prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/9272/1/Maqsood ur Rehm… · II CERTIFICATE OF APPROVAL This is to certify that the research work presented in this thesis,

FABRICATION AND EVALUATION OF SOLID LIPID

NANOPARTICLES FOR NICLOSAMIDE (BCS-II) AND

SULFASALAZINE (BCS-IV) DRUGS

By

MAQSOOD-UR-REHMAN

DEPARTMENT OF PHARMACY

UNIVERSITY OF MALAKAND

2012-2015

FABRICATION AND EVALUATION OF SOLID LIPID

NANOPARTICLES FOR NICLOSAMIDE (BCS-II) AND

SULFASALAZINE (BCS-IV) DRUGS

This dissertation is submitted

As partial fulfilment of the requirement for the

Degree of

Doctor of Philosophy in Pharmacy

By

MAQSOOD-UR-REHMAN

DEPARTMENT OF PHARMACY

UNIVERSITY OF MALAKAND

2012-2015

I

In the name of Allah

The Most Gracious,

Merciful and Compassionate

II

CERTIFICATE OF APPROVAL

This is to certify that the research work presented in this thesis, entitled “ FABRICATION

AND EVALUATION OF SOLID LIPID NANOPARTICLES FOR NICLOSAMIDE

(BCS-II) AND SULFASALAZINE (BCS-IV) DRUGS ” was conducted by Mr. Maqsood

Ur Rehman under the supervision of Prof. Dr. Mir Azam Khan. No part of this thesis has

been submitted anywhere else for any other degree. This thesis is submitted to the Department

of Pharmacy, University of Malakand in partial fulfillment of the requirements for the degree

of Doctor of Philosophy in Field of Pharmacy (Pharmaceutics), Department of Pharmacy,

University of Malakand.

Student Name: Maqsood Ur Rehman Signature: __________

Examination Committee:

a) External Examiner 1: Signature:___________

Dr. Asad Ullah Madani Assistant Professor

Department of Pharmacy

Islamia University Bahawalpur

b) External Examiner 2: Signature:___________

Dr. Saeed Ahmad Khan Assistant Professor


Kohat University of Science and Technology, Kohat

c) Internal Examiner: Name Signature:___________

Dr. Waqar Ahmad

Professor


Supervisor

Prof. Dr. Mir Azam Khan Signature:____________

Professor


Co-Supervisor

Dr. Waheed S. Khan Signature:____________

Principal Scientist

NIBGE Faisalabad

Prof. Dr. Munasib Khan Signature:_____________

Chairman


University of Malakand

III

CERTIFICATE

It is certified that Maqsood Ur Rehman having enrolment number 20080030101 has

carried out all the work related to the thesis entitled "Fabrication and Evaluation of

Solid Lipid Nanoparticles for Niclosamide (BCS-II) and Sulfasalazine (BCS-IV)

Drugs" under my supervision at the Department of Pharmacy, University of Malakand

in partial fulfilment for the award of PhD degree.

Date: _________________ Supervisor: _______________________

Dr. Mir Azam Khan

Professor



_____________________________

Chairman



IV

AUTHOR’S DECLARATION

I Mr. Maqsood Ur Rehman hereby state that my PhD thesis titled entitled

"Fabrication and Evaluation of Solid Lipid Nanoparticles for Niclosamide (BCS-II)

and Sulfasalazine (BCS-IV) Drugs" is my own work and has not been submitted

previously by me for taking any degree from this University (University of Malakand)

or anywhere else in the country/world.

At any time if my statement is found to be incorrect even after my Graduate the

university has the right to withdraw my PhD degree.

Maqsood Ur Rehman:

Date:

V

PLAGIARISM UNDERTAKING

I solemnly declare that research work presented in the thesis titled Fabrication and

Evaluation of Solid Lipid Nanoparticles for Niclosamide (BCS-II) and Sulfasalazine

(BCS-IV) Drugs ” is solely my research work with no significant contribution from

any other person. Small contribution/help wherever taken has been duly

acknowledged and that complete thesis has been written by me.

I understand the zero tolerance policy of the HEC and University of Malakand

towards plagiarism. Therefore I as an Author of the above titled thesis declare that no

portion of my thesis has been plagiarized and any material used as reference is

properly referred/cited.

I undertake that if I am found guilty of any formal plagiarism in the above titled thesis

even after award of PhD degree, the University reserves the rights to withdraw/revoke

my PhD degree and that HEC and the University has the right to publish my name on

the HEC/University Website on which names of students are placed who submitted

plagiarized thesis.

Student /Author Signature:______________

Maqsood Ur Rehman

VI

DEDICATED TO MY FATHER

HAJI MIR HAIDER SHAH (LATE)

VII

ACKNOWLEDGEMENT

Millions of thanks to Almighty ALLAH – the most gracious, merciful and

beneficent, Who has blessed me with the knowledge and power to perform and

complete this PhD research study. In addition, Who has always guided me in difficult

times of which I have never imagined in my life.

I take this opportunity to express my gratitude towards my supervisor Prof. Dr.

Mir Azam Khan for letting me be a part of his research group. His encouragement to

think independently, motivation and guidance during my PhD at the University of

Malakand made this research project successful. I thank him for providing me with all

possible resources to accomplish this project.

I would like to express my heartfelt thanks to my co-supervisor Dr. Waheed. S.

Khan, Principal Scientist and Head of Nano-Biotech Group at National Institute for

Biotechnology and Genetic Engineering Faisalabad for all his support and valuable

advice during these years and his kind support during my stay at NIBGE Faisalabad.

I would like to thank my sister and brothers i.e. Amjad Ali Shah, Shehryar Khan

and especially Atiq ur Rehman without their enduring support, this stage of my carrier

is not possible.

I am very thankful to the members of Graduate Studies Committee, Department

of Pharmacy and members of Advanced Studies Research Board, University of

Malakand for their support and guidance.

I thank to Ferozsons Laboratories Nowshera and Shaigan Pharmaceutical

Rawalpindi, Mr. Zahir Rahman Dy. General Manager Mr. Nadeem Khan Q.C analyst

at Ferozsons Labs, Mr. Abdullah, Mr. Khan Malook of CRL (Centralized Research

Lab) University of Peshawar, Mr. Farooq Aslam of NIBGE Faisalabad.

I am very much thankful to my colleagues Prof. Dr. Waqar Ahmad, Prof. Dr.

Munasib Khan Dr. Muhammad Junaid, Dr. Mohammad Shoaib, Dr. Farhat Ullah, Dr.

Abdul Sadiq, Dr. Nasiara Karim, Dr. S.Wadood Ali Shah, Dr. Shahzeb Khan, Mr.

Jahangir Khan, Ms. Rukhsana Ghaffar Mr. Mubashir Ahmad, Mr. Jamil Anwar

Abbasi, Ms. Mehrin Ghias, Mr. Aziz Ur Rehman, Dr. Muhammad Ayaz and Mr.

Qamar Gul.

