transdermal drug delivery using transferosomes

8/10/2019 Transdermal drug delivery using Transferosomes

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Internship report on

Formulation and Evaluation of Transfersome Vesicles for enhanced delivery of

anti diabetic drug

Submitted by

J.Perinba Danisha

Jeppiaar Engineering College

Shozhinganallur

S.AsifSRM University

Chennai.

An R&D Division of BioLim Biosolutions (P) Ltd

Chennai


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BONAFIDE CERTIFICATE

This is to certify that J.Perinba Danisha, B.Tech, Biotechnology, Jeppiaar Engineering College,

S.Asif, SRM University, Chennai, has undergone an internship program in the research project

Formulation and Evaluation of Transfersome Vesiclesperformed under nano research group of

BioLim Centre for Life Science, BioLim Biosolutions (P) Ltd, for a period of 70 days from

05.01.2014 to 18.03.2014.

GUIDE & SUPERVISOR

Baskar Viswanathan

Principal Investigator

Nano Research Group

BioLim Centre for Life Sciences

BioLim Biosolutions (P) Ltd.


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Dedicated to science


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Acknowledgement

First and foremost, I would like to thank the Almightyfor giving me good health and strength to

complete this internship successfully.

I would like to express my special thanks to BioLim Biosolutions (P) Ltd. for providing the

opportunity and facility to pursue an internship program in their esteemed organization.

I express my gratitude to Mr. Srinivasan Govardanan, Chief Executive, BioLim Centre for Life

Science, BioLim Biosolutions (P) Ltd., for accepting my request and allowing me to take up the

internship programme at their esteemed organization.

I indeed very much delighted to express our feelings of gratitude and indebtedness to my guide

and supervisor, Mr. Baskar, Principal Investigator, Nano Research Group, BioLim Centre forLife Science, BioLim Biosolutions (P) Ltd., for his motivation and remarkable guidance in the

success of the internship programme.

I owe my special depth of gratitude to Mr. Madhan, Principal Investigator, Microbiology and

Fermentation Research Group, BioLim Centre for Life Science, BioLim Biosolutions (P) Ltd.,

and Mr. Selvakumar, Principal Investigator, Phytomolecular Research Group, BioLim Centre

for Life Science, BioLim Biosolutions (P) Ltd., for their consistent encouragement and external

supervision throughout the internship tenure.

I extend my thanks to all members of BioLim Centre for Life Science, BioLim Biosolutions (P)

Ltd, Ayanavaram, Chennai.

Last but not least, I express my immense thanks to all of my friends and family members for

their necessary support throughout the tenure of this internship.


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Table of contents

CHAPTER CONTENTS PAGE NO

1 INTRODUCTION

Nano vesicles in drug delivery

Improvements in nano vesicles

Different types of vesicles

Invention of transfersome

Impact of variation in composition

Fate of Transfersome

2 BACKGROUND OF RESEARCH

3 OBJECTIVES OF RESEARCH

4 MATERIALS AND METHODS

Formulation of transfersome

screening of best formulation

stability study

penetration study

characterization study1. FTIR2. Zeta potential

5 RESULTS AND DISCUSSION

6 CONCLUSION

7 APPENDIX

8 REFERENCE


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Chapter 1

Introduction

Many drugs having good therapeutic values are failed due to the improper delivery system.

Transdermal drug delivery system is such a boon that overcomes the drawbacks of conventional

delivery methods. They are avoidance of first pass metabolism, predictable and consistency of

drug activity, low side effects, utility of short half-life drugs, high physiological and

pharmacological response, inter-and intra-patient variations and it is very convenient to patients.

However difficulty in penetration of drug through the stratum corneum stops the efficiency of

transdermal delivery system for commercial usage. [Shailesh T. Prajapati et al, 2011].So many

physical (iontophoresis, and sonophoresis) and chemical methods (enhancers, nano particles like

liposomes, niosomes, ethosomes, dendrimers, nano emulsion and transfersomes) are in use to

enhance the permeation rate. [ Hiren J. Patel et al,2011]

Nano vesicles in drug delivery:

Among all methods vesicle formulation is a promising technique for enhanced delivery. Vesicles

acts as effective carrier system and it is able to penetrate the skin and allows the drugs to

systemic circulation. At the starting stage of vesicles invention the various studies gives

conflicting results over vesicles usage in transdermal delivery. The controversial results proposed

two different ideas about the nano vesicles. They results are 1. Vesicles are only used for topical


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application 2.It may be also used in drug delivery by the combination of physical and chemical

methods. [Jos Juan Escobar-Chvez et al,2012]

