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PROJECT TOPIC

“DRUG RELEASE FROM SILK FIBROIN MICROPARTICLES”

By

Nikita Dewangan

B. Tech, 3rd Year

Department of Chemical Engineering

National Institute of Technology, Rourkela, Odisha

Guided By:

Dr. V. Premnath

Principal Scientist

Polymer Science and Engineering

National Chemical Laboratory, Pune, Maharashtra

&

Anuya Nisal

Scientist

Polymer Science and Engineering

National Chemical Laboratory, Pune, Maharashtra

At

National Chemical Laboratory, Pune, Maharashtra

Project Duration:

5th December 2013 to 5th January 2014

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Acknowledgement:

Words are only representation of my regards and gratitude that I have towards my mentor.

As a matter of fact, without co-operation no thought could be coined into action. Consistent motivation and invaluable support throughout the project is an issue that cannot be quantitatively measured.

I sincerely thank my Supervisor, Dr. V. Premnath for guiding me throughout the project and correcting the project report with attention and care.

It gives me immense pleasure to express my sincere and deepest sense of gratitude that I had an opportunity of working under the guidance of Mrs. Anuya Nisal, an eminent and renowned scientist from NCL, Pune.

I am also grateful to Miss. Bhakti Khude and Miss Chaitali S Deshpande, Project Assistant, for helping me with great effort throughout this project.

Lastly I would like to sincerely thanks NCL administration for allowing me work in the campus with good co-operation and care for me.

-Nikita Dewangan

- 03-01-2014

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Content

SR NO Title Page No 1 Introduction 4-6 2 Materials Required 6 3 Instruments used and their use in this study 6-7 4 General method 7-8 5 Calculations 8-10 6 Observations 10-19 7 Result and Discussion 19 8 Future work 20 9 Conclusion 20 10 References 20-21

Graphs

Sr No Title Page No 1 UV spectra of Cephalexin in Water at different concentration

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2 Calibration curve for Cephalexin

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3 UV-Vis Spectra of Methanol annealed particles 13 4 UV-Vis Spectra of Water annealed particles 13 5 UV-Vis Spectra of Autoclave annealed particles 14 6 Concentration vs time curve of water annealed particles

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7. Concentration vs time curve of water annealed particles

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8. Comparison of amount of drug release from water annealed and methanol annealed particles.

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9. Fitting curve for water annealed particles 18 10. Fitting curve for methanol annealed particles 19

Tables

Sr no Title Page no 1 ATR analysis of with drug particles 10 2 ATR analysis of without drug particles 11

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3 Drug release data- water annealed particles 14 4 Drug release data- methanol annealed particles 16

ABSTRACT

Silk fibroin has been used in various biomedical as well as tissue engineering application. The main goal of this project was to study the possibility of the use of silk derived fibroin as microsphere for the release of drug prepared under various annealed condition. And also to observe the amount of drug release as compare to the amount of drug loaded on each particle. The result of this project was the particles with different annealed condition shown different release concentration of drug also depending upon the crystallinity of the particles.

Keywords: B.mori, Regenerated Silk Fibroin (RSF), drug release.

1. Introduction A wide range of polymeric materials are now-a-days available for biomedical applications. The number of materials that are approved for human use however is small. They are recently receiving much attention mainly as a means of drug delivery system over a prolonged period of time enlarging the drug to specific sites as it is very necessary to show its reaction fast and steadily for the complete relieve of the problem in human body. One of the material which can be used to make drug delivery system is Silk. The silkworm chosen for this project is the larva or caterpillar of the domesticated silk moth. Being an economically important insect, it is a primary producer of Bombyx Mori silk (B. Mori) B. Mori consist of: Fibroin Sericin (70-80 %) (20-30 %)

B. Mori silk fibroin can be made into different

• Films

• Gels

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• Porous material

• Non-woven mats

Uses of silk fibroin in various fields such as:

Controlled drug delivery system

Anti coagulant blood material

Biosensors

Artificial ligaments

Artificial skin

Here in this project we have used microparticles as a drug delivery system.