VIII

I am very much thankful to my friend Dr. Abdullah for his moral support

throughout my academic and professional career, my lab fellows Mr. Muhammad

Shafique, Mr. Muhammad Ibrar, Mrs. Mehwish Kamran, Mr. Rizwan and Mr. Ihsan

for helping me in my research project, all the supporting staff of Department of

Pharmacy especially Mr. Rahim Khan, my friends Dr. Salman Zeb, Dr. Adnan Khan,

Mr. Asad Muhammad, Mr. Jahanzeb Khan, Mr. Muhammad Farooq, Mr. Ibrahim,

Mr. Ikram Ullah, Mr. Muhammad Hussain and Mr. Muhammad Ashfaq Khalil for

their moral support.

Last but not the least I am very much thankful to my mother, wife, my son

Abdul Moiz Rehman, my daughter Maham Gulalai and my family members including

Mr. & Mrs. Dr. Habib Khan, Engr. Zulqarnain Habib, Dr. Furqan Habib and Mehtab

ur Rehman for their love, support and encouragement and for making this possible.

IX

ABSTRACT

New drug entities with poor aqueous solubility are becoming more prevalent as result

of high-throughput screening in drug discovery. Poor aqueous solubility presents

significant challenges, as it reduces the absorption and oral bioavailability. Several

formulation approaches have been employed to overcome the limitations of low

dissolution rate and/or solubility including; pH-adjustment, co-solvents, surfactants,

inclusion complexes, lipid-based formulations i.e. Solid lipid nanoparticles (SLNs)

and Nanostructured lipid carriers (NLCs), and nano-suspensions. In this study efforts

are made for the selection of formulation approach based on the drug properties and

the required specifications of the final dosage form. Among these formulation

approaches solid lipid nanoparticles were selected with the aim of improving

solubility/bioavailability of the poorly water-soluble drugs; BCS-II (Niclosamide) and

BCS-IV (Sulfasalazine).

Two different techniques i.e. Micro-emulsion Technique and Solvent

Emulsification Diffusion Technique were used to fabricate SLNs. The SLNs

formulations were characterized by Scanning Electron Microscopy (SEM),

Differential Scanning Calorimetry (DSC), Powder X-ray Diffraction (P-XRD) and

Fourier Transform Infrared (FT-IR). The SLNs formulations loaded with Niclosamide

and Sulfasalazine were successfully converted to solid dosage form followed by

similarity study. In-vitro studies of SLNs formulations in comparison with marketed

dosage form showed improvement in solubility and dissolution while the in-vivo

studies confirmed improved oral bioavailability.

Niclosamide loaded SLNs fabricated by Micro Emulsion Technique having

particle size 204.2 ± 2.2 nm, polydispersity index 0.328 ± 0.02, zeta potential -33.16 ±

2mv, entrapment efficiency 84.4 ± 0.02%, and drug loading capacity 5.27 ± 0.03%

X

were obtained. Different kinetic models showed zero order kinetics and Case-II

transport mechanism. In-vivo pharmacokinetic study showed 2.15-fold increase in

peak plasma concentration for Niclosamide loaded SLNs while relative bioavailability

(Fr) of 11.08.

Fabrication of Niclosamide loaded SLNs using Solvent Emulsification

Diffusion Technique showed particle size 208.6 ± 2.2 nm, polydispersity index 0.376

± 0.04, zeta potential -34.11 ± 1.2 mv, entrapment efficiency 85.4 ± 0.04% and drug

loading capacity 3.18 ± 0.04 %. Observed zero order kinetics with case-II transport,

the range in the parenthesis might be helpful for the drug release mechanism. 2.04-

fold increase in peak plasma concentration was observed in pharmacokinetic study

with relative bioavailability (Fr) of 10.59.

In case of Sulfasalazine-SLNs prepared by Micro Emulsion Technique having

particle size 217.2 ± 3.2nm, PDI 0.373 ± 0.02, zeta potential -35.26 ± 2mV,

entrapment efficiency 89.1 ± 0.03% and drug loading capacity 2.87 ± 0.05% were

obtained. Kinetic modelling studies showed mixed order kinetics for drug release.

Release exponent was more than 0.89, regarded as Super Case-II diffusion

mechanism. In-vivo pharmacokinetic study showed 2.43-fold increase in oral

bioavailability of sulfasalazine as SLN formulation compared to commercial product.

Solvent Emulsification Diffusion Technique was used to fabricate

Sulfasalazine loaded SLNs, showed particle size 202.3 ± 2.2 nm, PDI 0.376 ± 0.02,

zeta potential -35.82 ± 2 mV, entrapment efficiency 86.3 ± 0.02% and drug loading

capacity 3.03 ± 0.04%. Zero order kinetics and Case-II transport mechanism for drug

release was observed with 1.86-fold increase in peak plasma concentration during

pharmacokinetic study.

XI

These studies validated that, SLNs as nanoparticulate drug delivery system

enhanced oral bioavailability of Niclosamide and Sulphasalazine. Hence, these studies

provide new strategies for the oral bioavailability of hydrophobic drugs.