Improvements in nano vesicles:

In vesicle via drug delivery the state of the vesicles is very important. The results of the study in

various states shows, the liquid state vesicles are more efficient than gel state vesicles. So the

need of the developments of proper vesicles which is in liquid state and has capability of

penetrating across the stratum corneum is very important in pharma industry. Over many

decades many different nano vesicles are identified for drug delivery and so many improvements

are made to increase the quality of the nano vesicles.[ Joke A. Bouwstra et al 2005]

Different types of vesicles:

Liposomes:

Liposomes are the first invented nano vesicles and still it is mainly used in cosmetic industries.Alec Bangham invented liposomes and he found that phospholipids in aqueous system creates a

hollow cylindrical structure and it accommodate hydrophilic structure inside the core and

lipophilic drug between the bilipid layer. In recent years many drugs are approved by using this

vesicles. [Jos Juan Escobar-Chvez et al,2012] These vesicle is made up of cholesterol and

phosphotidyl choline and the physio chemical property is depends on the fabrication and

methods used in production. The efficiency of the vesicles depends on particle size ,the presence

of penetration enhancers and the physical state of the vesicle, lamellarity, composition of lipid,

liposomes charge , mode of application, and are the important variables to analyze. Even though

transport across the skin is efficient through the liposomes ,When high-pressure homogenization

is used, it decreases the stability of high-weight molecules and Lipid crystallization leads to a lot

of polymorphic issues and they are susceptible to physical instability. [P.K.Lakshmi et al,2012]

Ethosomes:

To make further improvement and increase the flexibility of liposomes ethosomes are developed

by Touitou et al . It is the bilipid layer containing alcohol in the bilipid layer. [Nikalje Anna

Pratima et al,2012] Softness and malleability are the main characteristics of ethosomes and it

accommodates wide range of drugs. A characteristic feature of ethosomes is their small size

relative to the liposomes. This could be due to the presence of high ethanol concentration, it

possess a surface negative net charge to the liposome which causes the size of vesicles to

decrease. Various study reports shows that ethosomes have high invitro and invivo drug release


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efficiency. [Hiren J. Patel et al 2011].When the media is changed from organic to water the

vesicles stability decreases so loss of product may occurs. This is the important drawback of

ethosomes. [Nikalje Anna Pratima, 2012]

Niosomes:

Non ionic surfactants and lipids are the main constituent in the niosomes and toxic range of

niosomes is low and it is easily biodegradable. Niosomes are less rigid than liposomes and it can

penetrate where the liposomes and ethosomes cannot go. Compared to the liposomes and

ethosomes niosomes are more stable. [Jos Juan Escobar-Chvez et al, 2012]. The important

features are Low formulation cost, long shelf life, better drug targeting at specified site in a

sustained manner, better permeation and overcomes stability problems such as fusion, leaking,

aggregation. Compared to the liposomes and ethosomes it is more stable. [Yasam Venkata

Ramesh et al,2013]Invasomes:

Vesicles with terpenes as penetration enhancers were termed as invasomes. Verma and Fahrs

group created invasomes. Invasomes composed of phosphatidylcholine, ethanol and a mixture of

terpenes. Terpene disrupts the lipid layer for the penetration of the drugs. Minoxidil, Diclofenac

and Temoporfin are the drugs that are using invasomes as the carriers. [P.K.Lakshmi et al,2005].

Many highly hydrophobic drugs are currently in use by invasomes vesicles.[Eder Lilia

Romero,2013]

Dendrimers:

Dendrimers are nonpeptidic fractal 3-D structures consists of numerous micro molecules. The

dendrimer is a Greek word. It means dendra- tree and meros-part. This name was coined

in the late 1970s by a research group formed by Vgtle, Denkewalter, Tomalia, and Newkome.

These shows uniformity in their weight, shape and structural characteristics. Dendrimers as

transdermal drug-delivery systems are relatively new in pharma industry. They are a very good

alternative for drug-delivery systems; dendrimers can be used in antiviral and anticancer

pharmaceutical therapies, including vaccines. Amidoamine is the most commonly used material

in dendrimer production. These nano carriers have been used to transport photosensitizers for

photochemical therapy and antifungal molecules. [Jos Juan Escobar-Chvez,2012]

Nano Emulsions:


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Nano emulsions are isotropic dispersed systems of two immiscible liquids. It is suitable for both

hydrophilic and hydrophobic drugs because of the oil in water and water in oil emulsions.

