonyl

C=O stretching). The same peaks were observed at 3325, 2954, 1733, 1418,

1234, and 719 in F6 at 3302, 2955, 1471, 1299, and, 718 in F7 at 3320, 2954,

1733, 1467, 1257, 1260 and 719 cm–1 in F8. All the principle characteristic

bands of the drug were also observed in the respective formulations. The

Formulation code Mean particle size (μm)

F1 28.2±0.31

F2 14.2±.61

F3 56.8±.0.32

F4 42.9±0.43

F5 22.3±0.12

F6 85.2±0.32

F7 14.2±0.12

F8 14.2±1.22

F9 42.9±0.65

F10 42.9±0.46

F11 85.2±0.98

F12 96.2±0.12



findings were suggestive of the absence of any major incompatibility between

aceclofenac and the components employed in the preparation of dermatological

bases.

Fig. 16: FTIR of pure drug

Fig. 17: FTIR of formulation (F6)

Fig.18: FTIR of formulation (F7)



Fig. 19: FTIR of formulation F8

8.4.2. Differential scanning calorimetry (DSC)

Figure 20, 21 and 22 showed the results of the DSC analysis of

aceclofenac, and the physical mixture of drug loaded liposphere formulation.

From the DSC thermograms, it was observed that melting point depression

occurred The DSC thermogram of aceclofenac showed an exothermic peak

at 160.44 °C, which is the reported melting point of the aceclofenac. Drug-

loaded liposphere showed a large endothermic peak at 89.07 °C (F6) and

76.22 (F8). It was observed from the DSC thermogram that the exothermic

peak of aceclofenac at about 160.4 °C no longer exists in the DSC, traces of

the drug-loaded liposphere. Taking into consideration the drug-crystal-free

particle surface, it is apparent that aceclofenac is amorphously dispersed

within the liposphere, which is preferable for a controlled release system.

Furthermore, the inclusion of drug molecules in the lipid is normally

accompanied by a depression in the lipid’s melting point.



0.00 1.00 2.00 3.00 4.00

Time [min]

-40.00

-30.00

-20.00

-10.00

0.00

mW

DSC

100.00

150.00

200.00

C

Temp

154.10 x100COnset

170.49 x100CEndset

151.40 x100CStart

172.85 x100CEnd

160.44 x100CPeak

-754.14 x100mJ

-106.82 x100J/g

Heat

-38.56 x100mWHeight

File Name: Pure Drug.tadDetector: DSC60Acquisition Date 13/03/01Acquisition Time 14:49:51Sample Name: Pure DrugSample Weight: 7.060[mg]Annotation:

Thermal Analysis Result

Fig.20: DSC of pure aceclofenac drug

0.00 2.00 4.00 6.00 8.00

Time [min]

-15.00

-10.00

-5.00

0.00

mW

DSC

100.00

200.00

C

Temp

76.22 x100COnset

97.91 x100CEndset

68.71 x100CStart

101.32 x100CEnd

89.32 x100CPeak

-414.84 x100mJ

-56.75 x100J/g

Heat

-13.58 x100mWHeight

File Name: F8.tadDetector: DSC60Acquisition Date 13/03/01Acquisition Time 15:28:40Sample Name: F8Sample Weight: 7.310[mg]Annotation:


Fig.21: DSC of Drug loaded liposphere (F8)



0.00 2.00 4.00 6.00 8.00

Time [min]

-10.00

-5.00

0.00

mW

DSC

100.00

200.00

C

Temp

75.80 x100COnset

98.04 x100CEndset

71.47 x100CStart

102.07 x100CEnd

89.07 x100CPeak

-316.77 x100mJ

-41.03 x100J/g

Heat

-9.92 x100mWHeight

File Name: F6 NEW.tadDetector: DSC60Acquisition Date 13/03/01Acquisition Time 15:43:15Sample Name: F6 NEWSample Weight: 7.720[mg]Annotation:


Fig.22: DSC of Drug loaded liposphere (F6)

8.4.3. Scanning electron microscopy

Figure 23, 24 illustrates the SEM of optimized F8 batch of prepared

aceclofenac lipospehers. The particles are spherical in shape with irregular

surface as usually obtained when egg phosphatidylcholine is used as the

coat.