Some of the superior characteristics of B. mori:

Biocompatible

Biodegradable

Moisture and oxygen permeability

Promoting proliferation of human keratinocytes

Human skin fibroblasts ( it plays an important role in wound healing)

Silk Fibroin has several unique properties making it favourable matrix for incorporation and delivery of various range of drugs. The drug release from biodegradable microparticles is governed by various properties of the polymer, drug and carrier system. Polymer dependent factors include molecular weight and crystallinity.

There are two types of structure present in silk fibres:

Silk-I which is referred to as complex helix-dominated structure before spinning.

And Silk-II which is insoluble beta sheet crystals formed after the spinning.

The unique feature of the hydrophobic blocks is that they have highly repetitive sequences of glycine and alanine, which are responsible for the formation of tightly bonded anti-parallel β-sheet structure.

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The transition of silk fibroin can be induced to change from a random coil to a β-sheet structure by some treatments such as heating, stretching, or immersion in polar solvents. This transition makes silk fibroin attractive as a biomaterial because silk fibroin with a β-sheet structure is resistant to water and has good mechanical properties.

There is a critical need in medicine to develop simple and versatile methods to assemble robust, biocompatible and functional biomaterial coatings that direct cellular outcomes. So in this project we have used silk fibroin in the form of micro spheres which can be used as drug carriers and delivery vehicles. Silk material exhibit drug release behaviour dependent on diffusion of the drug through the polymer matrix, the degradation of the matrix or a combination of both mechanisms. Hydrophobic interactions were shown to be the main cause of interactions between Silk fibroin and drugs with hydrophobic moieties due to the hydrophobic blocks in the heavy chain of Silk Fibroin. (REF-2)

2. Material Required: 2.1 Raw material: Cocoons 2.2 Chemicals and Reagents used: NaHCO3, De-ionized water, LiBr, liquid

nitrogen, methanol 2.3 Drug Used: Cephalexin (antibiotic)

Cephalexin has a detectable range in UV-Vis Spectrophotometer is 261-262nm. It is often used in infection. It is very heat sensitive and must be kept in temperature range of 25-35°C.

2.4 Other Requirements: Cellulose acetate dialysis tubings, Syringe pump, electronic balance machine, magnetic strirrer.

3. Instruments used and their use in this project: 3.1 Freeze Dryer: Freeze drying, also known as lyophilisation is a

dehydration process. Freeze dryer works on the principle of freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase.

Use in this project: It is used to sublimate the frozen water in the particles.

3.2 Attenuated Total Reflectance (ATR): It uses the property of total internal reflection resulting in the evanescent wave. The beam of infra red is passed through the ATR crystals it then reflects at least once off the internal surface in contact with the sample. The

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penetration depth into the sample is between 0.5-2 micrometers. The number of reflections can be varied by varying the angle of incidence. The beam is then collected by the detector.

Use in this project: It is used to determine the crystallinity index of the particles with and without drug in microparticles.

3.3 UV-Vis Spectrophotometer: It is used to determine the amount of desired compound present in the solvent. It has its detectable range of wavelength between 200-1200nm.

Use in this project: It is used here to determine the amount of drug released from different annealed particles by measuring the highest value of absorbance at the detectable range of drug (Cephalexin) used here.

4. GENERAL METHOD: Processing of Silk

4.1 Preparation of regenerated silk fibroin 1. DEGUMMING: B. Mori silk cocoons were firstly boiled in 5 liters of a 0.5 wt-%

solution of NaHCO3 for half an hour for two batches. This process is known as degumming of silk, in which sericin is removed from the silk.

2. Degummed fibroin was rinsed thoroughly and dried at ambient condition overnight.

3. The fibroin obtained after degumming process was slowly dissolved in 9.3M LiBr solution and stirred continuously at a constant temperature of 60 °C for about 4 hours.

4. Dialysis of RSF is done by keeping it in cellulose acetate bag and then this bag is kept in a large beaker of volume 5lit in a cold room at a temperature between 4-5 ° C for 48 hours and water is changed firstly for 3 hours then after 6 hours and then after the interval of 12 hours to complete the total of 48 hours. And the solution obtained from this process is known as regenerated silk fibroin (RSF).