Current drugs using nanoemulsions for transdermal drug delivery are gamma tocopherol,

caffeine, plasmid DNA, aspirin, methyl salicylate, insulin and nimesulide. These nano carriers

are non irritant and non toxic molecules.[Jos Juan Escobar-Chvez,2012]

Invention of transfersome:

Transfersomes are molecules composed of phospholipid, surfactant and solvent. The

characteristic nature of the transfersomes shows many advantages over the other nano vesicles.

This is more commonly called as ultra deformable vesicles.

So analyzing the pros and cons of all nano vesicles transfersomes is a promising nano vesicle

because of its ultra deformability, high adaptability and stress-responsive nature. [Nathji Dhaval

et al, 2013]. The name derived from Latin and Greek word that is transferre (to carry across) andsome (body).In 1991 Gregor first invented transfersome. [Pushpa Anand et al, 2012].

Interdependency of local composition and shape of the bilayer makes the vesicle both self-

regulating and self-optimizing.

How transfersomes differs from other vesicles:

Transfersomes have recently been introduced, which are capable of transdermal delivery of low

as well as high molecular weight drugs. Transfersomes are specially optimized, ultra flexible

lipid supramolecular aggregates, which are able to penetrate the mammalian skin intact and then

act as a drug carrier for non-invasive targeted drug delivery and sustained release of therapeutic

agents. Transfersomes can change its structure to pass through narrow pore range(from 5 to 10

times less than their own diameter) without measurable loss[Hiren J. Patel et al,2011].They have

high entrapment efficiency, in case of lipophilic drug the entrapment efficiency value is near to

90%.They protect the encapsulated drug from metabolic degradation. They act as depot,

releasing their drugs slowly and gradually. They can be used for both systemic as well as topical

delivery of drug. The procedure for preparation of transfersome is simple and it doesnt need any

additives like oral and other type of medication. [Shailesh T. Prajapati et al, 2010].

Comparing the overall values of encapsulation efficiency, stability study, particles size of the

different type of vesicles shows transfersome have better characteristics over the other vesicles.

[Saeed Ghanbarzadeh and Sanam Arami et al ,2013].

Impact of variation in composition:


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The different formulation of transfersome (concentration of phospholipid, surfactant, solvent and

drug) has different unique characteristics like entrapment efficiency variation and different

penetrating capacity. Increase in the content of surfactant may have led to pore formation in the

bilayers. And different types of surfactant also show different results. This is calculated

according to the HLB values of surfactant. Likewise increasing in the lipid concentration also

decrease the encapsulation efficiency.[Marwa H. Abdallah,2013]. So formulation of

transfersome and evaluation using various test like Entrapment efficiency, vesicle size, stability,

zeta potential, drug release study gives the result of the transfersomes efficiency in a better

manner.

Fate of Transfersome:

Transfersome penetrated to the deepest dermis skin layer and reaches through the lymph it was

taken to the blood circulation. The kinetics of the transferosome is depends on the vesiclepenetration and carrying efficiency. The distribution capacity of the drug loaded also affects the

kinetics. Three important steps in the transferosome fate are

1. Penetration of transfersome2. Accumulation of transferosome at target site3.

Elimination of transfersome

The vesicle driving force is depends on the solvent used and before penetration of the

transfersomes the solvent completely evaporated. [Kombath Ravindran Vinod et al, 2012].

Thus from the study it is proved that transfersomes will act as effective vesicle for delivering the

drug through the skin.


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Chapter 2

Background of research

Transdermal delivery system is very useful area in pharma industry. However limited numbers of

products are only available in the current market. Transdermal delivery system overcomes the

drawbacks of conventional delivery system but the main problem prevents the efficiency in

transdermal delivery method is the penetration of the skin. So using permeation enhancers are

causing irritation and some nano vesicles are not capable of penetrate the macro molecules. So

research for improving the penetration will leads to find the better alternative for the

conventional methods.

And diabetes is the universal disease that spread all over the country. But the treatment methods

are not so efficient. For type 2 diabetes the drug must reaches the systemic circulation to increase

the insulin holding capacity. Comparing these factors the efficient delivery method throughtransdermal route will treat the disease in a better manner. Many works deal with same area

reveals that ultra deformable vesicles are nano vesicles which possess good characteristics. So

we mainly aimed to enhance the transfersome formulation for anti diabetic care.


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Chapter 3

Objectives of research

To formulate and optimize ultraflexible lipid nano vesicles for enhanced transdermal drug

delivery of antidiabetic drug.

Work plan:

Formulation of transferosome: To analyse the impact of different composition of

transferosome and to find the best transferosome which possess good entrapment efficiency and

stability test transfersomes are prepared in four different formulations using different phospho

lipid and different surfactantin different ratios.