Fig.23: Scanning photomicrographs of aceclofenac lipospehers with carnauba

wax: under high magnification (1000X)



Fig.24: Scanning photomicrographs of aceclofenac lipospehers with

Carnauba wax: under low magnification (350X)

The SEM photomicrographs in figure (23 and 24) showed that the

lipid lipospheres of batch have a spherical morphology. In SEM

photomicrographs, the lipid lipospheres were observed at different

magnification value i.e. 350 X, 1000 X which showed surface texture of

lipospehers. Surface texture of the lipospheres is rough because of presence

of crystals of drug on the surface of lipid lipospheres.The lipospheres were

found to be spherical, rounder, free flowing and of the monolithic matrix

type. The SEM photomicrographs of drug-loaded lipospheres showed that

the lipospheres were almost spherical in shape with rough and nonporous

surface and it also indicated that the drug was dispersed at amorphous state

in the lipid matrices.



8.5.4. In vitro release of aceclofenac from lipospehers (F1-F4)

Table 14: Percentage drug release from various formulations of Liposphers

S.No. Cumulative percent drug release (%)

Time

(min)

F1 F2 F3 F4

1. 0 0 0 0 0

2. 30 4.087 14.86 23.23 38.53

3. 60 8.974 21.31 40.54 46.85

4. 120 18.72 38.01 45.59 50.58

5. 180 20.01 52.23 49.22 57.95

6. 240 24.96 66.36 57.53 61.92

7. 300 26.43 80.24 62.41 65.80

8. 360 37.48 94.20 67.39 72

9. 420 47.27 -- 77.46 75.11

10. 480 59.88 -- 79.23 79.48

11. 540 68.13 -- 82.00 82.16

12. 600 75.31 -- 92.52 83.81

(Mean, n=3)

Fig.25: in vitro drug release of formulation batches F1, F2, F3, F4

0

10

20

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700

Cu

mu

lati

ve %

dru

g R

ele

ase

Time (min)

F1

F2

F3

F4




Table 15: Percentage drug release from various formulations of liposphers


Time

(min)

F5 F6 F7 F8

1. 0 0 0 0 0

2. 30 29.96 25.57 16.94 18.58

3. 60 33.82 27.60 32.76 29.96

4. 120 38.75 31.02 38.02 38.35

5. 180 42.78 32.76 47.27 52.14

6. 240 43.94 37.69 55.34 66.36

7. 300 51.33 48.96 66.36 75.28

8. 360 67.47 49.96 75.36 83.72

9. 420 69.23 56.28 83.81 91.68

10. 480 72.33 58.97 91.68 92.52

11. 540 80.57 69.05 -- --

12. 600 82.88 81.11 -- --

(Mean, n=3)

Fig.26: in vitro drug release of formulation batches F5, F6, F7, F8

0

10

20

30

40

50

60

70

80

90

100

0 100 200 300 400 500 600 700

Cu

mu

lati

ve %

dru

g R

ele

ase

Time (min)

F5

F6

F7

F8




Table 16: Percentage drug release from various formulations of liposphers


Time

(min)

F9 F10 F11 F12

1. 0 0 0 0 0

2. 30 8.52 12.72 13.47 17.16

3. 60 16.92 17.1 16.92 25.41

4. 120 18.88 27.93 25.33 31.10

5. 180 27.29 33.81 42.22 36.94

6. 240 37.73 43.91 43.94 40.54

7. 300 46.76 50.63 51.33 42.23

8. 360 55.00 59.13 67.47 50.64

9. 420 66.04 67.38 69.23 59.04

10. 480 75.28 68.22 72.33 62.32

11. 540 80.07 79.23 83.01 73.63

12. 600 83.64 82.60 83.90 83.89

(Mean, n=3)