XII

LIST OF ACRONYMS

ACRONYMS EXPLANATION

AUC Area Under Curve

BBB Blood Brain Barrier

BCS Biopharmaceutical Classification System

BP British Pharmacopeia

Cmax Maxium plasma concentration

DLC Drug Loading Capacity

DSC Differential Scanning Calorimetry

EDTA Ethylene Di-amine tetra-acetate

EE Entrapment Efficiency

FDA Food and Drug Administration of United States

FT-IR Fourier Transformed Infrared Spectroscopy

GIT Gastrointestinal Tract

HPH High Pressure Homogenization

HPLC High Performance Liquid Chromatography

HSH High Shear Homogenization/High Speed Homogenization

MDR Multiple Drug Resistance

NIC Niclosamide

NINT National Institute for Nanotechnology-Canada

NNI National Nanotechnology Initiative

PBS Phosphate Buffer Saline

PDI Poly Dispersity Index

PLA Poly Lactic Acid

PXRD Powder X-ray Diffractometry

SD Standard Deviation

SEM Scanning Electron Microscopy

SLNs Solid Lipid Nanoparticles

SZN Sulfasalazine

TEM Transmission Electron Microscopy

Tmax Maximum/Peak plasma concentration time

USP United States Pharmacopeia

UV Ultraviolet

XIII

TABLE OF CONTENTS

CHAPTER 1 INTRODUCTION .................................................................. 1

1.1 SOLUBILITY, DISSOLUTION AND BIOAVAILABILITY ........................................... 1

1.2 SOLUBILITY AND BIOAVAILABILITY EFFECTING FACTORS ............................... 2

1.2.1 PHYSICAL FACTORS ......................................................................................... 2

1.2.2 PHYSIOLOGICAL FACTORS ............................................................................... 3

1.3 APPROACHES TO FORMULATE LOW SOLUBLE DRUGS ...................................... 4

1.3.1 ADJUSTMENT OF PH ........................................................................................ 4

1.3.2 CO-SOLVENCY ................................................................................................. 4

1.3.3 SURFACTANTS ................................................................................................. 4

1.3.4 CYCLODEXTRIN COMPLEXES ........................................................................... 5

1.3.5 LIPID-BASED APPROACHES ............................................................................. 5

1.3.6 NANO-SUSPENSIONS ........................................................................................ 5

1.3.7 COMBINATION OF LIPID BASED AND NANOTECHNOLOGY .............................. 6

1.4 CLASSIFICATION OF NANOPARTICLES ................................................................ 6

1.4.1 METAL BASED NANOPARTICLES ..................................................................... 7

1.4.2 POLYMER BASED NANOPARTICLES ................................................................. 7

1.4.3 LIPID-BASED NANOPARTICLES ....................................................................... 7

1.5 NANOPARTICLES AS DRUG CARRIER .................................................................. 8

1.6 SOLID LIPID NANOPARTICLES ............................................................................ 9

1.6.1 DEFINITION AND ADVANTAGES ...................................................................... 9

1.6.2 ADVANTAGES OF SLNS OVER OTHER FORMULATIONS ................................. 11

1.7 SOLID LIPID NANOPARTICLES PRODUCTION TECHNIQUES ............................. 12

1.7.1 HIGH SHEAR HOMOGENIZATION .................................................................... 13

1.7.2 ULTRA-SONICATION TECHNIQUE .................................................................. 13

1.7.3 MICRO EMULSION TECHNIQUE ...................................................................... 14

1.7.4 SOLVENT EMULSIFICATION DIFFUSION TECHNIQUE ..................................... 14

1.8 SEPARATION AND PURIFICATION OF SOLID LIPID NANOPARTICLES ............... 15

1.9 STABILITY OF SLNS AND LYOPHILISATION ..................................................... 15

1.10 STABILITY OF SLNS AND SPRAY DRYING ......................................................... 18

1.11 APPLICATIONS OF SLNS IN DRUG DELIVERY SYSTEM .................................... 19

1.12 LIMITATIONS OF SOLID LIPID NANOPARTICLES .............................................. 20

1.13 NICLOSAMIDE .................................................................................................... 21

XIV

1.14 SULFASALAZINE ................................................................................................. 22

1.15 BACKGROUND OF THE STUDY ........................................................................... 23

CHAPTER 2 LITERATURE REVIEW .................................................. 25

CHAPTER 2 .................................................................................................. 33

2.1 SOLID LIPID NANOPARTICLE PRODUCTION TECHNIQUES ............................... 33

2.2 SOLVENT EMULSIFICATION DIFFUSION TECHNIQUE ....................................... 34

2.3 MICRO EMULSION METHOD ............................................................................. 35

2.4 STABILITY OF SLNS .......................................................................................... 38

2.5 DRUG LOADING AND RELEASE FROM LIPID NANOPARTICLES ........................ 42

2.6 ENHANCED SUSTAINED DRUG RELEASE ........................................................... 42

2.7 MECHANISM OF ACTION OF LIPID-BASED DELIVERY SYSTEMS ..................... 43

2.8 ENHANCEMENT OF BIOAVAILABILITY BY LIPID NANOPARTICLES .................. 44

CHAPTER 3 MATERIALS AND METHODS .......................................... 46

3.1 MATERIALS ........................................................................................................ 46

3.1.1 CHEMICALS ................................................................................................... 46

3.1.2 INSTRUMENTATIONS ...................................................................................... 46

3.2 METHODS ........................................................................................................... 49

3.2.1 FABRICATION OF BLANK SLNS BY MICROEMULSION TECHNIQUE ............... 49

3.2.2 FABRICATION OF BLANK SLNS BY SOLVENT EMULSIFICATION DIFFUSION

TECHNIQUE .................................................................................................... 51

3.2.3 FABRICATION OF NICLOSAMIDE LOADED SLNS BY MICROEMULSION

TECHNIQUE .................................................................................................... 52

3.2.4 FABRICATION OF NIC-SLNS BY SOLVENT EMULSIFICATION DIFFUSION

TECHNIQUE .................................................................................................... 53

3.2.5 FABRICATION OF SULFASALAZINE LOADED SLNS BY MICROEMULSION

TECHNIQUE .................................................................................................... 54

3.2.6 FABRICATION OF SZN-SLNS BY SOLVENT EMULSIFICATION DIFFUSION

TECHNIQUE .................................................................................................... 54

3.2.7 LYOPHILIZATION ........................................................................................... 56

3.2.8 CALIBRATION CURVE OF NICLOSAMIDE ........................................................ 56

XV

3.2.9 CALIBRATION CURVE OF SULFASALAZINE .................................................... 56

3.2.10 ENTRAPMENT EFFICIENCY ............................................................................. 57

3.2.11 DRUG LOADING CAPACITY ........................................................................... 57

3.3 CHARACTERIZATION ......................................................................................... 57

3.3.1 PARTICLE SIZE AND PDI ................................................................................ 57

3.3.2 ZETA POTENTIAL ........................................................................................... 58

3.3.3 SCANNING ELECTRON MICROSCOPY ............................................................. 58

3.3.4 DIFFERENTIAL SCANNING CALORIMETER ..................................................... 58

3.3.5 POWDER X-RAY DIFFRACTOMETRY .............................................................. 58

3.3.6 FT-IR STUDIES .............................................................................................. 59

3.3.7 IN-VITRO DRUG RELEASE ................................................................................ 59

3.3.8 DRUG RELEASE MECHANISM......................................................................... 59

3.3.9 STABILITY ...................................................................................................... 60

3.4 IN-VIVO STUDY .................................................................................................. 60

3.4.1 DOSE ADMINISTRATION ................................................................................ 60

3.4.2 QUANTIFICATION OF NICLOSAMIDE BY HPLC ............................................. 60

3.4.3 QUANTIFICATION OF SULFASALAZINE BY HPLC .......................................... 61

3.4.4 ANALYSIS OF DATA ....................................................................................... 61

3.5 GRANULATION ................................................................................................... 62

3.5.1 STATIC BED DRYING ..................................................................................... 62

3.5.2 COMPARATIVES STUDY OF LYOPHILIZED AND STATIC DRIED SLNS ............ 62

3.5.3 WET-GRANULATION ..................................................................................... 62

3.5.4 COATING OF GRANULES ................................................................................ 63

3.5.5 CAPSULE SHELLS FILLING ............................................................................. 64

3.6 SIMILARITY STUDY ............................................................................................ 64

3.7 STATISTICAL ANALYSIS .................................................................................... 65

CHAPTER 4 RESULTS AND DISCUSSION ............................................ 66

4.1 MICRO EMULSION TECHNIQUE ........................................................................ 66

4.1.1 PARTICLE SIZE AND PDI OF BLANK SLNS BY MICRO EMULSION TECHNIQUE .

..................................................................................................................... 66

