Development, Optimization and Characterization of

Mupirocin ethosomes gel for the treatment of skin infection

A Synopsis Report submitted

In Partial Fulfilment of the Requirements for the Degree of

MASTER OF PHARMACY
in
PHARMACEUTICS
By

POOJA SHARMA
(ROLLNO.1901280566004)
Under the supervision of
ANKIT KUMAR
(ASSISTANT PROFESSOR)

DR. APJ ABDUL KALAM TECHNICAL UNIVERSITY

(Formerly Uttar Pradesh Technical University) LUCKNOW
UTTAR PRADESH, INDIA

1
Dr. SACHIN TYAGI
Director ,
School of pharmacy,
Bharat Institute of Technology
Meerut-250001
CERTIFICATE BY THE DIRECTOR OF INSTITUTION

This is to certify that this synopsis work entitled “Development, Optimization and Characterization of
Mupirocin ethosomes gel for the treatment of skin infection”submitted in partial fulfilment of the
requirement for the Degree of Master of pharmacy of Dr.A.P.J. Abdul kalam Technical
University(APJAKTU) Uttar Pradesh, India done at School of pharmacy, Bharat institute of
technology, Meerut-250001, a bonafied record of work carried out by POOJA SHARMA, under the
guidance of SACHIN TYAGI during the academic session 2019-2021. The thesis embodies result of
original work of studies carried out by student himself and the contents of the thesis do not form the
basis for the award of any other degree to the candidate or to anybody else.

Date: DR. SACHIN TYAGI

Director
Place: Meerut

2
ANKIT KUMAR
Assistant Professor
School of pharmacy,
Bharat Institute of Technology
Meerut-250001
CERTIFICATE

This is to certify that this synopsis work entitled ““Development, Optimization and Characterization
of Mupirocin ethosomes gel for the treatment of skin infection” submitted in partial fulfilment of the
requirement for the Degree of Master of pharmacy of Dr.A.P.J. Abdul kalam Technical
University(APJAKTU) Uttar Pradesh, India done at School of pharmacy, Bharat institute of
technology, Meerut-250001, a bonafied record of work carried out by POOJA SHARMA, under the
guidance of ANKIT KUMAR during the academic session 2019-2021. The thesis embodies result of
original work of studies carried out by student himself and the contents of the thesis do not form the
basis for the award of any other degree to the candidate or to anybody else.

Date: Ankit kumar

Assistant professor
Place : MEERUT

3
SURYKANT KUMAR VERMA
Assistant Professor
School of pharmacy,
Bharat Institute of Technology
Meerut-250001

CERTIFICATE

This is to certify that this synopsis work entitled ““Development, Optimization and Characterization
of Mupirocin ethosomes gel for the treatment of skin infection” submitted in partial fulfilment of the
requirement for the Degree of Master of pharmacy of Dr.A.P.J. Abdul kalam Technical
University(APJAKTU) Uttar Pradesh, India done at School of pharmacy, Bharat institute of
technology, Meerut-250001, a bonafied record of work carried out by POOJA SHARMA, under the
guidance of SURYAKANT VERMA during the academic session 2019-2021. The thesis embodies
result of original work of studies carried out by student himself and the contents of the thesis do not
form the basis for the award of any other degree to the candidate or to anybody else.

Date: Suryakant Verma

Assistant professor
Place : MEERUT

4
 ABSTRACT

Over the previous decades, the production of controlled drug delivery has become important in the
pharmaceutical era. The pharmacological action depends on the concentration of drug administered at
the action site in body which depends on the dosage forms and the rate extent of absorption of the
drug at the site of action. Physiochemical parameters such determination of solubility, melting point,
partition coefficient, drug-excipient interaction, λmax scan using UV-spectrophotometry, FT-IR
spectrophotometry were performed in this study.xThe regression coefficientx(R2) was 0.999xwhich
wasxshows the linearityxof curve. Thexline of equationxfor the standardxcurve was yx= 0.041x
+x0.001. The drugxexcipient interaction study wasxperformed toxcheck in interactionxbetween drug
andxother formulationxexcipients by spectrophotometrically. Therexwas noxinteraction was
foundxbetween drug andxexcipients and it wasxclearly seen and confirmedxby UV
spectrophotometricallyxscan graph ofxdrug solution andxmixture of drugxand excipients inxfig 6.3
andx6.4. There wasxno fluctuation inxwavelength of mupirocinxcalcium.Preformulation of drug and
excipient was performed in which physiochemical properties and other parameters of drug were
studied. Physiochemical parameters such determination of solubility, melting point, partition
coefficient, drug-excipient interaction, λmax scan using UV-spectrophotometry, FT-IR
spectrophotometry were performed in this study.

5
 TABLE OF CONTENTS

SR.NO TOPIC PAGE NO

1 INTRODUCTION 1-9

2 LITERATURE REVIEW 12-18

3 AIM AND OBJECTIVE

4 PLAN OF WORK 20

5 RESULT & DISCUSSION 22-45

6 REFERENCE 48-51

List of Tables
6
Table 1.1: Different additives employed in formulation of ethosomes
Table 1.1: Different additives employed in formulation of ethosomes
Table 6.1 List of Sensory characters
Table 6.2 Solubility of Mupirocin calcium
Table 6.3x Melting point ofxthe Mupirocin calcium
Table 6.4 pH of the Mupirocin calcium
Table 6.5 Partition coefficient of Mupirocin calcium
Table 6.6 Determination of moisture content
Table 6.7: Calibration curve of Mupirocin calcium
Table 7.1: Different composition of ethosomes formulation
Table 7.2: Result for Vesicle size and Entrapment efficiency of drug loaded Ethosomes
Tablex7.3 Vesicle sizexand Entrapment efficiencyxof Optimized Ethosomes
Table 7.4: Results of evaluation of gelxformulation
Cumulative % drugxrelease of Mupirocinxcalcium fromoptimized ethosomes
Table 7.5:
formulation
Cumulative % drugxrelease of Mupirocin calcium from optimized ethosomes
Table 7.5:
formulation
Table 7.6: In-Vitro drug release data for F2
Table 7.7: Regression analysis data of ethosomal gel formulation

7
List of Figures

Simplified diagramxof skin structurexand macro routesxof drug penetrationx(1) via

Figurex1.1: thexsweat ducts; (2)xacross the continuousxstratum corneum orx(3) through the hair
follicles with their associated sebaceous glands
Figure 1.2: The skin
Figure 6.1: FT-IR Spectrum of Mupirocin calcium (Standard)
Figure 6.2: FT-IR Spectrum of Mupirocin calcium (Sample)
Figure 6.3: λmax scan graph of Mupirocin calcium in phosphate buffer pH 7.4
Figure 6.4: Calibration curve of Mupirocin calcium in phosphate buffer pH 7.4 at 227nm
Figurex6.5: UV spectraxof Mupirocin calcium
Figurex6.6: UV spectraxof mixture of Mupirocin calcium and Excipients
Figure 7.2: Vesicle size andxEntrapment efficiency ofxdrug loaded ethosomes
Figure 7.3: Vesicle size of optimized formulation
Figure 7.4: Entrapment efficiency of Optimized formulation
Figure7.5: Cumulative Percent Drug Released Vs Time (Zero Order Plots)
Figure7.6: Log Cumulative Percent Drug Remaining Vs Time (First Order Plots)