5. After dialysis the concentration of RSF was measured using Gravimetric method and it was obtained as 4.2 wt/wt. NOTE: All the above steps followed standard protocol as set previously (REF: 1).

4.2 Preparation of particles with and without drug

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1. Preparation of stock solution of drug: 10mg of drug (Cephalexin) was measured in electronic balance it then dissolved in 1 ml of de-ionized water and mixed properly so that drug is completely dissolved in it.

2. Preparation of with drug solution of RSF: 6ml of RSF was taken in 3 vials (each) and 240 µl of drug were put in each vials from 10mg/ml of stock solution of drug. And in another vial 6ml of only RSF is taken as a without drug solution.

3. With the flow rate of 0.1 ml/min with drug was dropped from the height of 92cm using syringe pump into Dewar flask containing liquid nitrogen. 16 no. of particles are formed per minute from each batch, hence 960 particles were finally collected in liquid nitrogen from each batch using syringe pump after every 1 hour. Similarly 960 were particles obtained from without drug solution.

4. Lyophilization: Particles thus obtained were then lyophilized in freeze dryer to sublimate the frozen water in the particles and this was done for about 15-16 hours.

5. The beads formed were white in colour, spherical in shape.

4.3 Annealing of particles with and without drug

Annealing of the freeze dried particles was done using following protocol:

Methanol Annealing: Around 900 particles (with drug) were kept in 10ml of methanol for 1 hour. Methanol was then removed and particles were kept to dry at room temperature for overnight. Similar procedures were carried out with 10 particles (without drug) but in 1ml of methanol.

Water Annealing: Around 900 particles (with drug) were spread out in a petri dish. 200ml amount of water is taken in 2litre beaker and inside it, kept a 400ml beaker containing 100ml of water with 10-15 small size ceramic rings of less than 1cm in diameter in order to provide better heating by giving rise to bubble formation. At the top of small beaker, petri dish containing particles was kept and the bigger beaker was covered with thin cloth to prevent the water vapour from condensing and falling into the particles (if any non-porous material is used like aluminium foil). The whole set up is kept in a heater for 60-90min at a temperature of 80°C. Similarly 10 particles (without drug) were also annealed.

Autoclave Annealing: Around 900 particles (with drug) were kept in autoclave at 121°C and at high pressure for 15-20min. Similarly 10-20 particles without drug is also annealed.

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5. CALCULATIONS: 5.1 : Calculation of RSF concentration using Gravimetric analysis

Let, W1=weight of empty petri dish W2=weight of petri dish with RSF (upto> 0.2 increase in decimal of W1) before heating W3= weight of petri dish with RSF after heating Heating Conditions: Temperature= 60°C Time= 1hour Vacuum supply of -25to -30 of mercury Wt% = ((W3-W1)/ (W2-W1))*100 = 4.2wt/wt % (for the RSF obtained)

5.2: Calculation of amount of drug loaded in particles

1. 16 particles are obtained in 1min with the flow rate of 0.1 ml/min thus 0. l ml of RSF solution with drug gives 16 particles per min .

2. 10mg of drug is dissolved in 1ml or 1000µl of water thus o.1 mg of drug is dissolved in 10µl of water. So in order to obtain 0.1 ml of water will contain 0.01mg of drug. Hence the amount of drug present in 16 particles =0.01mg

3. With drug RSF solution:

0.1ml RSF sol of concentration 4.2wt/wt (42mg/ml) will contain 4ul of 10mg/ml drug solution

10µl drug solution contains 0.1mg drug in 1ml water

Thus, 0.1 ml of water will contain 0.01mg drug

So 4µl of drug solution will contain 0.04mg of drug

So weight fraction of drug in RSF (FR) = weight of drug/( weight of drug+ weight of fibroin)

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=0.04/ (0.04+4.2)