4.2 FABRICATION OF NICLOSAMIDE BY MICRO EMULSION TECHNIQUE .............. 67

4.2.1 PARTICLE SIZE, PDI AND ZETA POTENTIAL OF NICLOSAMIDE SLNS ........... 67

4.2.2 ENTRAPMENT EFFICIENCY OF NICLOSAMIDE LOADED SLNS ....................... 69

4.2.3 DRUG LOADING CAPACITY OF NICLOSAMIDE LOADED SLNS ...................... 69

XVI

4.2.4 SCANNING ELECTRON MICROSCOPY OF OPTIMIZED NICLOSAMIDE LOADED ..

SLNS ............................................................................................................. 70

4.2.5 DSC THERMOGRAM OF NICLOSAMIDE SLNS OPTIMIZED ............................. 71

4.2.6 PXRD OF OPTIMIZED NICLOSAMIDE LOADED SLNS ..................................... 71

4.2.7 FT-IR STUDY OF OPTIMIZED NICLOSAMIDE LOADED SLNS ......................... 72

4.2.8 IN-VITRO DRUG RELEASE OF OPTIMIZED NICLOSAMIDE LOADED SLNS ........ 73

4.2.9 DRUG RELEASE MECHANISM OF OPTIMIZED NICLOSAMIDE LOADED SLNS . 73

4.2.10 STABILITY STUDY OF OPTIMIZED NICLOSAMIDE LOADED SLNS .................. 75

4.2.11 IN-VIVO STUDY OF OPTIMIZED NICLOSAMIDE LOADED SLNS ...................... 76

4.3 FABRICATION OF SULFASALAZINE BY MICRO EMULSION TECHNIQUE .......... 78

4.3.1 PARTICLE SIZE, PDI AND ZETA POTENTIAL OF LOADED SULFASALAZINE

SLNS ............................................................................................................. 78

4.3.2 ENTRAPMENT EFFICIENCY OF SULFASALAZINE LOADED SLNS ................... 79

4.3.3 DRUG LOADING CAPACITY OF SULFASALAZINE LOADED SLNS .................. 79

4.3.4 SCANNING ELECTRON MICROSCOPY OF OPTIMIZED SULFASALAZINE LOADED

SLNS ............................................................................................................. 80

4.3.5 DSC THERMOGRAM OF SULFASALAZINE SLNS ............................................ 81

4.3.6 PXRD OF OPTIMIZED SULFASALAZINE LOADED SLNS ................................. 82

4.3.7 FT-IR STUDY OF OPTIMIZED SULFASALAZINE LOADED SLNS...................... 82

4.3.8 FT-IR STUDY OF OPTIMIZED SULFASALAZINE LOADED SLNS...................... 83

4.3.9 IN-VITRO DRUG RELEASE OF OPTIMIZED SULFASALAZINE LOADED SLNS .... 84

4.3.10 DRUG RELEASE MECHANISM OF OPTIMIZED SULFASALAZINE LOADED SLNS .

..................................................................................................................... 85

4.3.11 STABILITY STUDY OF OPTIMIZED SULFASALAZINE LOADED SLNS .............. 86

4.3.12 IN-VIVO STUDY OF OPTIMIZED SULFASALAZINE LOADED SLNS ................... 87

4.4 SOLVENT EMULSIFICATION DIFFUSION TECHNIQUE ....................................... 90

4.4.1 ..................................................................................................................... 90

4.4.1 PARTICLE SIZE AND PDI OF BLANK SLNS .................................................... 90

4.5 FABRICATION OF NIC-SLNS BY SOLVENT EMULSIFICATION DIFFUSION

TECHNIQUE ........................................................................................................ 91

4.5.1 PARTICLE SIZE, PDI AND ZETA POTENTIAL OF LOADED NICLOSAMIDE SLNS .

..................................................................................................................... 91

4.5.2 ENTRAPMENT EFFICIENCY OF NICLOSAMIDE LOADED SLNS ....................... 92

4.5.3 DRUG LOADING CAPACITY OF NICLOSAMIDE LOADED SLNS ...................... 93

4.5.4 SCANNING ELECTRON MICROSCOPY OF OPTIMIZED NICLOSAMIDE LOADED

SLNS ............................................................................................................. 94

XVII

4.5.5 DSC THERMOGRAM OF NICLOSAMIDE SLNS OPTIMIZED ............................. 94

4.5.6 PXRD OF OPTIMIZED NICLOSAMIDE LOADED SLNS ..................................... 95

4.5.7 FT-IR STUDY OF OPTIMIZED NICLOSAMIDE LOADED SLNS ......................... 95

4.5.8 IN-VITRO DRUG RELEASE OF OPTIMIZED NICLOSAMIDE LOADED SLNS ........ 96

4.5.9 DRUG RELEASE MECHANISM OF OPTIMIZED NICLOSAMIDE LOADED SLNS . 97

4.5.10 STABILITY STUDY OF OPTIMIZED NICLOSAMIDE LOADED SLNS .................. 98

4.5.11 IN-VIVO STUDY OF OPTIMIZED NICLOSAMIDE LOADED SLNS ...................... 98

4.6 FABRICATION OF SZN-SLNS BY SOLVENT EMULSIFICATION DIFFUSION

TECHNIQUE ...................................................................................................... 100

4.6.1 PARTICLE SIZE AND PDI OF LOADED SULFASALAZINE SLNS ..................... 100

4.6.2 ENTRAPMENT EFFICIENCY OF SULFASALAZINE LOADED SLNS ................. 102

4.6.3 DRUG LOADING CAPACITY OF SULFASALAZINE LOADED SLNS ................ 102

4.6.4 SCANNING ELECTRON MICROSCOPY OF OPTIMIZED SULFASALAZINE LOADED

SLNS ........................................................................................................... 103

4.6.5 DSC THERMOGRAM OF SULFASALAZINE SLNS OPTIMIZED SLNS.............. 104

4.6.6 PXRD OF OPTIMIZED SULFASALAZINE LOADED SLNS ............................... 104

4.6.7 FT-IR STUDY OF OPTIMIZED SULFASALAZINE LOADED SLNS.................... 105

4.6.8 IN-VITRO RELEASE FROM OPTIMIZED SZN LOADED SLNS .......................... 106

4.6.9 DRUG RELEASE MECHANISM OF OPTIMIZED SULFASALAZINE LOADED SLNS .

................................................................................................................... 107

4.6.10 STABILITY STUDY OF OPTIMIZED SULFASALAZINE LOADED SLNS ............ 107

4.6.11 IN-VIVO STUDY OF OPTIMIZED SULFASALAZINE LOADED SLNS ................. 108

4.7 SIMILARITY FACTOR ....................................................................................... 111

4.8 CALCULATION OF F1 AND F2 FOR NICLOSAMIDE NANOFORMULATION ......... 111

4.9 CALCULATION OF F1 AND F2 FOR SULFASALAZINE NANOFORMULATION ...... 112

CONCLUSION ................................................................................................ 114

REFERENCES ................................................................................................ 116

PUBLICATIONS ................................................................................................ 154

XVIII

LIST OF TABLES

Table 1. Types and Terms of Nano Particulate Drug Delivery Systems ........... 10

Table 2. Lipids and Surfactants used in SLNs Fabrication ............................... 17

Table 3. Properties of Niclosamide ................................................................... 22

Table 4. Properties of Sulfasalazine .................................................................. 23

Table 5. Emulsifiers used for the production of lipid nanoparticles ................. 37

Table 6. Types of Lipids use in preparation of Lipid based nanoparticles ........ 38

Table 7. Blank SLNs formulations for Micro-emulsion Technique. ................ 50

Table 8. Blank SLNs for Solvent Emulsification Diffusion method ................. 52