Fig. 27: in vitro drug release of formulation batches F9, F10, F11, and F13

8.6. Drug release kinetics

The cumulative percent drug release curve of the drug loaded lipospehers. it

is obvious that with all lipospheres prepared using egg phosphatidylcholine,

the percentages of drug released after 8 h (T8hr) were overall significantly

0

10

20

30

40

50

60

70

80

90

0 100 200 300 400 500 600 700

F9

F10

F11

F12



lower than with those prepared with soybean phosphatidylcholine under the

same conditions. In addition, soybean phosphatidylcholine membranes are

known to be more fluid than egg phosphatidylcholine membranes resulting

in faster drug release. The highest T 8h value was obtained with carnauba

wax, formulation F8 (92.52 %) and F7 (91.68 %) formulation, with stearyl

alcohol. The lowest T 8h value was obtained with Bees wax, formulation F6

(58.97 %). The optimized batchs F8 and F7.

Drug release kinetics for formulations F1-F12 was shown in table (18)

shows First Order, Higuchi's and Peppas Korsmeyer's plot respectively.

Table 17: drug release kinetics for the various formulations of lipospheres

Formulation

Code

R2

n Zero

order

equation

First

order

equation

Higuchi’s

equation

Korsmeyer

Peppas

equation

F1 0.9852 0.9321 0.9557 0.9866 0.9364

F2 0.9929 0.8748 0.9571 0.9970 0.7696

F3 0.7947 0.9682 0.9389 0.9787 0.4105

F4 0.4777 0.9515 0.8751 0.9892 0.2745

F5 0.8383 0.9695 0.9591 0.9464 0.3677

F6 0.8855 0.9303 0.9425 0.9287 0.3847

F7 0.9396 0.9712 0.9891 0.9881 0.5719

F8 0.9440 0.9578 0.9925 0.9960 0.6054

F9 0.9934 0.9641 0.9855 0.9874 0.7842

F10 0.9743 0.9790 0.9935 0.9964 0.6625

F11 0.9673 0.9783 0.9917 0.9908 0.6732

F12 0.9364 0.9329 0.9592 0.9771 0.4947



Result: The correlation coefficient (R2) values showed that formulations

follow Korsmeyer peppas model for drug release.

8.6.1. Zero order, First order, Higuchi’s equation,and Korsmeyer

Peppas equation plot of drug release of Lipospheres F7, F8 (a, b)

Optimized batch

a) Fig.28: F7

b) Fig.29: F8

0

20

40

60

80

100

120

0 100 200 300 400 500 600

% D

rug

Re

lea

sed

Time

Release Profile

Zero

1st

Matrix

Peppas

Hix.Crow.

0

20

40

60

80

100

120

140

0 100 200 300 400 500 600

% D

rug

Re

lea

sed

Time

Release Profile

Zero

1st

Matrix

Peppas

Hix.Crow.



The drug release kinetics showed in (Table 17), where majority of the

batches governed by peppas model. All the batches showed release up to 8 h

and above 40-90% of drug released with each formulation. Formulation F8

(92.52%) showed maximum release while other formulation showed less

amount of drug release in 8 h. Formulation of F7, F8 and F11 has highest

correlation coefficient (R2=0.9970, 0.9960, 0.9908) respectively and follows

drug release by peppas model. The drug release from lipospheres depends on

many factors including the composition of lipospheres, the type of drug

encapsulated and nature of the cell. Once released, drug that normally

crosses the membrane of a cell will enter the cell. The drug is released from

lipospheres by one of the possible mechanism i.e., Endocytosis, fusion and

adsorption. Orally administered lipospheres release the drug by endocytosis

as gut epithelial cells take up intact lipospheres by absorptive endocytosis.

8.7. Formulations of lipospheres based gel

Lipospheres based gel was prepared according to the formula (Table 8).

8.8. Characterization of lipospheres based topical gel

Table 18: Result of pH, Viscosity, Drug content and Spreadability

S.

No.