8
 Chapter -1

Introduction

9
 1. INTRODUCTION

Transdermal Drug Delivery

The techniques which used for a drugs delivere to site of action can significantaly
effect on its efficacy. Appropriate drug selection with effective drug delivery can produce
optimum drug therapeutic outcomes. Over the previous decades, formulation of controlled
drug delivery has become essential in the pharmaceutical era. The pharmacological action
is depending on the drug concentration administered at the action site in body which
depends on the dosage forms and the rate extent of absorption of the drug at the site of
action1. Skin is a most eaisly available organs of the body of human. Adult body occupies a

acquire of approximately 2 of skin and have aproximately 33% of the blood circulation
in the body. Epidermis, uppermost layer of skin has morphologically particular regions as
basal layer, spiked layer, stratum granulosum or stratum corneum, which consist of lipidic
membranous sheets. These membraneous sheets have unique compositions which are made
out of cholestrol, ceramides, and free fatty acids2. The surface of human skin contains 200-
250 sweat ducts and 10-70 hair follicles on each centimeters square area of body skin. The
human skin has been recognized as having the capability of using it as the port of drug
administration But the body skin is a very complex barrier to entry all types of materials
because skin allows just little amounts of a drug to penetrate over a time periods. Definition
of the Transdermal delivery system is the delivery of drugs across the skin into Blood
circulation.
Transdermal delivery system has benefit of the relative approachability of the skin 3.
Transdermal drug delivery has been use in cosmetics and in pharmaceutical preparations
in the forms of topical formulations which provide a faster release of drug in to systemic
circulation. Gels are generaly used in transdermal formulation for topical use which have
several meritorious properties e.g- thixotropy, easily removable, greaseless, readily
spreadable, emollient, non-coloring and compatible with numerous additives and water
soluble/miscible. Transdermal drug delivery system have many advantages as compare to
conventional drug delivery like evasion of first pass metabolism, predictable and
extended duration of drug action, eliminate of side effects, increase in application of

1
short half-life drugs, reforming pharmacological and physiological action avoiding the
fluctuation in drug levels.

Ethosomes has permeation efficiancy of drug molecules (relatively small vesicle size) by
the skin to the blood circulation. Ethosomes have better stability, solubility and patient
compliance over other vesicular system but have poor yield. In ethosomes, ethanol
interacts with lipid atoms in the polar head region which results in decreaing the rigidity
of the stratum corneum fluids and leads to increase in fluidity. The ethosomes itself can
combine with the stratum corneum barrier. Various procedures have been targeted to
weaken and disrupt the highly organized intercellular lipids which may improve drug
transportation which may increase the penetration of drugs into the skin barrier. Ethosomes
are basically used for drug delivery by transdermal route. this drug delivery is the better
significant routes of drug administration but permeation of drugs into the skin is the major
factor which limits the use of transdermal drug delivery. Transdermal drug delivery may be
benefited for those lipophilic drugs which have molecular weight less than 500dalton
because human skin has selective permeability for drugs. Hence to conqure the stratum
corneum barrier, different mechanisms have been researched, as hep ofxchemical or
physical properties enhancers, such as sonophoresis, iontophoresis, etc.

Liposomes, niosomes, transferosomes and ethosomes in novel drug delivery system

further have the capability of defeating the skin barrier and have been expressed to
increase permeability of drug through the stratum corneum barrier. So the research on
ethosomes was designed to enhance the delivery of drugs into the deep parts of the skin
and through the skin. Depends on the formulation, delivery can be targeted for local
delivery or for systemic use. Incorporation of drug in to the ethosomes can be proved as a
potential formulation for cross the drug barrier so the research has been done on ethosomes.

For the administration of drug via skin there are several route of penetration as human skin
directly comes into contact with microorganisms, sebum, cellular debris, and other
compounds, which directly/indirectly influence the permeation of vesicles. The drug can be
penetrates by three routes to the applicable tissue via hair follicles which are associated
with sebaceous glands, through sweat ducts, and from continuous stratum corneum

1
between these appendix. These mentioned routes are significant for ions and large polar
atoms that effort to cross intact stratum corneum.

Figure 1.1: Simplified diagram of skin structure and macro routes of drug penetration
(1) via the sweat ducts; (2) across the continuous stratum corneum or (3) through the hair
follicles with their associated sebaceous glands

THE SKIN
The surface of the skin is entirely covered by the skin. It is one of the most extensive an
eaisly available organ of the human body. Adult body occupies a acquire of approximately

2 of skin The skin functions as a protective barrier that interface with the sometimes -
hostile environment.

It is also involved in maintaining the important temperature for the body to

function well, to eliminate wastes and also protect tissues from infection. Understanding of
how the skin function in these many ways starts with understanding the structure of the
3 layers of the skin -the epidermis, dermis and subcutaneous tissue.(fig.1.2.

1
 Figure 1.2: The skin

Selection of the Drug for Transdermal Drug Delivery System

Before the development ofxthe transdermal delivery system of any drug various
physiochemical properties, pharmacokinetics and pharmacodynamic properties are taken
under consideration. Typical requirement for transdermal delivery of drug includes.

 Low molecular weight ranging for 500-1000.

 Low melting characters (150-200º).
 Aqueous solution neither too acidic nor basic (between 5 and 9 pH units).
 Preferable liquid/water co-efficient i.e. partition co-efficient.
 The very important requirement of the drug to be administered transdermally is
demonstrated by need for controlled delivery, such as short half-life and adverse
effects included with other routes or complex oral route or IV dose regimen.
 Drug which gets extensively metabolized in the hepatic, first pass effect.

1
 ETHOSOMES

Ethosomes are referred as ethanolic liposomes. These are noninvasive drug delivery
carriers which allow the drugs to deliver deeply into the skin layers. These are soft, flexible
vesicles used to increase the delivery of drug. Ethosomal vesicles allows to take control
the release rate ofxdrug over an enhanced time period, keeping the drug protected from
immune response thus capable to release just real quantity of drug and keep that
concentration constant for longer time periods. Ethosomes are having varous advantages
as it avoid the 'first-pass' metabolism of drugs.Reduced plasma concentration levels of
drugs, with decreased side effects, Reduction of fluctuations in plasma levels of drugs.
utilization of drug with short half-life and low therapeutic index, easly elimination of
drug delivery in case of drug poisonous and reduction ofxdosing frequency which
increases the patient compliance.3 with many advantages ethosomes are having many
disadvantages also as drugs with heavy drugs molecules (>500 Da) are generally
difficult to penetrate in the stratum cornea, very low/ high partition coefficient ofxdrug
fail to arrive in systemic circulation, Highly melting drugs can be administered by topical
routes due to low solubility both in water and fat1. Many novel approaches has been
attempted to deliver drugs across skin barrier and enhance the efficacy.

Optimization of Transdermal Drug Delivery

Drug administration through Transdermal route offers a few potential influences

over conventional routes like evasion of first pass metabolism, predictable and extended
time periods of activity, Eleminating under capable drug reactions, usage of short half-
life drugs, improving physiological and pharmacological action, avoiding the rise and fall
in drug levels, inter and intra patient valuations, and most importantly, it provides patient
complines. But one of the serious issues in transdermal drug delivery is the low
penetration rate through the outer most layer of skin5.

The non- invasive approaches for giving rout of transdermal drug delivery of many
therapeutic substances are:

(1) Drug and vehicle interactions

1
(a) choice of right drug or prodrug

(b) Chemical potential chages

(c) Ion pairs and complex coacervates

(d) Eutectic systems

(2) Stratum corneum modification

(a) Hydration

(b) Chemical penetration enhancers

(3) Stratum corneum bypassed or removed

(a) Microneedle array

(b) Stratum corneum ablated

(c) Follicular delivery

(4) Electrically assisted methods

(a) Ultrasound (Phonophoresis, Sonophoresis)

(b) Iontophoresis

(c) Electroporation

(d) Magnetophoresis

(e) Photomechanical wave

(5) Vesicles and particles

(a) Liposomes and other vesicles

(b) Niosomes

(c) Transfersomes

1
Ethosomes as Novel Vesicle Carriers

Ethosomes are very soft, maliable vesicles which are madeup of mainly water,
phospholipids and ethanol. In deep skin strata and systemic cerculation ethosomes can
eaisly deliver to drug or penetrate to drug. The soft, flexible vesicles then permiate
disorganized lipid bilayers. These “maliable vesicles” describe recent vesicular carriers for
increase delivery to/through skin6-7.The vesicle has been notable for their significance in
cellular liaison and molecule transportation for many years.

scientists have understood the properties of vesicles structure for use in beter drug
delivery at site of action within their pits. As oppose to liposomes, ethosomes have been
appeared to display high encapsulation efficiency for a wide limitation of molecules
including lipophilic drugs, and are specific at delivering molecules to and via skin as
discussed8.Review literature shows a hopeful future of ethosomes in formulating
transdermal delivery more effective. Ethosomes also offer a better chance for the non-
invasive delivery of little, medium, and large sized drug molecules. For the formation of
ethosomes no complicated equipments are required. These systems were seen to be highly
old carriers for the delivery of drug with different lipids into and via skin, in in-vivo and in-
vitro, in animal and clinical studies9. There are several benefits of ethosomes drug delivery as
ethosomes enhance penetration of drug by skin, ethosomes can be utilized for the distribution of
drugs across the skin as peptides, protein molecules, increases patient attractiveness for products.
The ethosomal system is passive, non-intrusive and is easily available for fast commercialization2.