Table 9. Different NIC loaded SLNs formulations by Microemulsion Method 53

Table 10. NIC loaded SLNs by Solvent Emulsification Diffusion Method ...... 53

Table 11. SZN loaded SLNs formulations by Microemulsion Method ............ 55

Table 12. SZN loaded SLNs by Solvent Emulsification Diffusion Method ..... 55

Table 13. Excipients used during wet granulation method ............................... 63

Table 14. Excipients used during Coating ......................................................... 64

Table 15. Size and PDI of blank SLNs formulations Mean±SD (n=3) ............. 67

Table 16. EE% and DLC% of Niclosamide Loaded SLNs Formulation .......... 70

Table 17. Cumulative Percent Drug Release from NIC-SLNs Formulations ... 74

Table 18. Kinetic Models for Niclosamide-SLNs ............................................. 75

Table 19. Stability study of NIC-SLNs (NME-3) ............................................. 76

Table 20. Pharmacokinetic parameters of NIC-SLNs (NME-3) & Mesan® .... 77

Table 21. EE & DLC of SZN loaded SLNs formulations ................................. 80

Table 22. Different Kinetic Models for Sulfasalazine ....................................... 86

Table 23. Pharmacokinetic parameters of SME−3 & Marketed drug ............... 89

Table 24. Particle size and PDI of unloaded SLNs ........................................... 91

Table 25. EE% and DLC% of Niclosamide Loaded SLNs Formulation .......... 93

Table 26. Different Kinetic Models for NIC loaded SLNs ............................... 98

Table 27. Stability study of NIC-SLNs (NSE-2) ............................................... 99

Table 28. Pharmacokinetic parameters of NSE-2 & Marketed Drug .............. 100

Table 29. Different Kinetic Models for SZN loaded SLNs ............................. 107

Table 30. Pharmacokinetic parameters of SSE−2 & Marketed drug .............. 110

XIX

LIST OF FIGURES

Figure 1. Biopharmaceutical Classification System for Drugs ....................... 2

Figure 2. Formulation approaches for poorly soluble drugs ........................... 5

Figure 3. Top-down and bottom-up approaches ............................................. 6

Figure 4. Types of pharmaceutical nano-systems ........................................... 9

Figure 5. SLNs Production Methods............................................................. 14

Figure 6. Metabolism of lipid in body .......................................................... 20

Figure 7. Chemical Structure of Niclosamide ............................................... 22

Figure 8. Chemical Structure of Sulfasalazine .............................................. 23

Figure 9. ZS-90, Malvern Instruments .......................................................... 47

Figure 10. Scanning Electron Microscope .................................................... 47

Figure 11. Differential Scanning Calorimeter ............................................... 48

Figure 12. Infra-Red Spectroscope ............................................................... 49

Figure 13. Schematic diagram of Micro-Emulsion Method ......................... 51

Figure 14. Schematic diagram of Solvent-Emulsification-Diffusion ........... 54

Figure 15. Average Particle size of NME−3 formulation ............................. 68

Figure 16. Zeta Potential of NME−3 formulation......................................... 68

Figure 17. SEM image of Niclosamide Loaded SLNs (NME-3) .................. 70

Figure 18. Thermograms of Pure Niclosamide and NME-3 ......................... 71

Figure 19. P-XRD Spectra of Pure Niclosamide and NME-3 ...................... 72

Figure 20. FT-IR Spectra of Pure Niclosamide (A) and NME-3 (B) ........... 73

Figure 21. Percent Drug Release from NIC-SLNs Formulations ................. 74

Figure 22. Comparative in−vivo drug release from NME−3 &

marketed drug ................................................................................................ 77

Figure 23. Particle size of SME-3 Formulation ............................................ 78

Figure 24. Zeta Potential of SME-3 Formulation ......................................... 79

Figure 25. SEM image of Sulfasalazine Loaded SLNs (SME-3) ................. 81

Figure 26. DSC thermogram of Pure Sulfasalazine and SME-3 formulation

........................................................................................................................ 82

Figure 27. P-XRD Spectra of Pure Sulfasalazine and SZZ−SLNs (SME-3) 83

Figure 28. FT-IR Spectra of Pure Sulfasalazine (A) & Processed SZN-SLNs

(B) .................................................................................................................. 84

Figure 29. Percent Drug Release from SZN loaded SLNs formulations ..... 85

XX

Figure 30. Particle size analysis of optimized formulation (SME-3) ........... 87

Figure 31. PDI analysis of optimized formulation (SME-3) ........................ 87

Figure 32. Comparative in−vivo release from SZN-SLNs (SME−3) &

Marketed Drug ............................................................................................... 88

Figure 33. Average Particle size of NSE−2 formulation .............................. 92

Figure 34. Zeta Potential of NSE−2 formulation .......................................... 92

Figure 35. SEM micrograph of Niclosamide Loaded SLNs (NSE-2) .......... 94

Figure 36. DSC Thermograms of Pure Niclosamide and NIC-SLNs (NSE-2)

........................................................................................................................ 95

Figure 37. P-XRD Spectra of Pure Niclosamide and NSE-2 ........................ 96

Figure 38. FT-IR Spectra of Pure Niclosamide (A) and NSE-2 formulation

(B) .................................................................................................................. 97

Figure 39. Percent Drug Release from NIC-SLNs Formulations ................. 97

Figure 40. Comparative in-vivo drug release from NSE-2 & Marketed Drug

...................................................................................................................... 100

Figure 41. Particle size of SSE-2 Formulation ........................................... 101

Figure 42. Zeta Potential of SSE-2 Formulation ......................................... 101

Figure 43. Entrapment Efficiency & Drug Loading Capacity of SZN−SLNs

...................................................................................................................... 103

Figure 44. SEM micrograph of Sulfasalazine Loaded SLNs (SSE-2) ........ 103

Figure 45. DSC thermogram of Pure Sulfasalazine and SSE-2 formulation

...................................................................................................................... 104

Figure 46. P-XRD Spectra of Pure Sulfasalazine and SZZ−SLNs (SSE-2)105

Figure 47. FT-IR Spectra of Sulfasalazine (A) and SZN−SLNs (SSE-2) (B)

...................................................................................................................... 106

Figure 48. Percent Release from SZN loaded SLNs formulations ............ 106

Figure 49. Change in size of SSE-2 during stability ................................... 108

Figure 50. Change in PDI of SSE-2 during stability................................... 108

Figure 51. Comparative in−vivo release from SSE−2 & Marketed Drug .. 110

Figure 52. Dissolution Profiles of NME-3 and Marketed Drug ................. 112

Figure 53. Dissolution Profiles of SME-3 and Marketed Drug .................. 112

CHAPTER 1 INTRODUCTION

1

Chapter 1 INTRODUCTION

1.1 Solubility, Dissolution and Bioavailability

The concentration of a solute in solvent that remains in contact with an excess

quantity of solute or in other words the maximum quantity of a solid substance that

can dissolve in a specific solvent at specific temperature and pressure is known as

solubility [1, 2]. According to United States Pharmacopeia & British Pharmacopoeia

the drug with solubility below 0.1 mg/ml in solvent are considered practically

insoluble drugs and will face significant solubilization problems [3-5]. Poor aqueous

soluble drugs present major challenges because it decreases bioavailability due to low

absorption [6]. However, the necessary solubility to get decent bioavailability should

be evaluated by considering the dose as well as the gastrointestinal tract (GIT)

permeability of the drug. Maximum absorbable dose (MAD) shows the required

permeability and solubility of the drug to obtain maximum oral absorption. If the

MAD value is lesser than dose, then incomplete absorption is expected [7].