=0.009434

Weight each particle made from 0.1 ml of RSF (Wt)= 4.2/16 = 0.2625

Thus the amount of drug present in each particle= FR *Wt

=0.2625*0.009434

= 0.00248

Thus the amount of total drug present in 16 numbers of particles

=16*0.00248

= 0.03960

6. OBSERVATIONS: 6.1 ATR analysis 6.2 ATR analysis of as is (without annealed) particles and annealed particles (with and

without drug). 6.3 Table .1

ATR analysis of with drug particles

Sr.No Sample Type Crystallinity Average

value Standard deviation

1 Without annealed 0.899029 0.866 0.053 0.893118 0.661037 2 Methanol

annealed 1.229113 1.2 0.032

1.133377 1.177132 3 Water annealed 1.080796 1.05 0.033 1.015747 1.057938

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4 Autoclaved

annealed 1.233091 1.3 0.07

1.46246 1.137125

Table.2

ATR analysis of without drug particles

Sr.No Sample Type

Crystallinity Average Value Standard Deviation

1 As is 0.884752 0.85 0.06 0.803854 0.761363 2 Methanol 1.208189 1.30 0.09 1.382727 1.349291 3 Water 1.078478 1.07 0.02 0.966849 1.058592 4 Autoclaved 1.390915 1.32 0.06 1.270785 1.294962

6.2: UV-Vis Spectra

Graph .1

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Graph.2 CALIBRATION CURVE FOR CEPHALEXIN

The equation of the straight line obtained from the graph is y=17.4*x-0.00007

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

200 250 300 350 400

Abso

rban

ce(A

)

Wavelength(nm)

UV-Vis Spectra of Cephalexin in water

0.05mg/ml

0.04mg/ml

0.03mg/ml

0.015mg/ml

0.01mg/ml

0.005mg/ml

0.003mg/ml

Peaks at 262nm for different concetrations of cephalexin

Absorbance-3y = 17.40x - 7E-05

R² = 0.997

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 0.01 0.02 0.03 0.04 0.05 0.06

Abso

rban

ce

Conc (mg/ml)

Calibration curve for Cephalexin

Absorbance-3

Linear (Absorbance-3)

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UV-VIS Spectroscopy of particles with drug

1. Methanol annealed particles: Graph.3:

2. Water annealed particles

Graph -4:

Graph-5

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

200 250 300 350 400

Abso

rban

ce(A

)

Wavelength(nm)

UV_Vis Spectra of Methanol annealed particles

0min

5min

15min

30min

45min

60min

90min

120min

180min

260min

peaks at 262nm of cephalexin

0

0.5

1

1.5

2

2.5

200 250 300 350 400

Abso

rban

ce(A

)

wavelength(nm)

UV-Vis spectra for water annealed particles

0min

5min

15min

30min

45min

60min

120min

180min

260min

peaks at 262nm of cephalexin

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It can be seen from the above graph that there is no peak at 262nm which is a detectable value of Cephalexin. Hence we can interpret that Cephalexin present in autoclaved annealed particles may have degraded due to exposure to very high temperature of 121°C in an autoclave as it is found from the literature that Cephalexin is very heat sensitive and can only be kept at 25-35°C above which it has a great chance to degrade.

6.4 Drug Release Data : 6.4.1: Table-3 Water annealed particles Time (min) Concentration(mg/ml) Standard deviation 0 0 0 5 0.0040 0.0011 15 0.0075 0.0026 30 0.0106 0.0015 45 0.0126 0.0010 60 0.0117 0.0006 120 0.0129 0.0005 180 0.0150 0.0009 260 0.0150 0.0007 Concentration value at different time can be calculated using the equation y=17.4*x-0.00007, from the Calliberation curve. Where:

0

0.5

1

1.5

2

2.5

200 250 300 350 400

Abso

rban

ce(A

)

Wavelength(nm)

UV-Vis Spectra of Autoclave annealed particles 20

min

30min

40min

50min

60min

120min

210min

No peak at 262nm

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y= Absorbance value x=concentration And, x=(y+0.00007)/17.4; Curve of concentration of drug released from the particles at different time Graph-6

Graph-6:

Data given in the table-3 can be fitted using Origin Pro-8 and it is shown as

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

0 50 100 150 200 250 300

Conc

entr

atio

n of

dru

g re

leas

ed fr

om th

e pa

rtic

les

(mg/

ml )

time(min)

concentration vs time curve of water annealed particles

Series1

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DATA SET : Equation: Y= (1-(1/x^A) Where: x=time in min Y=concentration A=constant =0.00281 Adj. R-square=0.92627