Table 1.1: Different additives employed in formulation of ethosomes10

Class Example Uses

Soya phosphatidyl
Soya phosphatidyl choline
choline, Egg phosphatidyl
Phospholipid Egg phosphatidyl choline
choline Dipalmityl
Dipalmityl phosphatidyl choline
phosphatidyl
Choline
Polyglycol Polyglycol As a enhancer

1
 For providing softness for
Ethanol
Alcohol Vesicle membrane As a peemeation
Isopropyl alcohol
enhancer

1
 For providing the stability to
Cholesterol Cholesterol
vesicle membrane
Rhodamine-123 Rhodamine
red Fluorescene
Dye For characterization study
Isothiocynate (FITC) 6-
Carboxy luorescence
Vehicle Carbopol 934, HPMC As a gel former

Table 1.2: Ethosomes as a carrier of v arious drug molecules has

been listed below
Drug Use Comments
Improved dermal
Ammonium deposition Exhibiting
Anti-inflammatory
glycyrrhizinate sustained release Improved
biological anti-
inflammatory activity
Azelaic acid Azelaic acid Prolong drug release
Improved drug delivery Increase
skin permeation Improved in
Acyclovir Acyclovir
biological activity Improved in
Pharmacodynamic profile.

Treatment of Reduced drug toxicity Improved

Bacitracin dermal Deposition
dermal infection Improved intracellular
delivery
Prevents inflammation Significant accumulation of the
Cannabidol drug in the skin Improved
and edema

1
 PLAN OF WORK

1. Literature Review
2. Selection ofxdosage form
3. Selection ofxdrug candidate
4. Procurement of drug & excipients
5. Identification & characterization of API
 UV estimation
 IR Study
6. Preformulation studies
 Physical Appearance
 Melting Point
 Determination of Absorption Maxima (max)
 Solubility study
 Partition coefficient
 Drug –excipients compatibility study
 Calibration curve of drug
7. Formulation development of ethosomes
8. Evaluation of ethosomes
 Measurement of pH
 Determination of Viscosity
 Spreadability
 Drug Content
 In vitro drug release of drug
9. Stability studies
10. Result and discussion
11. Summary and Conclusion
12. Compilation and Submission of thesis

1
Chapter -2

Literature
Review

2
 3. REVIEW OF LITERATURE

Ahmed et al., (2016) developed ethosomal gel formulation of lornoxicam, stand for its
the transdermal delivery. The ethosomal formulation was maked by hot method utilizing
phospholipid and ethanol and then after evaluated for entrapment efficiency and
vesicular size, shape, in-vitro skin permiation, skin retention and drug molecule
interaction and stability.The FT-IR studies manifest no interaction among the drug,
excipients. Transmission electron microscopy affirmed the three dimensional structure of
ethosomes. The sonicated drug ethosomal formulation ET7 were elected for further skin
penetration studies as it show highest percentage of drugs entrapment (93.96%) and very
small particles size (100±3.9 nm). Formulation ofxET7 containing 2% weight by weight
phospholipid and alcohol 30% exhibited highest % ofxdrug penetration (74.18%) at the
finish of 24 hours. The ethosomal vesicle was incorporated in carbopol gel base and the
anti-inflammatory efficasy was accord with the plain lornoxicam gel. The
pharmacodynamic studies exhibited the increase anti-inflammatory action of ethosomal
gel correlated to the plain gel formulations. Stability studies bring out at two variant
temperatures, showed no any significant change in entrapment efficiency ofxvesicles at
the end of 3 months, it is indicating that all formulations were physiochemically
stable11.

Indora and Kaushik, (2015) thosomes were upgraded with changing centralization
ofxthe phospholipid and ethanol. Ethosomal plan (F8) by soya phosphatidylcholine (3%)
with likewise ethanol 20 percent was advanced. On portrayal sphericals, unilamellar
vesicles with smooth surfaces were seen by transmission electron microscopy in the
scope ofx5 to 200 nm. Zeta capability of The F8 plan was resolved to be - 4.16 mv.
Medication capture effectiveness ofxthe F8 detailing was resolved to be 90 %.
The improve definition show the pH (8) and thickness (73,200 cps). Physical
assessment of ethosomal gel was done12.

Rathore et al., (2015) worked onfluconazole loaded ethosomes gel and liposomes gel:
an updated review for the treatment ofxdeep fungal skin infection13.
2
Vijayakumar et al., (2014) developed and portray a vesicular medication transporter
framework (ethosomes) for topical conveyance of Gliclazide to conquer the
issues related with oral course. Ethosome of Gliclazide was maked by thin film hydration
method by different composition of drug, soya lecithin, propylene glycol. Ethosomes
formulation were portrayed for the similarity, Vesicle size, level of Drug content, level of
Entrapment proficiency, Surface morphology, Surface charge, stability studies and in vitro
medication discharge. The ethosomal gels were maked for develope the ethosomal
definition number F3 by included into 1% Carbopol gels. The in vitro medications
discharge and in vivo the skin disturbance study or hypoglycemic movement were discover
for the gel F3-G. The FTIR was utilized for drugs and physical blend was described, the
consequence of IR study indicated that no cooperation among medication and polymers and
other plan parameters of defined ethosomes and ethosomal gel are assessed which
demonstrated better results14.

Sujitha et al., (2014) Developed and assessed of ethosomes containing piroxicam by

employments of phospholipid (1-3%), ethanol (20-40) percent, propylene glycol (10%)
with refined water by chilly strategy. Planned ethosomal vesicles assessed represent
vesicular size, shape, tranquilize getting effectiveness; in vitro skins dissemination,
aggravation of skin and security contemplates were discover. Checking electron
microscopy and size analyser results show that ethosomes were in circular, unilamellar,
nanometric shape and size. The plan EF5 showed most noteworthy medication
ensnarement proficiency of 73.59 percent. At that point, created definition of ethosomal
vesicles were additionally planned as gel by utilizing Carbopol15.

Preeti and Kumar, (2014) delivery of Celecoxib and to assess the impact on
centralization of Soya and Sodium deoxycholate. Transfersomal vesicles contain
Soya PC blended in with Sodium deoxycholate and Celecoxib were maked by ordinary
rotatory vanishing (Film hydration strategy) and portray for various parameters including
vesicle shape, particle size and size circulation, surface morphology, capture proficiency,
in-vitro and ex-vivo medicine discharge and in-vivo calming exercises. Vesicles were
additionally cheaked for skin disturbance and penetration considers. After effects of the
considerable number of studies decleared that Celecoxib Transfersomal gels detailing was

2
restoratively viable transdermal medication conveyance framework for fix of Rheumatoid
Arthritis16.

Abdallah, (2013) the vesicle was round fit as a fiddle as decided through Transmission
Electron Microscopy. Iin the vesicles the EE% of was in scope of 50-70 percents. The
outcome show that the Nystatin in the all of plans were effectively captured with
indistinguishable medication content. In vitro skin infiltration contemplates were discover
by stomach hare skin utilizing a disintegration dialysis mechanical assembly manufactured
in our research facility. The qantity of medication convey in the profound skin and the
amount of medication infiltration were most noteworthy if there should be an occurrence of
transfersome with an expanded proportion of 2.59, when contrasted with liposomes and the
business product17.