Where Cs is the solubility (mg/ml) of the drug; ka (rate constant for absorption

from intestine); SIWV (volume of fluid) & SITT (transit time) in minutes.

Dose, intestinal permeability and solubility are important parameters in

Biopharmaceutical classification system (BCS). Two classes i.e. BCS-II & BCS-IV

facing the solubility problem which intern lead to low bioavailability of these drugs

[8]. If the maximum amount of drug is soluble in ≤250mL in pH-range

(physiological) 1.0 to 7.5, is considered more soluble. If the drug has absorption >90

% after administration is consider to be highly permeable [9-15].


2

Figure 1. Biopharmaceutical Classification System for Drugs

1.2 Solubility and Bioavailability Effecting Factors

1.2.1 Physical factors

a. Melting Point and Lipophilicity

The following equation proposed by Yalkowsky et. al shows the effect of

m.p & lipophilicity on solubility in water [16].

S (aqueous solubility); log Pow (water partition coefficient); MP (melting

point).

According to Yalkowsky et. al aqueous solubility reduces 10 times as m.p

rises by 100 degree or log Pow rises by one [17, 18].

b. pKa

Henderson-Hasselbalch equation shows the effect of pKa on solubility in

water.


3

S (aqueous solubility) at definite pH and S0 (intrinsic-solubility). Various

solid material like amorphous, crystalline, polymorphs, solvates and hydrates etc.

have different values of solubility and molecules with higher energy incline to

have more solubility than the molecules possess low energy [16, 19, 20].

c. Molecular Weight

High molecular weight through the GIT and BBB (blood brain barrier) led to

decrease permeability [16, 21, 22]. Lipinski’s rules demonstrates, the low bio-

availability is expected when the drug molecule has more than five donors, ten

acceptors bonds and M.Wt higher than 500 [17]. Veber’s rules says, high

bioavailability is expected for the molecule it has more than ten rotatable bonds

and polar surface area is greater than 140 A˚ [23].

d. Polymorphism

This is the property of compound/molecule to available in two or more than

two crystalline lattices. They have diverse physicochemical properties (melting

point, density, vapour pressure, X-ray, colour, crystal shape, hardness, solubility,

dissolution rate and bioavailability). During pre-formulation, it is vital to recognise

the polymorph, which is stable at 25 °C. For examples: Chloromphenicol occur in

three different form i.e. A-form, B-form & C form, among these B-form is highly

stable [24, 25]. Enantiotropic polymorphs can be inter converted below the melting

point of either polymorph and the conversion is reversible at a define temperature

[26].

1.2.2 Physiological factors

a. pH

The change in pH gradient of GIT has main effect on ionisable drugs

solubility. The pH at which the basic & acidic drugs are present in ionized form

may be highly soluble [27, 28].


4

b. Stomach Emptying

Gastric emptying is an important factors, which affects the permeability of

drug molecule from the GIT & then bioavailability [9, 29, 30].

c. GIT Mobility

The GIT mobility is different among species. Food in the GIT can enhance

the absorption and solubility of hydrophobic drugs by postponing stomach

emptying, stimulating secretions of bile salt & changing GIT pH [16, 31, 32].

1.3 Approaches to Formulate Low Soluble Drugs

1.3.1 Adjustment of pH

Solubility of low water-soluble molecules having ionisable molecule can be

improved through pH adjustment of the solution which facilitate ionized form. For

example Dilantin® (Phenytoin) Injection by Pfizer is pH adjusted formulation

containing co-solvents [33-36].

1.3.2 Co-solvency

Co-solvency is the phenomenon to use mixture of water miscible solvents, to

enhance solubility of low aqueous soluble molecule through decreasing the polarity of

water [37]. For example Nimotop® (Nimodipine) IV injection by Bayer is co-solvent

formulation.

1.3.3 Surfactants

When the concentration of surfactants surpasses the critical micelle

concentration, increase water-solubility of drugs. Examples of low water soluble

drugs that use micellar solubilization are mostly antidiabetic drugs like gliclazide,

glimepiride, repaglinide, glipizide and rosiglitazone [16, 36, 38].

.


5

Figure 2. Formulation approaches for poorly soluble drugs

1.3.4 Cyclodextrin complexes

These are macro-cyclic oligo-saccharides containing a lyophobic external

surface and a lipophilic internal cavity where lipophilic drug molecules can be

encapsulated [39]. So compound are encapsulated into the cavity which results in

better water solubility [16, 36, 40, 41].

1.3.5 Lipid-Based Approaches

In these type of approaches the drug is dissolved in a mixture of two or more

formulation excipients such as lipid, surfactant and co-surfactant [42]. Lipid-based

formulation would be emulsions, micro-emulsions, nano-emulsions, suspensions,

micelle-solutions, liposomes and nanoparticles. Among these lipid-based approaches

the solid-lipid nanoparticles (SLNs) and nano-structured lipid carriers (NLC) are

paying attention a noteworthy level during last decades [36, 43-46].

1.3.6 Nano-suspensions

According to Noyes Whitney equation as the particle size is reduced the suface

area would be increases which lead to enhance dissolution-rate [47]. The effect of


6

reduced particle size on dissolution-rate & oral-bioavailability enhancement has been

reported previously for large number of low water soluble drugs [48, 49]. For

example Aptivus® (HIV-protease inhibitor tipranavir) a poor water soluble drug that

use nano-suspension [50].

Figure 3. Top-down and bottom-up approaches

1.3.7 Combination of Lipid Based and Nanotechnology

Both lipid-based technologies and nano-technology approaches are used to

augment solubility/bioavailability of poor water-soluble drugs [51]. In Nano

technological-based approaches, the solubility and dissolution rate of compounds can

be improved by decreasing the particle size or increasing the surface area. Ultrafine

particles of drugs can be shaped either top-down or bottom-up techniques. On other

hand, lipids have also twisted further profitable & academic consideration as a

favourable approach to enhance bioavailability of lipophilic drugs [52].

1.4 Classification of Nanoparticles

For decades, different pharmaceutical dosage forms are being used as drug-

delivery-systems to treat diseases. Nano-particles are colloidal system in solid form in


7

which the drugs are either entrapped or adsorbed. Nano-particles offer many plusses

in delivery of drug because of their small size, huge surface-area & have the ability to

change their surface morphology. Based on the type of the inactive ingredient used,

there are four classes of nanoparticles: Metal based nanoparticles, Polymer based

nanoparticles and Lipid based nanoparticles [53-55].