Identification of best formulation:Using quantification test the amount of drug entrapped is

found from the four different formulations. Because of the variation in the concentration, type of

the surfactant and phospho lipid concentration possess different properties. So from the result thetransfersome which have good entrapment efficiency will be identified.

Stability Study:The impacts of the environmental stress must be considered in the transfersome

studies because transferosome vesicles are mainly designed for transdermal delivery.

Temperature and pH change tends to weaken the vesicles and disintegrate the structure and leads

to leakage of drug. So study of the stability of the vesicles at different temperature and pH will

gives the stable level for the vesicles.

Characterization studies: Characterization of transferosome by Entrapment efficiency, Vesicle

size, Zeta potential and FTIR studies.

Permeation Study: The permeation through the skin is also a important factor. The study is

done by using a artificial membrane which act similar to skin. The in-vitro release of drug loaded

transferosomes through an artificial cellophane membrane was determined by a simple dialysis

method.


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Chapter 4

Materials & Methods

Reagents and chemicals:

S.NO Reagents and chemicals Manufacturer

1 Hexane RFCL chemicals

2 Silica gel MERCK specialty private limited

3 Phosphate buffer

KH2PO4

K2HPO4

Merck specialty pvt ltd

4 Folin-ciocalteu reagent LOBA chemie pvt ltd

5 Sodium bicarbonate LOBA chemie pvt ltd

6 Sodium deoxycholate TITAN biotech

7 Span 60

8 Gingerol (reference drug)9 Cholesterol LOBA chemie pvt ltd

Apparatus used:

S.No Apparatus Manufacturer

1 Flash chromatography

apparatus

2 Rotary shaker sudhakar biologicals and chemicals

3 Water bath

5 Cooling centrifuge REMI


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6 Weighing machine Infra values

7 UV- visible spectroscopy


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Methodology

Formulation Of transfersome:

In order to find the best constituents and the quantity different formulations are made. This

provides detail about the formulation which have better efficiency.

Compositional variation in the preparation of transfersomes to find the formulations which have

better entrapment efficiency.

Content Phospholipid Surfactant Solvent

Soyalecithin

Cholesterol

Span 60 Sodium

deoxycholate

Chloroform

T1 1 - 1 - 1

T2 2 - - 1 1T3 - 1 1 - 1

T4 - 2 - 1 1

Different methods in preparation of transferosomes:

Method 1:

Thin film hydration technique in normal shaking condition: (room temperature)

1. Vesicles forming ingredients listed above (phospholipids and surfactant) and the drug aredissolved in volatile organic solvent.

2. Organic solvent is then evaporated by gentle shaking of the formulations in rotary shaker. This

continues until the solvent completely evaporated and a thin film forms on the round bottom

flask.

3. A prepared thin film is hydrated with phosphate buffer of pH 7.4; the buffer is added gradually

in 20 minutes along with continuous shaking in water bath.

Method 2:

Modified hand shaking lipid film hydration technique: (600C)

1. Vesicles forming ingredients listed above (phospholipids and surfactant) and the drug are

dissolved in volatile organic solvent.


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2. Organic solvent was removed by evaporation while hand shaking above lipid transition

temperature (60C) in water bath. The shaking continues till all the solvent get evaporated and a

thin film is formed.

3. The film was then hydrated with phosphate buffer (pH 7.4) with gentle shaking for 20 minutes

at 600C temperature. [Roopesh Sachan et al ,2013]

4. The hydrated thin film was kept overnight in rotary shaker for complete evaporation of

solvent.

Screening of best formulation:

Entrapment Efficiency Calculation:

The total amount of drug encapsulated in the nano vesicles is defined as nano vesicles . This will

provide the encapsulating capacity of the vesicle.

EE% = Total drug concentrationFree drug concentration X 100Total drug concentration.

Steps followed:

2ml of transferosomes are centrifuged in cooling centrifuge for 30 minutes at 8000 rpm.

The supernatant that contains unentrapped drug is collected and the concentration isfound using quantification study.

The OD is calculated at 725 nm range.

Using the gallic acid calibration curve as standard the concentration of drug entrapped iscalculated by the above formula

The formulation with good entrapment efficiency is identified by analyzing this results .

Stability Study:

To analyze the stability of each formulation, it is kept in different temperature and pH range.

After a regular interval the entrapment efficiency is calculated using the above centrifugation

method followed by quantification study.