Dhiman and Singh (2013) the drug entrapment efficiency of developed ethosomes
formulated by use of phospholipid concentration of 2.5 gm and 30 ml ethanol heat for 12.5
minutes, were determine to be maximum i.e. 73.9 %. Lipid vesicular system of rutin
exhibited a negative zeta potential of -46.0 mV indicating a very high degree of stability for
rutin ethosomes. In vitro medication infiltration investigations of unadulterated rutin with
rutin ethosomes demonstrated that rutin ethosomes were acceptable ready to enter across
pig ear skin toward the finish of 120 minutes rather than pure rutin. So, the rutin ethosomes
were very good able to facilitate penetration of rutin deep into the pig ear skin when
compare with pure rutin18

Aute et al., (2013) Reviewed into ethosomes were noninvasive medication conveyance
transporters that ready to medications to came to in the deep skin and the fundamental
flow.Therefore, ethosomal delivery systems are conceptually complicated, they are eaisly
prepare, safe for use a combination that may be highly extand their application.
Ethosomes are very soft, flexible vesicles utilized for expanded conveyance of medication
dynamic operators. On account of their funny structure, ethosomes are empower to typify
and convey the dynamic specialists through the skin exceptionally lipophilic particles, for
example, cannabinoids, Minoxidil and testosterone, and just as cationic medication e.g-
propranolol, insulin, trihexaphenidyl, Cyclosporine and salbutamol and so forth. Upgraded
the conveyance of bioactive medication atoms by the skin or cell films by which methods

2
for an ethosomal bearers have numerous difficulties with open doors for the examination
and future advancement of novel improved therapies19.

Maheshwari et al., (2012) formulated, evaluated and looked at the transdermal potenty of
novel vesicular nanocarriers: ethosomes and ultradeformable liposomes, contain
clotrimazole (CLT), an enemy of parasitic bioactive operators. The ethosomal prepration
(ET4) and ultradeformable liposomal (UL) prepration (TT3) indicated most noteworthy
ensnarement 68.73 ± 1.4% and 55.51 ± 1.7%, ideal nanometric size range 132 ± 9.5 nm
and 121 ± 9.7 nm, and littlest polydispersity record 0.027 ± 0.011 and 0.067 ± 0.009,
continueously. The detailing ET4 gave increment the transdermal motion 56.25 ± 5.49
µg/cm2/h and diminished the slack time of 0.9 h in contrast with TT3 prepration (50.16
± 3.84 µg/cm2/h; 1.0 h). Skin connection and FT-IR examines show more
noteworthy infiltration upgrading impact of ET4 than TT3formulation. ET4
plan additionally had the most elevated zone of restraint (34.6 ± 0.57 mm), as opposed
to TT3 detailing (29.6 ± 0.57 mm) and advertised cream definition (19.0 ± 1.00 mm)
againstcandidal species20.

Prasanthi and Lakshmi, (2012) investigated efficiency of ethosomes as novel lipid

bearers for transdermal conveyance compound of Alfuzosin Hydrochloride (AH)
has been assessed. Taguchi hearty structured strategy was utilized for portrayal of
ethosomal definitions. Phospholipid type, centralization of the phospholipid and
convergence of ethanol was chosen as autonomous factors and their impact on
the reliant factors (ensnarement effectiveness and motion) was contemplated.
Ethosomal detailing (EA8) with soya phosphatidylcholine (3%) and ethanol 20% were
improved. Vesicles were round, unilamellar with smooth surface. The
enhanced definition displayed vesicle size (6.85 ± 1.35µm), zeta potential (- 8.14
± 0.62mv), capture productivity (91.86 ± 3.25%), transition (27.42 ± 0.04µg/cm2/hr),
slack time (0.26±0.20hr) and skin affidavit (298.01 ± 15.4µg/g). Transdermal
motion was expanded by 6.92 occasions over medication arrangement. Vesicles with
skin connection contemplates showed greasy change in the dermis. The ethosomes plan
were stables at 4°C for timeframe 120 days. Results recommended ethosomes as
proficient bearers for AH transdermal delivery21.

2
Alpana Ram et al., (2012) developed and evaluateed the potential employments of
transfersomes vesicles in the transdermal conveyance of Ibuprofen tranquilize. It was
checked by exemplifying the medication in various prepration of made out of various
proportions of the soya phosphatidylcholine , the range 80 or tween 80, defined by lipids
film hydration utilizing rotatary vanishing technique and assessed for atom size, shape, zeta
potential, entanglement adequacy (%EE),elasticity, dependability, and in vitro skin
tranquilize infiltration. The %EE of ibuprofen medicate in the vesicles were 47.8±2.2 and
the flexibility of both inhance by increment in surfactant focus was decide to be 34.4±1.4
and 26.5±1.6. The dependability reads for Transferosomes were discover for timespan of 5
weeks at 450C. In vitro skin tranquilize infiltration considers were discover by human skin
utilizing franz release cell, and medication discharge after time of 24 hrs and motion was
discovered 2.5824 and 1.9672 ug/cm2/hr respectively22.

Chandel et al., (2012) Ethosomal carriers are drug conveyance frameworks containing
adaptable vesicles, madeup ofxhydroalcoholic or hydro or glycolic phospholipids by
which the grouping of alcohols is moderately high. The high conc of ethanol present
improve in smoothness of lipids consequently upgrade in entrance capacity of the skin and
increment the medication infiltration. Ethosomal detailing can contain different
medications, for example, that acyclovir, fluconazole, salbutamol, Insulin, cyclosporine,
minodixil, and so on. Ethosomes are shaped by hot and cold techniques. The shape and size
of Ethosomal definition might be diminished by sonication/expulsion strategy. The high
centralization of ethanol makes the ethosomes unexplaned and helpful for trandermal
conveyance, conveyance of hormones medicate, hostile to joint inflammation, against HIV
operators and so on. These are best formulations developed23.

Sathali et al., (2010) investigate that ethosomes are very useful carrier for the
transdermal delivery of diclofenac potassium released from higher entrapment efficiency,
better stability profile and faster anti‐inflammatory efficiency. The increase distribution
ofxdiclofenac potassium by ethosomal carrier in the skin give help to develop targeting
of this drug to the epidermal and dermal sites, therefore, making new opportunities for

2
current topical application of drug like diclofenac potassium in inflammatory
conditions24.

Maurya et al., (2010) formulated transdermal conveyance arrangement of stavudine

tranquilize, a hydrophobic medication is utilized for the prescription ofxAIDS, by
ethosomes. All the medication conveyance framework were portray for
vesicle morphology, particle size and medication getting effectiveness by
Scanning Electron Microscopy , Transmission Electron Microscopy, Differential light
dissipating and centrifugation separately. The impact of different detailing variable on
human skin pervasion of medication stavudine was considered by engineered
semipermeable film/skin of recently conceived mice by utilization of dissemination cell.
The defined framework were consolidated by HPMC gel and the detailing assessed for
all medication entrance and mice skins affidavit. The create ethosomal definition
displayed transdermal motion 25.01±0.34 ìg/cm2/hr enter rodent skins as thought
about from 2.98±0.21ìg/cm2/hr for standard/plain medication arrangement, 4.28±0.54
ìg/cm2/hr for hydroethanolic arrangement and 9.7±0/21 ìg/cm2/hr for old style
liposome25.

Patel et al.,(2009) Research, The transdermal medication delivery of Curcumin for the
capability of tranfersomes plan. This formulation was figured by changed utilizing hand
shaking technique utilizing surfactant, for example, Tween 80 or Span 80 in different
fixations. The medication getting effectiveness was decide by PC (Lecithin): Edge
Activator on proportion subordinate. Most elevated capture was decide to be 89.6±0.049
inside T8 detailing. The normal vesicle size additionally associated into the ensnarement
viability of the plan is decide to be 339.9nm with T8 detailing. Entrance which was
additionally dependant on PC(Lecithin):Edge Activator proportion. The T8 detailing,
which showed higher entanglement proficiency, gives higher skin pervasion of medication
than transfersomal gel this information affirms the above said26.