1.4.1 Metal Based Nanoparticles

Currently these nanoparticles are developing as better drug delivery carriers. To

fabricate metallic nano-particles, various metals are explored but Ag and Au nano-

particles lead for biomedical use [56]. Functionalization of surface on metallic nano-

particles can be done easily [57]. Variety of ligands like carbohydrates, amino acids

and DNA etc. can be been linked to these nano-particles [58, 59].

1.4.2 Polymer Based Nanoparticles

These nanoparticles are composed of bio-degradable, bio-compatible and non-

toxic polymers [60]. Lately research discover few modification of naturally occured

polymers which comprises artificial poly-esters such as poly-lactide, poly-

cyanoacrylate and similar polymers [61]. In natural polymers chitosan is extensively

used polymer now-a-days [62, 63]. Polymeric nano-particles based on natural

polymer offer significance over traditionally used oral and intravenous drug delivery

systems in terms of safety and effectiveness [64].

1.4.3 Lipid-Based Nanoparticles

Lipid-based nanoparticles show interesting features concerning therapeutic

purposes [65]. In these type of approaches the drug is dissolved in a mixture of two or

more formulation excipients such as lipid, surfactant and co-surfactant [42]. Lipid-

based formulation would be emulsions, micro-emulsions, nano-emulsions,

suspensions, micelle-solutions, lipo-somes and nano-particles. Among these lipid-


8

based approaches the solid-lipid nanoparticles (SLNs) and nano-structured lipid

carriers (NLC) are paying attention a noteworthy level during last decades [36, 43-

46].

1.5 Nanoparticles as Drug Carrier

Nano-particles are being increasingly used as drug-delivery systems. In

scientific terms, the word nanoparticle refers to a structure in 1-100nm size range

[66]. However, more commonly the term is applied to any particle within the

nanometer size range. Possibly of more importance is the fact that materials at this

scale frequently display different properties than those of the bulk material. A large

number of nano-particle based drug-delivery systems have been established which

include solid-lipid nanoparticles (SLNs), liposomes (self-assembled lipid bilayers),

micelles (self-assembled amphiphilic molecules) and dendrimers (repeatedly

branched spherical polymers). Nanoparticles are capable of achieving enhanced

solubility [67]. This is critical in an age of increasingly hydrophobic drugs, where

new methods are continually required for solubility enhancement.

Depending on the type and composition, nanoparticles may also be able to

provide this enhancement with considerably decreased toxicity as compared to earlier

methods (e.g., Cremophor) [68]. Secondly, nanoparticles have the capacity for

controlled and/or bioresponsive drug release. Formulation could be designed so that

drug is released gradually for prolonged period of time, generally by a diffusion

process [69, 70]. Alternatively, systems can be developed that achieve a rapid release

of drug upon the addition of a biological stimulus, such as a change in pH, redox

potential, temperature or the presence of a relevant enzyme [71-74]. Thirdly,

nanoparticles have been reported to evade MDR mechanisms. This can be a result of

their cellular internalization pathway [69, 75] or P-gp inhibition [76]. Fourthly,


9

nanoparticles have been shown to protect loaded molecules from enzymatic

degradation [77]. This advantage is particularly relevant for proteins and other

enzymatically labile compounds that would quickly become inactivated in the absence

of a protective drug carrier.

Figure 4. Types of pharmaceutical nano-systems

1.6 Solid Lipid Nanoparticles

1.6.1 Definition and Advantages

Solid lipid nanoparticles lead rapidly emerging branch of nano-technology with

many potential applications in delivery of drugs. Because of their exceptional size

dependent properties, solid lipid nanoparticles offer the option to design new

therapeutics. Solid lipid nanoparticles have the ability to achieve the goal of sustained

and targeted drug delivery and attracted attention of researchers.


10

Table 1. Types and Terms of Nano Particulate Drug Delivery Systems

Term Particle Size (nm) Reference P

oly

mer

ic S

yst

ems

Dendrimers 1–10 [78, 79]

Polymer micelles 10–100 [78]

Niosomes 10–150 [78]

Nanoparticles 50–500 [78, 80-83]

Nanocapsules 100–300 [78, 84, 85]

Nanogels 200–800 [78]

Polymer–drug nanoconjugates 1–15 [86, 87]

Chitosan polymers 100–800 [88, 89]

Methacrylate polymers 100–800 [90]

Pro

tein

/pep

t

ide

nan

otu

bes

Peptide nanotubes 1–100 [91]

Fusion proteins and

immunotoxins

3–15 [86]

Met

al

nan

ost

ruct

ure

s

Metal colloids 1–50 [92]

Carbon nanotubes 1–10 (diameter) & 1-1000 (length) [78]

Fullerene 1–10 [78]

Gold nanoparticles 100–200 [86]

Gold nanoshells 10–130 [86]

Silicone nanoparticles 100–600 [92]

Magnetic colloids 100–600 [93]

Lip

id b

ase

d s

yst

ems

Solid lipid nanoparticles 50–400 [94]

Lipid nanostructured systems 200–800 [95]

Cubosomes 50–700 [78]

Liposomes 10–1000 [96]

Polymerosomes 100–300 [86]

Immunoliposomes 100–150 [86]

Several reports exist that drugs can be loaded into nanoparticle systems,

including polymeric nanoparticles, polymer-drug conjugates and liposomes[97, 98].

Unfortunately, these systems may be limited by a number of disadvantages, including


11

toxicity, low drug encapsulation efficiency, unknown or unproven safety of some of

the materials used in their preparation, and laborious or high-cost production methods

[99-101].

SLNs are characterized by a solid lipid core with stabilizing surfactants and/or

polymers on the particle surface [102, 103]. They have gained increasing attention

since their development in the early 1990s based on their reported ability to combine

the advantages of several nanoparticle systems while negating some of their

disadvantages [104, 105]. First, they can be prepared from inexpensive, readily

available materials. A wide variety of both lipids (e.g., triglycerides, partial

glycerides, waxes, PEGylated lipids, fatty acids/alcohols, steroids) and

surfactants/polymers (e.g., polysorbates, brijs®, lecithin, bile acids) have been used in

their preparation [106]. Secondly, because the lipid matrix can be prepared from

biocompatible lipids and the surfactants and/or polymers used can be chosen based on

FDA approval status, these drug delivery systems typically exhibit very low toxicity

[107]. Coupled with the low toxicity of the materials used in SLN preparation is the

fact that most SLN preparation methods do not rely on the use of organic solvents,

eliminating that as a possible toxicity concern. Thirdly, and possibly of most

importance, is the fact that SLNs can be prepared using simple, scalable production

methods, a requirement for translation from academic labs to industrial labs [108-

112].

1.6.2 Advantages of SLNs over other formulations

a. Smallest blood capillaries in body are approximately 5-6 μm and hence particles

should be less than 5 μm in the blood stream without forming aggregates to

minimize embolism. Therefore, SLNs are better suited for I.V. delivery.