Formulation pH Viscosity

(Cps)

Drug

content

(%)

Time(sec) Spredability

(g.cm/sec)

1 F7 6.5 29600 83.33 20 47.27

2 F8 6.9 30200 96.51 20 37.14

3 LF7 6.9 29200 100.317 20 40

4 LF8 7.0 31000 115.73 20 34.66



Fig.30: Lipospheres based gel of optimized batch

All the Lipospheres based topical gel formulations were having good feel

and showed no clogging and lumps which indicate good texture of system.

pH of lipospshere based topical gel was around the neutral pH and in the

range of 6.5-7.0.

All the formulations showed no significant skin irritation on intact skin (Fig

32).Thus, indicating skin acceptability of these formulations for topical

application.

Viscosity is an important parameter for characterizing the gels as it affects

the spreadibility, and release of the drug. Viscosity of formulations was

ranging between 29000-32000 cps.

Easy spreadability is one of the important characteristics of any topical

preparation as far as patient compliance is concerned. Liposphere based gel

is considered to be good if it takes minimum time to spread on the surface.

Among the various gels studied LF8 aceclofenac liposphere gel was find to

show better spreadability. The values of spreadability indicated that the gel

is easily spreadable by small amount of shear. Drug content uniformity of all

formulations were observed and F7, F8 without permeation enhancer and

LF7,LF8 with permeation enhancer batch showed the 83.33 %, 96.51 %,

100.317 % and 115.73 % drug content respectively. Finally selected

formulations were subjected to a stability testing for three months and drug

content of batch F7, F8, LF7 and LF8 was found to be 83.33 %, 96.51 %,

100.317 % and 115.73 % respectively. Depending upon different evaluation



parameters made on all formulations, batch LF8 was declared as an

optimized batch.

8.8.1. In vitro permeation of liposphere based gel

The release profile of a drug predicts how a delivery system might function

and gives valuable insight into its in vivo behavior. The optimized (F8 and

LF8) were subjected to in vitro release studies. These in vitro release studies

were carried out using simulated tear fluid (STF) of pH 7.4 as the dissolution

medium. The drug release data obtained for formulations shows the

cumulative percent drug released. It was found that cumulative percent drug

release was 82.42 %, 91.68 % for F8 and LF8 formulation respectively.

Table 19: Result of cumulative percent (%) drug release of optimize batch

(F8, LF8)

S.NO. Cumulative percent (%) drug release

Time (hr) Batch F8 Batch LF8

1 0 0 0

2 1 25.33 24.84

3 2 33.74 35.03

4 3 43.85 47.32

5 4 55.69 58.49

6 5 64.12 66.02

7 6 71.12 75.40

8 7 79.32 79.23

9 8 82.42 91.68



Fig.31: in vitro release profile of optimized formulation F8 and LF8

8.8.2. Skin irritation testing (Draize patch test)

The results of the skin irritation study revealed that following 72 h

application of LF7, LF8 and marketed preparation (AUDIGEL) and blank

gel, there was no reaction found on the skin. Therefore, it can be assured that

the gel formulation can be used for topical application.

Fig.32: The skin irritation results of lipospheres based topical gel to skin of

four rabbits (number A,B,C,D) after administration of 72 h (1) No application

(2) Marketed aceclofenac gel (AUDIGEL) (3) LS based topical gel (LF7) (4)

LS based topical gel (LF8)

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10

cum

ula

tive

Pe

rce

nt

dru

g re

leas

e

time (hr)

F8

LF8



8.8.3. In vivo anti-inflammatory study of lipospheres

Table 20: Result of Percentage inhibitions for anti-inflammatory activity

Edema degree (ml) at different movement (h)

Group

0

1

2

3

4

Control

0.7±00.12 1.28±0.48 1.32±0.043 1.33±.09 1.46±0.266

Marketed

0.77±0.22

0.9±0.40 0.91±0.41 0.86±0.4 0.84±0.46

LF7 0.89±0.24 0.98±0.36 1.08±0.37 1.12±0.3 1.06±0.40

LF8 0.65±0.077 0.82±0.075 0.84±0.077 1.02±0.19 0.72±0.36

Edema degree (ml) at different movement (h)