Jain et al., (2007) investigated that the component for adjusted intracellular medication
conveyance framework from ethosomes utilizing of perception strategies and cell line
study. Ethosomal details were made utilizing lamivudine as model medication and assessed

2
in vitro, ex vivo/in vivo. The motified ethosomal detailing showed multiple times higher
than transdermal transition (68.4±3.5 µg/cm2/h) across rodent skin was contrasted and the
lamivudine arrangement of 2.8±0.2 µg/cm2/h. specific region with lamellar stacks got from
vesicles were seen in intercellular territory of profound skin layers. Consequences of cell
take-up study showed altogether higher intracellular take-up of ethosomes (85.7%±4.5%)
as analyzed from medicate arrangement (24.9%±1.9%). Consequences of the assessment
examines indicated that lipid annoyance close by with versatility of ethosomes vesicles
gives off an impression of being the essential patron for improved skin

permeation27.

2
 Chapter-3

Plan of work

PLAN OF WORK

2
1. Literature Review
2. Selection ofxdosage form
3. Selection ofxdrug candidate
4. Procurement of drug & excipients
5. Identification & characterization of API
 UV estimation
 IR Study
6. Preformulation studies
 Physical Appearance
 Melting Point
 Determination of Absorption Maxima (max)
 Solubility study
 Partition coefficient
 Drug –excipients compatibility study
 Calibration curve of drug
7. Formulation development of ethosomes
8. Evaluation of ethosomes
 Measurement of pH
 Determination of Viscosity
 Spreadability
 Drug Content
 In vitro drug release of drug
9. Stability studies
10. Result and discussion
11. Summary and Conclusion
12. Compilation and Submission of thesis

2
 3. RESEARCH INVISTIGATED

The advantage ofxtransdermal drug delivery over other delivery systems are
as follows: Avoidance ofx'first-pass' metabolism ofxdrugs. Reduced plasma
concentration levels of drugs, with decreased side effects. Reduction ofxfluctuations in
plasma levels of drugs. Uses of drug candidates with short half-life and low therapeutic
index. Easily excretion of drug delivery in case of drug poisonous. Decrease ofxdosing
frequency an increase of patient compliance.

Ethosomes are the nano particle drug delivery system which improved forms of liposomes
that are contain more quantity of ethanol. The ethosomal delivery system is made-up of
water, phospholipid and ethanol. They can be easily across the skin and increase drug
delivery both to deep skin strata and Blood circulation. it is a soft, flexible vesicles which
penetrate by disorganized lipid bilayers. These “soft vesicles” express to novel vesicular
carriers for increase delivery to or through skin.

Mupirocin drug (pseudomonic acid A, or Bactroban or Centany) is an antibiotic initially

obtained from Pseudomonas fluorescens. It is applied topically, and is fundamentally
viable against Gram-positive bacteria. Mupirocin drug is bactericidal at high
concentrations and bacteriostatic at low concentrations. Mupirocin has a
unique mechanism of action, which is selective binding to bacterial isoleucyl-tRNA
synthetase, which halts the incorporation of isoleucine into bacterial proteins.

The subject of the present investigation was to statistically enhance the vesicular
formulations Ethosomes of Mupirocin.

To check the skin penetration power and amount of mupirocin drug reached to circulation
through ethosome by typically administration.

3
 4. DRUG PROFILE

5.1 Mupirocin

Mupirocin (pseudomonic acid A, or Bactroban or Centany) is an antibiotic initially

obtained from Pseudomonas fluorescens. It is applied topically, and is fundamentally
viable against Gram-positive bacteria. Mupirocin drug is bactericidal at high
concentrations and bacteriostatic at low concentrations. Mupirocin has a unique
mechanism of action, which is selective binding to bacterial isoleucyl-tRNA synthetase,
which halts the incorporation ofxisoleucine into bacterial proteins. Because this
mechanism of action is not shared with any other antibiotic, mupirocin has
few problems of antibiotic cross-resistance.

Chemical structure:

Figure 5.1: Structure of Mupirocin

Chemical Formula: C26H44O9

Molecular weight: 500.6222

3
IUPAC name: 9-{[(2E)-4-[(2S,3R,4R,5S)-3,4-dihydroxy-5-{[(2S,3S)-3-[(2S,3S)-3-
hydroxybutan-2-yl]oxiran-2-yl]methyl}oxan-2-yl]-3-methylbut-2-enoyl]oxy}nonanoic acid

Pharmacology

Indication: For the treatment of Staphylococci nasal carriers.

Pharmacodynamics: Mupirocin, an anti-microbial delivered from Pseudomonas

fluorescens, is structurally irrelevant to some other topical or systemic antibiotics.
Mupirocin is utilized to treat contamination brought caused by Staphylococcus aureus
and beta-hemolytic streptococci including Streptococcus pyogenes. This anti-microbial
has preety much nothing, if any, potential for cross-resistance with other anti-bacterial
agents.

Mode of action: Mupirocin reversibly ties to bacterial isoleucyl-tRNA synthesis, a

protein which improve the coversion ofxisoleucine and tRNA to isoleucyl-tRNA.
counteraction of this enzymes from functioning appropriately results in the inhibition of
bacterial protein and RNA synthesis.

Route of elimination: Following the application of mupirocin (mupirocin ointment),2% to

a 400 cm2 area on the rare of 23 healthy volunteers once day by day for 7 days, the
average (range) total urinary discharge of monic acid over 24 hrs following the last
administration was 1.25% (0.2% to 3.0%) of the administered dose ofxmupirocin.

Half life: 20 to 40 minutes

Uses

Mupirocin is used to treat certain skin infections (such as impetigo). It is an anti-bacterial

drug. It works by stopping the growth of specific bacteria.

Side effects

 severe stomach pain, diarrhea that is watery or bloody;

 severe itching, rash, or other irritation ofxtreated skin;

3
 unusual skin blistering or peeling; or.

 any signs of a new skin infection.

3
 5. PREFORMULATION STUDY

Preformulation is a period of innovation work process where the new medication

substances are characterize for their mechanical, chemical and physical properties in order
to establish, stable, safe and powerful dosage form. Preformulation may include
organolaptic properties, melting point, solubility, identification of drug by chemical and
spectrophotometric method.

Characterization for physiochemical properties of drug

A) Organoleptic Properties
Organoleptic properties of drug were determined by direct observation of drug sample
under optical microscope for its appearance color and crystal morphology.

Table 6.1 List of Sensory characters

S. No. Sensory characters Result

1.
Colour and Morphology White to grayish powder
2.
Odor Odorless

B) Solubility Study: The drug sample was subjectively tested for its solubility in
different polar, semi polar and non polar solvents. Solubility ofxthe drug was calculated
by taking about 10 mg of drug sample in a test tube containing 2.0 mL of solvent and
shaking for 10 minutes at room temperature and observed for its solubility.

Table 6.2 Solubility of Mupirocin calcium

S. No. Solvent used Observation

1 Distilled Water ----

2 0.1 N Hydrochloric acid ++--
3 Ethanol ++++
4 Methanol ++++

3
 5 Chloroform ++++
6 0.1 N NaOH ++--
7 pH 7.4 Phosphate Buffer +++-
++++= freely soluble; +++- = Soluble; ++ - - = Sparingly soluble; + - - - = Slightly
Soluble; = Insoluble

Same procedure was repeated to check drug solubility in other solvent like water, ethanol,
methanol, 0.1N HCL, 0.1N NaOH, Chloroform and 7.4 pH buffer )

Melting point:

Melting point is a parameters to check the drug's purity. if used pure chemicals, then
melting points are express dark and constant. Since the drugs contain mixed chemicals,
they are characterized with certain range ofxmelting point. The melting point was
determined by capillary tube method in which small quantity ofxfinely powder drug was
placed into a capillary tube (shut down twords one side) and placed in the melting point
determining apparatus (Chemline CL-725) containing silicon oil. The castor oil
temprature was gradual rise automatically upon increasing the temperature. The
temperature was noted down at which powder started to melt and the temperature at
which the drug powder was melted completely.