Temperature: Room temp, 60oC

pH:6.2(acidic),9.58(basic)

Permeation Study: The in-vitro release of drug loaded transferosomes through an artificial

cellophane membrane was determined by a simple dialysis method. Literally this value is similar

to the permeation through the skin.

Steps followed:


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Membrane Activation:The artificial membrane is activated by heating the membrane in buffer 1at 600C for 20minutes.The membrane is taken out and it is washed completely using distilled water.Again the membrane is activated by buffer 2 in the same procedure.

One side of the membrane is closed tightly using a thread, it is checked for leakage usingdistilled water

2 ml of transferosomes loaded with drug is loaded using pipette. Another end is also tied

with thread tightly.

Membrane with the transferosome dipped in 100 ml of PBS buffer.

It is placed in the magnetic stirrer and the 2ml of sample is taken and it is replaced withPBS buffer at every 20 minutes interval.

Again the drug concentration is calculated using Quantification test.

Characterisation Study:FTIR: Interference Study

The drug, transfersome and drug loaded transfersome samples are prepared separately.

FTIR is used for studying the types of chemical bonds between the drug and excipients,and the FTIR spectrum is obtained to investigate the interaction between the -Sitosteroland solid lipid in transfersomes.

Zeta potential:

Size and charge were measured using Zetasizer and Zeta potential respectively. SLN were

determined for their average size distribution and charge in the prepared sample by MalvernZetasizer DTS version 2.0. Average size of the particles was measured thoroughly from the

number distribution data.


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Chapter 5

Results and discussion

Result and Discussion:

Formulation of transfersome:

Table 1:Gallic acid standard values:

Optical Density value of total gingerol in 200 l sample is 1.267

Optical Density value of Unentrapped drug in 200 l sample: 0.662Standart gallic acid graph:

concentration OD

20 0.095

40 0.11

60 0.228

80 0.419

100 0.473

120 0.528

140 0.6

160 0.687

180 0.811

200 0.918


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Fig 1: Transfersome formulation using different constituents

Table 2:The spectrometric values and the corresponding entrapment efficiency values of

four formulation prepared by shaker method:

Formulation OD(at 725 nm) Entrapment Efficiency

T1 0.084 89.47%

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 50 100 150 200 250

ODat725nm

Conc g/ml

Gallic acid standard graph

OD

Linear (OD)


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T2 0.147 76.31%

T3 0.108 75.26%

T4 0.106 82.10%

Table 3:The spectrometric values and the corresponding entrapment efficiency values of

four formulation prepared by modified hand shaking water bath method:

Formulation OD(at 725 nm) Entrapment Efficiency

T1 0.093 84.21%

T2 0.129 79.47%

T3 0.112 81.26%

T4 0.148 78.94%

Effect of surfactant:

From the entrapment efficiency values of span 60 and sodium deoxycholate the formulation

contain span 60(T1) having high drug loading capacity and high stability in different pH and

temperature conditions. The concentration variation shows high concentration of surfactant leads

to pore formation it tends to leakage of drug so it decreases the entrapment efficiency. And also

Span 60 is bio compatible and it is accepted in pharma industry.[Ankit Gupta et al,2012].

Effect of phospho lipid:The transferosome formulation (T3,T4) with cholesterol as phospho lipid have less entrapment

efficiency and the transfersome prepared is not a clear solution ,it contains flakes. Unlike T3, T4

the formulation contains T1,T2 produce better entrapment efficiency and clear transfersome is

formed.

Stability Test:

Stability of transfersomes is found at room temperature and 600C. From the stability study of

seven days the results shows that the stability level of T1 nearly 80% in room temperature and

the stability decreases gradually. . And at 600C the stability range is 805%. The result shows

that the transfersomes are more stable even in normal room temperature and 60 0C condition. The

decrease in stability is due to the leakage or the weakening of the transfersome. But when

compared to the drug content level in different days this is negligible and the further studies in

stability by increasing the time level will gives the clear stability range of the transfersome.


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Table 4: Optical density values of four different formulations in seven days at room

temperature

T1 T2 T3p T4

Day1 0.073 0.082 0.081 0.076

Day2 0.076 0.093 0.076 0.084

Day3 0.077 0.127 0.084 0.105

Day4 0.089 0.110 0.114 0.115

Day5 0.101 0.131 0.123 0.127

Day6 0.092 0.136 0.127 0.154

Day7 0.084 0.157 0.187 0.221

Table 5: Entrapment efficiency values of four different formulations in seven days at room

temperature

T1 T2 T3 T4

Day1 88 83 84 86

Day2 86.5 80 86 81

Day3 85.6 68 82 75

Day4 81.9 75 73 73

Day5 76.6 66 69 68Day6 80.8 64 68 63

Day7 83 63 55 35

Stability values at 60oC

Table 7:Optical density values of four different formulations in seven days at

60oC

T1 T2 T3 T4Day1 0.103 0.120 0.084 0.096

Day2 0.105 0.096 0.093 0.074

Day3 0.106 0.132 0.087 0.101

Day4 0.113 0.126 0.119 0.124


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Day5 0.121 0.148 0.142 0.102