Group

5

6

7

8

% Inhibition after

8 hr

Control 0.7±00.12

1.28±0.48 1.32±0.043 1.33±.09 ----

Marketed 0.77±0.22

0.9±0.40 0.91±0.41 0.86±0.4 65%

LF7 (III) 0.89±0.24 0.98±0.36 1.08±0.37 1.12±0.3 81%

LF8 (IV) 0.65±0.077 0.82±0.075 0.84±0.077 1.02±0.19 96.78%

Data were expressed as mean±S.D and statistically assessed by one way

analysis of variance (ANOVA). Values for edema rate percentage for

lipospheres were compared to the saline control and the differences were

determined statistically using Dunnett’s t test. P<0.05 was considered

significant.



The anti-inflammatory activity of the optimized formulation was evaluated

by the carrageenan-induced hind paw inflammation method on wistar rats.

The percentage inhibition value of LF7 and LF8 was compared to marketed

gel. Both of the formulations LF7 and LF8 not only decreased the

inflammation by a larger magnitude, but also sustained the effect for a

prolonged period. After 8 h, the percent edema for LF7 and LF8 was found

to be 81 % and 96.78 %, respectively (as shown in Table 20). While in case

of marketed gel, percentage edema inhibition were found to be 65 %. Hence

the lipospheres based gel formulation of aceclofenac remained superior to

the marketed product in its ability to suppress edema and sustained the anti-

inflammatory activity.

8.9. Stability studies

All the selected formulations were subjected to a stability testing for

three months as per ICH norms at a temperature of 40 ºC ± 2 ºC / 75% ±

5% R H. All selected formulations (LF7 and LF8) were analyzed for the

change in appearance, pH and drug content by procedure stated earlier.

(Table: 21 stability studies)

Table 21: Result of stability study of optimized batch

S No. Month Appearance pH Drug content (%)

Batches Months Appearance pH Drug

content

(%)

1 LF7 Initial white 6.9 100.317

1 white 6.8 100.2

2 white 6.9 99.5

3 white 7.0 99.30

2 LF8 Initial white 7.0 115.73

1 white 6.9 105.21

2 white 6.8 106.2

3 white 7.0 102.1

Chapter No.10 Future scope


10. Future scope

we can Increase drug loading efficiency

We can improve the bioavailability of poorly water soluble drug by lipid

drug delivery system.

Increasing physical and chemical storage stability

Minimizing overall costs

Chapter No.9 Summary and Conclusion


9. Summary

The lipospheres system is a newly introduced lipid-based carrier system

developed for parenteral and topical drug delivery of bioactive compounds.

Lipospheres consist of water-dispersible solid microparticles of particle size

between 0.2–500 μm in diameter and composed of a solid hydrophobic fat core

stabilized by one monolayer of phospholipid molecules embedded in their

surface which is a potential group of penetration enhancers. Both egg and

soybean phosphatidylcholine contain unsaturated fatty acids which may be

responsible for the penetration enhancement. The packing nature of unsaturated

fatty acids disrupts the stratum corneum lipid structure and enhances the

percutaneous penetration of drugs, also they strongly raise the fluidity of the

stratum corneum. In addition, lecithin has a high affinity for epidermal tissue

and even seems to improve skin hydration. Being biodegradable and composed

of natural body constituents, topically administered phospholipids can be

generally considered as safe. Lipospheres like solid lipid nanoparticles are one

of the carriers of choice for topically applied drugs because their lipid

components have an approved status or excipients used in commercially

available topical cosmetic or pharmaceutical preparations. The small size of the

lipid particles ensures close contact to the stratum corneum and can increase the

amount of the drug penetrating into the mucosa or skin. Due to their solid lipid

matrix, controlled release from these carriers is possible which is important to

supply the drug over a prolonged period of time and to reduce systemic

absorption, increased drug stability can be achieved and finally lipospheres

possess a film forming ability leading to occlusive properties.