Table 6.3 Melting point of the Mupirocin calcium

S. Melting Point of Melting Point of Average Melting Point of
No. Standard Drug Sample Drug Sample Drug
1. 77ºC-78ºC
2. 77-78 ºC 76ºC-77ºC 77ºC-78ºC
3. 77ºC-78ºC

3
D) Determination of pH (1 w/v solution in water):
Dissolved in 10 mL of distilled water around 10 mg of the drug powder. The solution was
filtered after sonication for 60 sec. The pH of the drug solution was determined using
standard glass electrode pH meter.
Table 6.4 pH of the Mupirocin calcium
S. No. pH of the solution Average pH of the solution
1. 4.15
2. 4.20 4.17±0.021
3. 4.16

E) Detrmination of partition coefficient

The partition coefficient of drugs was examined in n-Octanol: pH 7.4 Phosphate Buffer, n-
Octanol: water framework. It was determined by taking 5mg ofxdrug separately in three
glass bottle containing, 10 mL of n-Octanol and Phophate Buffer pH 7.4 in 50:50 ratio. The
glass bottle containing mixture was shaken for 24 hour in a wrist activity shaker at room
temperature. Then mixture was transfer in separating funnel and two phases were isolated
and the amount ofxdrugs in aqueous phase was analyzed spectrophotometrically using UV-
spectrophotometer at λmax 227 nm after proper dilution with respective buffer. The
partition coefficient of drug was determined utilizing the following formula.

𝐴𝑚𝑜𝑢𝑛𝑡𝑜𝑓𝑑𝑟𝑢𝑔𝑖𝑛𝑜𝑟𝑔𝑎𝑛𝑖𝑐𝑝ℎ𝑎𝑠𝑒
𝑃𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛𝐶𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 (K) =
𝐴𝑚𝑜𝑢𝑛𝑡𝑜𝑓𝑑𝑟𝑢𝑔𝑖𝑛𝑎𝑞𝑢𝑒𝑜𝑢𝑠𝑝ℎ𝑎𝑠𝑒

Table 6.5 Partition coefficient of Mupirocin calcium

S.N. MEDIUM K
1. n-Octanol:PBS pH 6.8 2.24
2. n-Octanol:PBS pH 7.4 2.45
3. n-Octanol:0.1 N HCl 1.89

3
F) Identification
Test FT-IR
Spectroscopy
Infra- red spectrum equipment gives more adequate data about the structure ofxa
compound. This technique provides a spectrum containing an enormous number of
absorption band from which an abundance ofxdata can be derived about the structure of
an organic compound. The region from 0.8 µ to 2.5 µ is called Near Infra-red and that
from 15 µ to 200 µ is called Far infra-red region.

Approx 5 mg of drug was mixed with KBr and prepared their pallet. Pallet was analyse
using FT-IR spectrophotometer (Bruker, USA).

Figure 6.1: FT-IR Spectrum of Mupirocin calcium (Standard)

3
 60

%T 52.5

45

572.88
623.03
37.5

916.22
956.72
2362.88

852.56
2756.37

1176.62

1043.52
1020.38
3059.20

1305.85
2843.17

30

1589.40

723.33
1340.57

752.26
1217.12
1448.59

1099.46
1504.53

1255.70
3344.68

22.5
40003600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 40
carv dilol 1/cm

Figure 6.2: FT-IR Spectrum of Mupirocin calcium (Sample)

15

L) Determination of moisture content

Principle: The titrimetric assurance of water depends on the quantitative response of
7.5

water with an anhydrous solution of iodine and sulphur dioxide in the presence of a
buffer that reacts with hydrogen particles. Karl Fisher Reagents known as the
first titerimetric solution, the iodine and sulphur dioxide was dissolved in pyridine and
0

methanol. The test specimen might be titrated with the reagent legitimately, or the
analysis might be completed by a residual titration procedure. The stoichiometry of
the reaction is not correct, and the reproducibility of assurance realise on such factors as
the relative concentration of the reagent ingredients, the stoichiometry ofxthe inert
solvent used to dissolve the test specimen, and the method utilized in the specific
assurance.

Consequently, an experimentally standardized procedure is used in order to achieve the

ideal accuracy. Exactness in the method is administered to a great extent by the degree
to which atmospheric moisture is excluded from the system. The titration of water is
generally carried out with the utilization of anhydrous methanol as the solvent for
the test specimen; anyway other reasonable solvents might be used for unique
or unusual test specimens. (Note: Now-a-days pyridine free KF reagents are coming

3
in which pyridine is replaced by the imidazole, because pyridine has carcinogenic
effects).

Karl Fischer volumetry utilized for tests with high water content, i.e. 1-100 mg per
sample. An iodine-containing solution delivers as titrating agent. The water substance of
the sample is calculated using titration volume and titer of the titrating agent. One-
component reagents helpfully contain all reactants (iodine, sulfur dioxide and a base)
dissolved in a resonable alcohol in one solution, Though two-component reagents
contain all every single important reactants isolated in two different solutions to enhance
the speed of the Karl Fischer reaction and the titer stability of the titrating agent.

Karl Fischer coulometry is a micro-method and is particularly fit for samples with low
water content, from 10 µg up to 10 mg. Here, the necessary iodine is created
electrochemically in the titration vessel by anodic oxidation from iodide contained in the
coulometric reagents. The amount ofxexpended electric charge is utilized to determine
the consumption of iodine and therefore the amount of water in the sample.

Table 6.6 Determination of moisture content

S. No. Drug KF Factor Amount of KF Reagent Moisture content
consumed (mg)
1 Mupirocin 0.521 0.21ml 0.109
calcium

M) Determination of λ max of Mupirocin calcium

The λmax of Mupirocin calcium was determined by analyzing the drug solution in double
beam ultraviolet spectrophotometer. Accurately weighed 10 mg of mupirocin
calcium was dissolved in 10 ml ofx7.4 pH buffer solution in 10 ml of volumetric flask.
The resulted solution was 1000µg/ml of strength and from this solution 1 ml
solution was pipette out and transfer into 10 ml capacity of volumetric flask and volume

3
was made upto 10 mL with 7.4 pH buffer solution. This solution was scan at
wavelength 400-200 nm on UV spectrophotometer. The higher absorption peak was
obtained at 227 nm which was the λmax ofxdrug.

Figure 6.3: λmax scan graph of Mupirocin calcium in phosphate buffer pH 7.4

N) Preparation of calibration curve of Mupirocin calcium

Previously prepared stock solution (1000 µg/mL of strength) of mupirocin calcium was use
to prepare suitable dilution into concentration range of 5-25 μg/ml. 0.5, 1.0, 1.5, 2.0 and 2.5
mL of this solution was taken in different volumetric flask having 10 mL of capacity and
dilute upto 10 mL with phosphate buffer pH 7.4 to obtained 5, 10, 15, 20 and 25 μg/mL of
solution. The absorbance of these solutions was taken at 227nm using UV-
spectrophotometer (Labindia-3000 Plus). Graph between absorbance and concentration was
plotted folloed linearly regressed on Microsoft excel.

Table 6.7: Calibration curve of Mupirocin calcium

S. Concentration (µg/mL ) Absorbance
No. n=3

0 0
1

4
 5 0.205
2
10 0.412
3
15 0.615
4
20 0.825
5
25 1.021
6

1.2
y = 0.041x + 0.001
1 R² = 0.9999
Mean absorbance

0.8

0.6

0.4

0.2

0
0 5 10 15 20 25 30

Concentration (µg/ml)

Figure 6.4: Calibration curve of Mupirocin calcium in phosphate buffer pH 7.4 at

227nm

6.3 Compatibility studies of drug and excipients

In the similarity testing program mixes ofxdrug and excipients are set up by triturating
the medication with individual excipients. Mixture ofxMupirocin calcium (50 mg) and
excipients (50 mg) was prepared and transferred to inert glass vials. Vials were covered
with rubber closures followed by the aluminum seal caps. The vials were put away at
4°C (fridge) as control and at 40°C/75%RH for quickened temprature condition for about

4
a month. The visual observations (color, flow, & sticking) were recorded. Sample
were analyze for any interaction by using UV spectrophotometer and DSC.