6.4.2 Table-4

Methanol annealed particles

Time(min) Concentration(mg/ml) Standard Deviation 0 0 0.000 5 0.003662 0.003 15 0.005815 0.003 30 0.008117 0.002 45 0.008737 0.002 60 0.008579 0.001

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90 0.008959 0.001 120 0.009327 0.001 180 0.010196 0.002 260 0.011176 0.002 Concentration value at different time can be calculated using the equation

y=17.4*x-0.00007, from the Calliberation curve. Where: y= Absorbance value x=concentration And x=(y+0.00007)/17.4; Curve of concentration of drug released from the particles at different time Graph-7

Graph-8

Data given in the table-4 can be fitted using Origin Pro-8 and it is shown as:

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0 50 100 150 200 250 300

Conc

entr

atio

n of

dru

g re

leas

ed fr

om th

e pa

rtic

le(m

g/m

l)

Time(min)

Concentration vs time curve for methanol annealed particles

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DATA SET: DATA SET : Equation: Y= (1-(1/x^A) Where: x=time in min Y=concentration A=constant =0.00204 Adj. R-square=0.85245

6.5 Comparison of Drug release from water annealed particles and methanol annealed particles Graph-9:

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7. Result and Discussion: The ATR analysis was done to proof the same crystallinity in particles with and without drug. The ATR analysis of particles with and without drug also shows that the value of crystallinity index of autoclaved particles is highest which explain its high crystallinity as compare to rest others. Among water and methanol, methanol has greater value of crystallinity index and water has less. Since crystallinity plays an important role in drug delivery system the release of drug can be interpreted in relation with the crystallinity of particles in way such that the more crystallinity the particle has, drug release from it is slower and lesser as can be seen in data table-4 and table-3 that methanol annealed particles has lower concentration of drug released as compared to water annealed particle observed using UV-Vis Spectrophotometer. In the graph-9 it can be seen that initially there is not much difference in the value of concentration of methanol annealed and water annealed particles but for longer time the deviation is much larger. In case of autoclave annealed particles it is clearly observed that there is no peak at 262nm, detectable value of Cephalexin in UV-Vis spectrophotometer so

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

0 50 100 150 200 250 300

Conc

entr

atio

n (m

g/m

l)

Time(min)

Comparison of amount of drug release from water annealed and

methanol annealed particles.

Water

Methanol

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no data is collected for the amount of drug released from the autoclave annealed particles.

8. Future work: 1. Study of drug release in PBS to ensure the same trend of drug release as in

water. 2. Study of drug release from scaffolds made out of annealed particles. 3. And the same experiment can be carried out with different type of drug to

study the release of drug from autoclave annealed particles also. 4. The drug release observation can be taken for closer interval of time in order

to get best fitting curve for the concentration versus time curve.

Effective ways to increase the drug release from microspheres are:

• To increase the initial drug loading while making the particles with drug. • Prefer matrix with lower value of crystallinity index.

9. Conclusion:

Microspheres were successfully prepared both with and without drug. Microspheres as drug carriers have the advantage of sustained or controlled release. Therefore microparticles as a delivery system have drawn much attention in the pharmaceutical field and have been successfully applied in laboratory trials.

10. References: 1. Nairiti_report scaffold 2. Esther Wenk, Silk Fibroin as a vehicle for drug delivery in tissue regeneration,

ETH ZURICH. 3. Kaplan et.al, Effect of Silk Protein processing on Drug Delivery from Silk Film,

Macromolecualr Journals. 4. K.Z Gumargalieva et.al,Biodegradable polymeric microparticles in biomedical

applications; International Journal of Polymeric materials and Polymeric biomaterials

5. Lu Wang et.al, Preparation of Silk fibroin microparticles and its cytocompatibility, Journal of Biomaterials and nano-biotechnology, 2013, 4, 84-90.

6. Bessa et.al, Silk Fibroin microparticles as carriers for delivery of human recombinant BMPs, physical characterisation and drug release, Journal of tissue engineering and regenerative medicine.

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7. Alagusundaram.M et.al/Int.J Chem. Tech Res.2009, Microspheres as a novel drug delivery system-A review.

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