Day6 0.141 0.142 0.170 0.194

Day7 0.154 0.197 0.191 0.206


temperature 60oC

T1 T2 T3 T4

Day1 86.8 82.6 90 88.4

Day2 85.7 89.4 89 92

Day3 85.2 80.5 90.5 86.4

Day4 84.2 82.1 83.1 82

Day5 82.6 77.8 78.9 86

Day6 78.9 78.9 75.7 74

Day7 77.8 73.6 74 73

Size and shape:

Fig 2: Microscopical view of transfersome vesicles

From the microscopical analysis of transfersome the results indicates that the spherical shapedmulti lamellar vesicles are formed and the vesicles are uniformly dispersed without aggregation

irregularities.( SEM result)

Determination of drug content in transferosome: Entrapment Efficiency calculation

The total amount of drug in 1 ml sample= 220 g/ml


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The amount of drug entrapped in 1 ml sample = 160 g/ml

So from this result we interpret that 72% of the drug is entrapped into the transfersome. The

results indicates that this formulation is capable of encapsulate consisting amount of drug. Ankit

Gupta et al also done similar kind of work and the results confirming the ogod entrapment

efficiency of the transferosome.

The formulation containing span 60, soya lecithin and chloroform in 1:1:1 ratio shows 90%

entrapment efficiency. So from this we concluded that this is the best formulation that have good

drug encapsulating capacity.

Stability study:

Table 8: Optical density values of four different formulations in seven days at room

temperature

T1 T2 T3p T4

Day1 0.073 0.082 0.081 0.076

Day2 0.076 0.093 0.076 0.084

Day3 0.077 0.127 0.084 0.105

Day4 0.089 0.110 0.114 0.115

Day5 0.101 0.131 0.123 0.127

Day6 0.092 0.136 0.127 0.154

Day7 0.084 0.157 0.187 0.221

0.00%

10.00%

20.00%30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%

0 1 2 3 4 5

Entrapmentefficiency

T1,T2,T3,T4 formulation

Entrapment efficiency

entrapment

efficiency


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temperature

T1 T2 T3 T4

Day1 88 83 84 86

Day2 86.5 80 86 81

Day3 85.6 68 82 75

Day4 81.9 75 73 73

Day5 76.6 66 69 68

Day6 80.8 64 68 63

Day7 83 63 55 35

Stability study values at 60oC

Table 10:Optical density values of four different formulations in seven days at

60oC

T1 T2 T3 T4

Day1 0.103 0.120 0.084 0.096

Day2 0.105 0.096 0.093 0.074

Day3 0.106 0.132 0.087 0.101

Day4 0.113 0.126 0.119 0.124Day5 0.121 0.148 0.142 0.102

Day6 0.141 0.142 0.170 0.194

Day7 0.154 0.197 0.191 0.206

Table 11: Entrapment efficiency values of four different formulations in seven days at

room temperature 60oC

T1 T2 T3 T4

Day1 86.8 82.6 90 88.4Day2 85.7 89.4 89 92

Day3 85.2 80.5 90.5 86.4

Day4 84.2 82.1 83.1 82

Day5 82.6 77.8 78.9 86


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Day6 78.9 78.9 75.7 74

Day7 77.8 73.6 74 73

Stability of transfersomes is found at room temperature and 600C. From the stability study of

seven days the results shows that the stability level of T1 nearly 80% in room temperature and

the stability decreases gradually. . And at 600C the stability range is 805%. The result shows

that the transfersomes are more stable even in normal room temperature and 60 0C condition. The

decrease in stability is due to the leakage or the weakening of the transfersome. But when

compared to the drug content level in different days this is negligible and the further studies in

stability by increasing the time level will gives the clear stability range of the transfersome.

Drug Release Study:

The penetration capacity of the transfersome is studied by dialysis using a artificial membrane.

The penetration of transfersome through the membrane is very high and the release is much

sustained at regular interval. One of the main reasons for choosing transdermal delivery is

sustained release of drugs through the skin. This proves the penetration capacity and the

sustained releasing mechanism of the drug.