The purpose of this study was to prepare lipospheres containing

aceclofenac intended for topical skin delivery with the aim of exploiting the

favorable properties of this carrier system and developing a sustained release

formula to overcome the side effects resulting from aceclofenac oral



administration. Lipospheres were prepared using different lipid cores (carnauba

wax, bees wax, steryl alcohol) and phospholipids coats (egg phosphatidylchoine

and soya phosphatidylcholine) by melt dispersion technique. Characterization

of the prepared lipospheres formulation carried out through photomicroscopy,

scanning electron microscopy (SEM), particle size analysis, diffential scanning

caorimetry (DSC), and in vitro drug release and stability study. It was

uniformly dispersed after suitably gelled by Carbopol 940 preparation. The

characterization of the prepared lipospheres based topical gel rheological study,

pH, Spreadability, drug content, skin irritation test. No oedema and erythema

were observed after administration of lipospheres based aceclofenac gel on

rabbit skin, the anti-inflammatory effect of liposphere systems was assessed by

the rat paw edema technique and compared to the marketed product. Results

revealed that liposphere systems were able to entrap aceclofenac at very high

levels (101.65 %). The particle size of liposphere systems was well suited for

topical drug delivery. DSC revealed the molecular dispersion of aceclofenac

when incorporated in lipospheres. Lipospheres were very stable after 3 months

storage at 2–8 °C. Liposphere topical gel was found to possess superior anti-

inflammatory activity compared to the marketed product.



Conclusion

This present work indicates that the Lipospheres based aceclofenac gel could be

successfully prepared by the melt dispersion technique. The melt dispersion

method produced smaller particles. This study also indicates that the amount of

lipids and lecithin significantly affects the particle size as well as entrapment

efficiency. It can be concluded that the optimized lipospheres gel exhibit faster

onset and prolonged action as compared to the marketed product. Further, in

vivo anti-inflammatory and skin irritation studies are necessary to assess the

improvement of therapeutic efficacy of the LS gel compared to the marketed

product. Findings of this investigation suggest that lipospheres can be

considered a promising delivery system for topical aceclofenac delivery.

Lipospheres were able to entrap the drug at very high levels and sustained its

release over a prolonged time. Lipospheres possessed a suitable size for topical

route and being based on non irritating and non toxic lipids, lipospheres seemed

to be well suited for use on damaged or inflamed skin. Furthermore, lipospheres

possessed a very high stability as well as superior anti-inflammatory activity

compared to the marketed product.

Chapter 12 Errata


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Chapter No.11 References


11. References

1. Domb, A.J. et al., Degradable polymers for site-specific drug delivery,

Polymer Adv. Technol, (1992):3, 279.

2. Barke, S.A. and Khossravi, D., Drug delivery strategies for the new

millennium, Drug Del. Technol, (2001):6, 75.

3. Mizushima, Y., Lipid microspheres (lipid emulsions) as a drug carrier: an

overview, Adv. Drug Del. Rev, (1996):20, 113.

4. Ravi Kumar, M.N.V., Nano and microparticles as controlled drug delivery

devices, J. Pharm. Pharmaceut. Sci, (2000):3, 234.

5. Gombotz, W. et al., Prolonged release of GM-CSF, United States Patent,

(1995):5, 253.

6. Wake, M.C. et al., Effects of biodegradable polymer particles on rat marrow-

derived stromal osteoblasts in vitro, Biomaterials, (1998):19, 1255.

7. Cortesi, R. et al., Preparation of liposomes by reverse-phase evaporation

using alternative organic solvents, J. Microencapsul,( 1999):16, 251.

8. Vyas, S.P., Singh, R. and Dimitrijevic, D., Development and characterization

of nifedipine lipospheres, Pharmazie, (1997):52, 403.

9. Jenning, V., Thünemann, A.F. and Gohla, S.H., Characterization of a novel

solid lipid nanoparticle carrier system based on binary mixtures of liquid and

solid lipids, Int. J. Pharm,(2000):199, 167.

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preparation and evaluation of liposphere based topical drug delivery system containing nsaid drug

Documents

industrial pharmacy

institute of pharmacy

nsaid drug thesis

evaluation of liposphere

thesis work

topical drug delivery

leeladhar prajapati

degree of master