Figure 6.5: UV spectra of Mupirocin calcium

Figure 6.6: UV spectra of mixture of Mupirocin calcium and Excipients

4
 6. PREPARATION AND CHARACTERIZITION

Preparation of ethosomes
Preparation of Mupirocin calcium ethosomes
Formulations of Mupirocin loaded Ethosomes were prepared by using the cold method.
Ethanolic vascular system of ethosomes was made out of phospholipid, ethanol,
polyethyleneglycol, drug and distilled water to 100% volume by volume. In ethanol,
Phospholipid was dissolved along with the drug. Mixture of phospholipid, Ethanol and
drug was heated at 400 C ± 10C on Magnetic stirrer hot plate and a fine stream of
distilled water was added slowly-slowly, with constant mixing at 700 rpm by using a
mechanical stirrer. Mixing was continued for an additional 4-5 minutes, with maintaining
the system at 400C ± 10C. Then after mixture was left for 30 minutes to cool at room
temperature and then mixture was sonicated by using a probe sonicator at 40 C for
five cycles of 3 minutes each with a minute rest between cycle.
Table 7.1: Different composition of ethosomes formulation
F. Code Drug Phospholipid Ethanol PEG Water

F1 50 150 10 10 100

F2 50 150 20 10 100

F3 50 150 30 10 100

F4 50 250 10 10 100

F5 50 250 20 10 100

F6 50 250 30 10 100

4
 Evaluation of Mupirocin calcium loaded ethosomes
7.2.1 Microscopic observation of formulated ethosomes
An optical microscope (cippon, Japan) with a attached camera (Minolta) was used for
observe the shape ofxthe prepared ethosomes formulation.

F1 F2 F3

F4 F5 F6
Figure 7.1: Microscopic observations of prepared ethosomes formulations

Vesicle size and zeta potential

Vesicle size and zeta potential of the Ethosomes were measured by photon correlation
spectroscopy using a Malvern Zetasizer the results shown in table.

4
Entrapment efficiency
Entrapment efficiency was determined by measuring the concentration of unentrapped
free drug in aqueous medium. About 1 ml of the mupirocin loaded ethosomes dispersion
was placed in the Ependorfxtubes and centrifuged at 17000 rpm for 30 minutes. The
Ethosomes with encapsulated of mupirocin drugs were separated at the lowest part of
the Ependorf tubes. Plain ethosomes without drugs were used as blank sample and also
centrifuged at 17000 rpmfor 30 minutes. Measure the free drug focus by utilizing
The UV, absorbance of the supernatant was found at 227nm.

Table 7.2: Result for Vesicle size and Entrapment efficiency of drug loaded
Ethosomes
Formulation Code Vesicle size Entrapment Efficiency

F1 250.36±2.45 65.45±0.32

F2 232.25±3.25 72.12±0.41

F3 210.32±4.56 85.45±0.32

F4 356.56±3.41 65.47±0.74

F5 345.58±3.69 71.74±0.65

F6 365.45±1.25 76.65±0.25

4
Figure 7.2: Vesicle size and Entrapment efficiency of drug loaded ethosomes

Figure 7.3: Vesicle size of optimized formulation

4
 Figure 7.4: Entrapment efficiency of Optimized formulation

Table 7.3: Vesicle size and Entrapment efficiency of Optimized Ethosomes

Formulation Code Vesicle size Entrapment Efficiency Zeta potential
(nm)
F3 210.32±4.56 85.45±0.32 -32.45

Formulation of ethosomal gel

Preparation of Ethosomal gels: Mupirocin loaded ethosomes

(equivalent to 2%) into gels was achieved by slow mechanical mixing at
25rpm (REMI type BS stirrer) for 10 minutes. The developed
formulation was mix into three different carbapol gel with
concentration 0.5, 1 and 2% weight by weight.

4
EVALUATION OF GEL

Physical Characteristic

The Physical Characteristic was checked for gel formulations (homogeneity and texture)
and observations were shown in Table 7.4.

Determination of pH

The pH of the ethosomal gels were estimated by computerized pH meter. One gram of
ethosomal gel plan was broken down in 25 ml purified water and then the electrode was
dipped in formulation for 30 minutes until reading was constant then constant reading
was noted. The measurements of pH ofxeach ethosomal gels formulation were repeated
two times and average pH value was founded of each formulation.

Washability
Formulations were applied on the skin and then ease and extent of washing with water
were checked manually and Result was founded easly washed.

Extrudability study
Collapsible metal tubes or aluminium collapsible tubes was used for check Extrudability
study of gel. Firstly The ethosomal gel formulation was filled into collapsible metal tubes
and closed tightly. Then tubes were pressed to till extrude the gels and the extrudability
of the formulation was checked passed or failed.

Spreadability
Principle: In Spreadability test An important criteria for ethosomal gels is that it must
possess good spreadability. Spreadability is a word which communicated to signify the
extent of area by the gel which easly spreads on application to skin. The therapeutic
value of formulation is also depends on spreadability study.

For the spreadability study of the formulations a special appratus has been designed.
Spreadability is communicated in terms of time in seconds taken by two slides to slip
ofxfrom formulation, placed between, under the application of a specific burden. The
partition of two slides lesser time taken, better the spreadability.

4
Method:
Firstly we selected Two glass slides have standard dimensions (6×2). The gel
formulation whose spreadability must be determined was put over on one slides. The
second slide was kept over the first slide that the formulation was between them that
slides across a length of 6 cms. 100 grams weight were placed over the upper slide that
by which the ethosomal gel between the two slides was find out uniformly to form a thin
layer. Then 100grams weight was withdrawed and the greater amount of the gel
formulation holding to the slides was discarded. The first slide (Lower slide) was fixed
on the leading group ofxthe apparatus and one end ofxthe upper slide (second slide) was
tied to a string to which 20 gram load could be applied 50with the help ofxa simple
pulley. The time taken to travel distance of 6 cms by the upper slide and separated away
from lower slide under the direction of the weight was noted. The experiment was
repeated and the average of 6 such determinations was calculated for each
gel formulation.

Where,

m = weight attached to the upper slide (20

grams) l= length of glass slide (6cms).

t = time taken is seconds.

Viscosity
The Brookfield digital Viscometer used for the estimation of viscosity of the prepared
ethosomal gel. Viscosity was measured by using spindle no. 6 at 10 rpm and 250C.
Properly the adequate amount of ethosomal gel was filled in wide mouth container. The
shaft of the Viscometer was diped by the gel which was filled in the wide mouth container.

4
Before the measurements of viscosity Samples of the gels were left for 30 minutes to settle
at the constant temperature (25 ±/10C).

Table 7.4: Results of evaluation of gel formulation

Spreadability Viscosity % Assay

Homogenecity
Code
and Texture pH (gm.cm/sec.) (cps)

2265 98.89
F1 Good 7.2 14.58

2385 95.65
F2 Good 6.8 13.54

2478 97.85
F3 Good 6.9 12.45

In-vitro drug release studies using the prehydrated cellophane membrane

Preparation of cellophane membrane for the diffusion studies:

The cellophane membrane was taken, size approximately (25x2)cm and washed
by the running water. Then it was drenched for 24 hours in distilled water, Then after
remove to glycerin present on it before used to diffusion studies and it was filled on the
diffusion cell for further studies.
The prepared Ethosomes delivery system was evaluated by in vitro drug
release. The drug discharge examines were determined using modified franz diffusion
cell. The study ofxdissolution was carried out in 200 ml dissolution medium which was
stirred at 50 rpm and trmprature maintained at 37±0.2C.
Samples (10ml) were taken at different time interval and maintained volume with
the same amount ofxnew dissolution medium (sample was withdrawn 10 ml, then it was
made up to 10ml by PBS pH 7.4). The samples withdrawn were assayed
spectrophotometrically at 227.0 nm for Mupirocin calcium and using UV visible
spectrophotometer. The release of Mupirocin calcium was calculated with the help of
Standard curve of Mupirocin calcium.

5
 The observations of drug release for the drug in uncoated formulation and coated
formulation is tabulated in Table 7.5.

Table 7.5: Cumulative % drug release of Mupirocin calcium fromoptimized

ethosomes formulation

Time (hrs) % Cumulative Drug Release

0.5 19.98±0.45
1 32.25±1.25
2 49.98±0.98
4 65.58±0.32
6 79.98±0.41
8 93.12±0.75

Release kinetics
The drug development, In-vitro diffusion has been played as an important role. Under some
conditions it very well may be used as a substitute for the appraisal of bioequivalence.
Several theories/kinetic models express the drug dissolution from immediate and
modified release pharmaceutical dosage forms. To determine the dissolution profiles of
drug several models are present where ft is the function ofxt (time) identify with to
the quantity of drug dissolved from the pharmaceutical dosage system. When want to
compare of dissolution profiles between two different drug products dependent on model
method (curve fitting), then can used statistic analysis and model independent methods.