Fig 3: Invitro drug release study using artificial membrane to study the penetration capacity of

the transferosome

Table 12: Optical density values of dialysis study:

Time(minutes) OD at 725 nm

30 0.022

60 0.017


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90 0.018

120 0.015

150 0.019

180 0.010

Invitro release study graph with respect to time

Table 13:In-vitro Drug Release Profile of -Sitosterol Entrapped Transfersomes

0

0.005

0.01

0.015

0.02

0.025

0 50 100 150 200Opticaldensityvalueat725nm

Time(mins)

Invitro Drug release graph

OD at 725 nm


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In-vitro Release Profile of -Sitosterol and -Sitosterol Entrapped Transfersomes

FTIR STUDY: Compatibility study

A broad peak at 3569.91 cm-1 indicates alcohols, phenols with OH stretch, Hbonded. The

peak at 2920.56cm-1, 2851.70 cm-1 and 2377.02 cm-1 indicates alkanes CH stretch was

obtained for -Sitosterol in Fig. 5.2.4 (a). For -Sitosterol entrapped transfersomes peak at

3736.33 cm-1 indicates alcohols, phenols with OH stretch, and peaks at 2920.56 cm-1 and

2851. 70 cm-1 indicates alkanes CH stretch and sharp peak at 1640.93 cm-1 and 1085.68 cm-1

indicate C=O and C-O stretches, respectively in Fig. 5.2.4 (b). The data indicated that flexibility

of the transfersomes to load the -Sitosterol. Similar results were reported by Karan et al., (2012)

in which IR peaks were obtained at 3426.89, 2924.52, 2855.1, 1738.51, 1057.31 cm-1.


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Fig 4: FTIR for transferosome

Fig 5: FTIR for drug( sito sterol)


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Fig 6: FTIR for drug( sito sterol) loadedtransferosome

Zeta potential:

Zeta size Analysis for Empty Transfersomes


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Histogram of Cumulants Mean Particle Size Distribution of Empty Transfersomes

Zeta size Analysis for -Sitosterol Entrapped Transfersomes


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Histogram of Cumulants Mean Particle Size Distribution of -Sitosterol Entrapped

Transfersomes

Zeta Potential Distribution of -Sitosterol Entrapped Transfersomes


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Chapter 6

Conclusion or future perspectives

From the overall result from various studies we know that transfersome act as effective vesicle.

And its encapsulation capacity and penetration capacity is high. It has good compatibility with

the drug used. But the Zeta result shows the size of the transfersomes after loading the drug is

high. Even though the size doesnt affect the penetration if the future studies which helps to

decrease the size will improve the penetration more than 90%. Then it will be the best alternative

for conventional technique. So the further studies will improve this work and the vesicles have

capability to loaded in a patch and further it should be used for the diabetic treatment.


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Chapter 7

Appendix

(Standard operating procedures, media compositions, material safety data sheet, organism data

sheet, ethical certificates, patient compliance form, authentication certificate)


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Chapter 7

References

1. Anahita Rajoul Dezfuli, Aravindram A.S, M.Manjunath2, Ganesh N.S And Shailesh

,Development And Evaluation Of Transdermal Films Loaded With AntihypertensiveDrug, International journal of pharma and bio science, 2012

2.

Hiren J. Patel, Darshan G. Trivedi, Anand K. Bhandari, Dushyant A. Shah,Penetrationenhancers for transdermal drug delivery system: A review , IJPIs Journal ofPharmaceutics and Cosmetology,2011

3.

Jos Juan Escobar-Chvez,Roberto Daz-Torres,Isabel Marlen Rodrguez-Cruz,ClaraLuisa Domnguez-Delgado,Rafael Sampere Morales,Enrique ngeles-Anguian,LuzMara Melgoza-Contreras, Nanocarriers for transdermal drug delivery,2012

4. P. Loan Honeywell-Nguyen2, Joke A. Bouwstra,Vesicles as a tool for transdermal and

dermal delivery,drug delivery formulation and nano technology,2005.5. Prasanthi.D, P.K.Lakshmi,VesiclesMechanism Of Transdermal Permeation: A Review, Asian Journal of Pharmaceutical and Clinical Research,2011.

6. Touitou E, Alkabes M, Dayan N. Ethosomes: novel lipid vesicular system for enhanceddelivery.1997

7. Nikalje Anna Pratima, Tiwari Shailee,International Journal of Research in Pharmacy andScience,Ethosomes: A Novel Tool for Transdermal Drug Delivery,2012

8.

Yasam Venkata Ramesh*, N.Jawahar, Satya Lavanya Jakk,Proniosomes: A Novel NanoVesicular Transdermal Drug Delivery, Journal of pharmaceutical science,2011.

9. Eder lilia Romero and maria Jose Morilla, Highly deformable and highly fluid vesicles as

potential drug systems,international Journal of nano medicine.2013.10.

Nathji dhaval*, patni chandra, shah hira, dr. Chaudhary sunita, sanghavi kinjal, dr. Patelupendra, transferosome: an enhancement approach for transdermal drug deliverysystem,international journal of pharmacy and integrated life sciences.2013.

11.

Pushpa Anand*, A. Samnani, M. Bhoumick, B.K. Dubey,Ultra Deformable ElasticVesicles Transferosomes For Enhanced Skin Delivery- An Update,world Journal ofpharma,2012.

12.Saeed Ghanbarzadeh and Sanam Arami,Enhanced Transdermal Delivery of DiclofenacSodium via Conventional Liposomes, Ethosomes, and Transfersomes.Biomed researchinternational.2013.

13.

Marwa H. Abdallah, Transfersomes As A Transdermal Drug Delivery System ForEnhancement The Antifungal Activity Of Nystatin , International Journal of Pharmacyand Pharmaceutical Sciences,2013.

14.Subadhra Sandhya1, Parre Saikumar1, Reddy Tera Rohit1, David Banji.Critical IssuesRelated To Transfersomes Novel Vesicular System, Acta Sci. Pol., Technol.Aliment.2012.


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15.Roopesh Sachan, Tarun Parashar, Soniya, Vishal Singh1, Gaurav Singh1, SatyanandTyagi2, Chirag Patel, Anil Gupta, Drug Carrier Transfersomes: A Novel Tool ForTransdermal Drug Delivery System,International Journal Of Research And DevelopmentIn Pharmacy And Life Sciences,2012

16.Ankit Gupta , Geeta Aggarwal , Samita Singla , Ritika Arora Transfersomes: A Novel

Vesicular Carrier for Enhanced Transdermal Delivery of Sertraline: Development,Characterization, and Performance Evaluation,sciebtia pharmaceutica,2012.

Chapter 8

Curriculum vitae

J.Perinba Danisha

+91 9043930431

[email protected]

Career Objective

To Enhance and develop my technical skills and continually learn about the latestparadigm and processes and to fulfill my needs and aspirations along with theorganization with loyalty and honesty.

Academic Profiles

Examination Specialization College/School Board/

University

Year Of

Passing

Percentage

B.TECH Bio

Technology

Jeppiaar

Engineering

college

Anna

University

2014 75

HSC Maths

Biology

St.Josephs

Convent

Hr.Sec.Scl

Tamil Nadu

Board Of

Examinations

2010 85.5

SSLC SSLC St.Josephs

Convent

Hr.Sec.Scl

Tamil Nadu

Board Of

Examinations

2008 93.5


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Project Description

Project in Novel drug delivery system using Nano Technology regarding Diabetic

Treatment with the help of Transdermal patches.

Technical Proficiency

Operating System : Windows OS Languages : C (basics) Packages : MS Office.

Trainings

Completed training on Bioinformatics, Chromatography& Molecular Biology organizedby VIT University Vellore.

Completed training on techniques in biotechnology organized by Viveks lab Nagercoil. Successful completion of course in CMOA.

Achievements

Passed business English communication (BEC) vantage exam. Participated in international seminar on stem cell and regenerative medicine Participated in seminar on popularization of bio technology conducted by ocean research

centre, Sathyabama University. Worked as a secretary of RRC club in my school.

Extracurricular activities:

I have won inter school speech and essay competitions Led my school basket ball team and won prices.

Worked as an organizer in Jeppiaar Engineering college hostel day fest, Gazellina 2013.

Personal Traits

Disciplined & good etiquette. Willingness to work in team and hard worker. Goal Oriented Active Team Player Flexibility and Self-motivated person.

Area of Interest

Micro Biology Cancer Biology Bioinformatics

Hobbies

Listening songs


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Pencil sketching Gardening

Personal Profile

Name : Perinba Danisha.J

Father's Name : Mr.D.Jesubatham Date of Birth : 05-10-1992 Gender : Female Marital Status : Single Nationality : Indian

Address: Library street,Mission Compound,

Aralvoimozhi.Kanyakumari dist.629301.

Declaration

I hereby declare that the above-mentioned information is correct up to my knowledge and I bearthe responsibility for the correctness of the above-mentioned particulars.Date : 23/02/2014 yoursauthentically,Place :Chennai PerinbaDanisha.J

transdermal drug delivery using transferosomes

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