In order to illuminate mode and mechanism of drugs release, the invitro information was
coverted and clarified at graphical interface developed utilizing different dynamic
models. The zero request discharge Eq.(1) represent to the drug disintegration of
particular kinds of changed release pharmaceutical dose structures, as by virtue of
network tablets with low dissolvable medications, transdermal frameworks,, coated
forms, osmotic systems etc. where the drug release is not dependent on concentration.

5
 Qt = Qo + Kot eq.(1)

Where, Qt is the quantity of drug in amount released in time t, Qo is the initial quantity
of the drug in amount in the solution and Ko is the zero order release constant

The first order Eq. (2) express the release from the system where release is concentration
dependent. e.g.- containing water soluble drugs in permiable matrices of pharmaceutical
dosage forms.

log Qt =rffrrlog Qo + K1 t /2.303 eq.(2)

Where Qt is the quantity of drug in amount discharged in time t, Q is the initial qantity
ofxdrug in amount in the solution and K1 is the first order release constant.

Higuchi defined the release of drug from insoluble matrix as a square root of time
as given in Eq. (3)

Qt = KH √t eq. (3)
Where, Qt is the quantity of drug in amount released in time t, KH is Higuchi's
dissolution constant. The accompanying plots were made: combined % drug release vs.
time (zero order kinetic models); log combined of % drug remaining versus time (first
order kinetic model); combinede % drug release vs. square root of time
(Higuchi model).

5
 Table 7.6: In Vitro drugfrffrelease data for F2

Square Cumulative* Log Cumulative Cumulative Log cumulative

S. No. Time (H) Root of Log Time Percentage Drug Percentage Drug Percent Drug Percent Drug
Time Release±SD Release Remaining Remaining

1 0.5 0.707 -0.301 19.98 1.301 80.02 1.903

2 1 1 0 32.25 1.509 67.75 1.831

3 2 1.414 0.30103 49.98 1.699 50.02 1.699

4 4 2 0.60206 65.58 1.817 34.42 1.537

5 6 2.449 0.77815 79.98 1.903 20.02 1.301

6 8 2.828 0.90309 93.12 1.969 6.88 0.838

* Average of three determinations

40
 Figure7.5:Cumulative Percent Drug Released Vs Time (Zero Order Plots)

Figure7.6:Log Cumulative Percent Drug Remaining Vs Time (First Order Plots)

Table 7.7: Regression analysis data of ethosomal gel formulation

Formulation Zero order First order

F2 R² = 0.658 R² = 0.825

41
STABILITY STUDIES
Stability studies of ethosomal gel were carried out with optimized formulation of F3
which was stored for a Time ofx45 days at 4±1°C, RT and 40±1°C. Particle size of
ethosomal gel formulation was founded by optical microscopy by using a calibrated
visual micrometer. The particle size of the ethosomes was found to increase at
RT, which may be attributed to the aggregation of ethosomes at higher temperature. At
452°C the aggregate i.e. these ethosomes were unstable at higher temperature like
452°C .Percent efficiency of ethosomes also decrease at higher temperaturelike 452°C.

42
Chapter-4

Result &
Discussion

43
 7. Result and Discussion
In vitro drug release data of the formulation was formed to give good result of fit test by
straight relapse analysis according to zero order, first order kinetic equation and in order
to find the mechanism ofxdrug release data according to Korsmeyer's models. When the
relapse coefficient values of formulations were compared, it was observed that 'r' values
ofxformulation was maximum i.e 0.924 hence indicating drug release from formulations
wasfound to follow Pappas model of drug release kinetics.

 Organoleptic properties of muperocin was found to be

S. No. Sensory characters Result

1.
Colour and Morphology White to off white powder
2.
Odor Odorless

 Solubility of mupirocin calcium was found to be Freely soluble in Ethanol,

Methanol, chloroform and sparingly soluble in 0.1n NaOH and insoluble in
water.
 Average melting point of sample drug og mupirocin was found to be 77ºC-78ºC.
 Average pH of mupirocin solution was found to be 4.17±0.021

 Partition coefficient of Mupirocin calcium was found to be:

S.N. MEDIUM K

1. n-Octanol:PBS pH 6.8 2.24

2. n-Octanol:PBS pH 7.4 2.45

3. n-Octanol:0.1 N HCl 1.89

44
  FT-IR Spectroscopy
Approx 5 mg ofxdrug was mixed with KBr and prepared their pallet. Pallet was analyse
using FT-IR spectrophotometer (Bruker, USA).

 Determination of λ max of Mupirocin calcium

The higher absorption peak was obtained at 227 nm which was the λmax of drug.

 Preparation of Mupirocin calcium ethosomes

Ethosomal formulations were prepared by using the cold method. We make different 6
composition of ethosome formulation and Evaluated it.

 Vesicle size and Entrapment efficiency of Optimized Ethosomes: Entrapment

Efficiency of F3 Formulation code was found very high.
Formulation Code Vesicle size Entrapment Efficiency Zeta potential
(nm)

F3 210.32±4.56 85.45±0.32 -32.45

45
  Results of evaluation of Ethosomal gel formulation

Spreadability Viscosity % Assay

Homogenecity
Code
and Texture pH (gm.cm/sec.) (cps)

2265 98.89
F1 Good 7.2 14.58

2385 95.65
F2 Good 6.8 13.54

2478 97.85
F3 Good 6.9 12.45

 Stability studies

F3 Formulation was stored for carried out of stability study for a time ofx45 days at
4±1°C, RT and 40±1°C. These ethosomes were unstable at higher temperature like
452°C .Percent efficiency ofxethosomes also decrease at higher temperature like
452°C.

46
 8. SUMMARY AND CONCLUSION

Preformulation of drug and excipient was performed in which physiochemical

properties and other parameters of drug were studied. Physiochemical parameters such
determination of solubility, melting point, partition coefficient, drug-excipient interaction,
λmax scan using UV-spectrophotometry, FT-IR spectrophotometry were performed in this
study. The obtained data from these studies were matched with the data given in standard
monographs to confirm the accuracy of procured drug.

Procured drug was odorless and white crystalline in nature. In solubility study it
was found that drug was freely soluble in ethanol, methanol and soluble in chloroform
and phosphate buffer pH 7.4 and sparingly soluble in 0.1 N NaOH and 0.1N HCl. It
was completely insoluble in distilled water. Melting point of drug was found 77ºC-
78ºC while it was 77ºC reported in standard monograph. The pH ofxdrug solution was
found to be 4.7±0.021 The partition coefficient (K) value was found to be 2.24, 2.45,
1.89 in n-Octanol:PBS pH 6.8, n-Octanol:PBS pH 7.4 and n-Octanol:0.1 N HCl
respectively. The obtained FT-IR characteristic peaks ofxdrug was matched with the
peaks of drug given in standard monograph was revealed similar. Moisture content
ofxmupirocin calcium was found 0.109 mg.

The drug solution was scan on UV-spectrophotometer at 200-400 nm in

wavelength range to determine the maximum absorbance (λ max) and it was found at
227nm. The calibration curve was prepared in pH 7.4 Phosphate Buffer. The regression
coefficient (R2) was 0.999 which was shows the linearity of curve. The line of
equation for the standard curve was y = 0.041x + 0.001. The drug excipient interaction
study was performed to check in interaction between drug and other
formulation excipients by spectrophotometrically. There was no interaction was
found between drug and excipients and it was clearly seen and confirmed by UV
spectrophotometrically scan graph ofxdrug solution and mixture of drug and excipients
in fig 6.3 and 6.4. There was no fluctuation in wavelength of mupirocin calcium. All

47
the data of preformulation study was found similar as given in standard
monograph which confirmed that the drug was authenticate and pure in form and it
could be used for formulation development of mupirocin calcium loaded ethosomes.

48
 Chapter -5

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