12

b. Size of the micro particles is a limitation to cross the intestinal lumen into

lymphatic system following oral delivery of vaccines, peptides, and other bio

macromolecules. Micro particles remain in Peyer’s patches while SLNs are

disseminated systematically.

c. Avoidance of organic solvents when desired

d. Excellent reproducibility and feasible large scale production

e. Unique ability to create controlled release and drug targeting by coating/attaching

ligands to SLNs [113].

f. Increased product stability of about 1 year

g. Lipids are biodegradable and hence have better biocompatibility [114].

h. Avoidance of organic solvents when desired

i. Feasibility of large scale production and sterilization

j. Increased stability of the active ingredient [115].

1.7 Solid Lipid Nanoparticles Production Techniques

For several years, solid lipids have been used in the form of pellets to achieve delayed

drug release. In the early 80s, Speiser and co-workers developed spray dried and

congealed micropellets and nanopellets of lipids for oral administration [116, 117].

Nanopellets developed by Speiser often contained high amounts of microparticles.

Domb produced lipospheres by high shear mixing or ultra-sonication [118]. However,

both the nano pellets and lipospheres produced by Speiser and Domb respectively

were contaminated by microparticles. Since the last decade, several scientists have

realized the potential of SLNs technology and their research efforts have brought

about improvement in solid lipid nanoparticles synthesis. Following 4 methods are

mostly used for the production of SLNs.


13

1.7.1 High shear homogenization

High shear homogenization technique were initially used for the production of solid

lipid nano-dispersions [119-122]. Olbrich et al. studied the effect of various process

parameters like emulsification-time, temperature and stirring-rate on the particle size and

zeta-potential.

Homogenizers have been used commercially for several years now for the

production of nano-emulsions for parenteral nutrition, such as Intralipid® and

Lipofundin® [123]. Thus, scaling up represents fewer problems when compared to

other techniques and it is cost effective. Naturally, a lot of research has been done

utilizing this method to produce better solid lipid nanoparticles by several research

groups. A homogeneous dispersion with small particle size is desirable to increase the

physical stability of aqueous dispersion. In this technique, the liquid is forced at a

high pressure of 100-2000 bar through a narrow gap of few microns. High shear

pressure and cavitation powers reduce the particle size [124, 125]. The two HSH

production techniques are the hot homogenization techniques and cold

homogenization techniques.

1.7.2 Ultra-sonication Technique

SLNs can be developed by ultra-sonication (high speed stirring) [126]. The

main disadvantage of this technique is large sized particle in the range of micrometer.

This lead physical instabilities likes particle growth upon storage. So for making a

stable formulation, studies have been performed by various research groups that high

speed stirring and ultra-sonication are used combined and performed at high

temperature [127].


14

1.7.3 Micro emulsion Technique

Gasco et al. developed micro-emulsions technique for the production of SLNs

[66]. Micro emulsion are fabricated by stirring a mixture composed of a low-melting

lipid like stearic acid, an surfactant like Tween-20, Tween-60 co-surfactant like Na

mono octyl-phosphate) and water. The hot micro emulsion is dispersed in cold water

under stirring with different ratios 1:25 to 1:50. The dilution process is critically

determined by the composition of the micro emulsion [128, 129]. SLNs were

produced only with solvents which distribute very rapidly into the aqueous phase,

while larger particle sizes were obtained with more lipophilic solvents [130].

Figure 5. SLNs Production Methods

1.7.4 Solvent Emulsification Diffusion Technique

This method is derived from solvent-evaporation technique, the water miscible

solvent (ethanol) is used as an oil phase. During the diffusion process an interfacial


15

turbulence is generated between two phases which may ultimately leads to the

formation of nano-particles. Smaller particle size can be achieved by increasing the

concentration of water miscible solvent increases [112, 131, 132].

1.8 Separation and Purification of Solid Lipid Nanoparticles

Depending on the method of preparation, potentially toxic impurities such as

surfactant micelles, residual monomers, polymers, metallic impurities and organic

solvents can be present in the SLN dispersion. For an effective SLN drug delivery

system, it should be free from any unencapsulated drug or impurities. SLNs can be

separated and purified using diafiltration, ultracentrifugation, dialysis, gel filtration

and crossflow microfiltration [112, 133, 134].

1.9 Stability of SLNs and Lyophilisation

Lipid crystallization is important for the stability of lipid nanoparticles [44]. It

significantly affects the drug incorporation and release rates. Polymorphic transition is

the ability to form a different unit cell structure in crystals due to different molecular

conformations and packing patterns. SLNs do not completely crystallize during their

storage and contain various polymorphic forms such as α, β’ and β [135]. The main

difference between the polymorphic forms is the molecular distance. “α” form is

unstable and is characterized by the hexagonal structure with the largest molecular

distance. “β” form is stable and is characterized by the tightest triclinic packing

pattern. Presence of residual liquids in lipid nanoparticles promote the crystallization

of the stable form because unstable crystals may redissolve and recrystallize to the

more stable form [135]. Increase in particle size, change in particle shape, and drug

expulsion occurs when lipids undergo polymorphic modifications. An increase in

thermodynamic stability and decrease in the drug incorporation rate was observed in


16

the following order [2]: supercooled melt < α-modification < β’-modification < β-

modification. Differential scanning calorimetry (DSC) and X-ray scattering are

widely used to study lipid polymorphic transitions [136]. Different lipid forms possess

different melting points and enthalpies and thus can be detected by DSC. X-ray

scattering can be used to detect the length of long and short spacings of the lipid

lattice. The stability of SLN dispersions has been reported to be in the range of 12 to

36 months [137]. But in most formulations the particle size increases within a short

period of time and hence lyophilisation is a way to increase the stability of SLNs

[138]. Ostwald ripening as well as hydrolysis can be avoided by lyophilisation.

Moreover it also makes SLNs feasible to be incorporated into various dosage forms

such as tablets, capsules, pellets, parenteral redispersion, etc. Lyophilisation involves

freezing the SLN dispersion followed by the evaporation of the water under vacuum.

The lyophilisation parameters to be considered are freezing out effect which leads to

changes in osmolality and pH. Low water and high particle content produces high

osmotic pressure which in turn favours particle aggregation and hence the lipid

content of the SLN dispersion should not exceed 5% [139] . Cryoprotectants such as

mannitol, sorbitol, trehalose, fructose, glucose and polyvinylpyrrolidone are usually

added to decrease particle aggregation and to obtain better redispersion of the

lyophilizates [140]. Cryoprotectants help in SLN stability by decreasing osmotic

activity of water and crystallization and favoring the formation of glassy state of the

frozen sample [139, 141, 142]. They prevent direct contact between lipid particles and

they also interact with the polar groups of the surfactants and serve as a pseudo

hydration shell [143]. Trehalose has been reported to give the best results as

cryoprotectant for SLN lyophilization. Cryoprotectants are usually used in a

concentrations of 10-15% [135].


17

Schwarz et al. reported that the particle size of reconstituted lyophilizates of

Compritol® SLN was 330 nm when compared to 160 nm prior to lyophilization of the

liquid dispersion [144]. Increase in particle size of approximately 1.5-2.4 times has

been observed following lyophilization with the particles still in the submicron range.

Table 2. Lipids and Surfactants used in SLNs Fabrication

LIPIDS SURFACTANTS

Triacylglycerols:

Tricaprin

Trilaurin

Trimyristin

Tripalmitin

Tristearin

Phospholipids:

Egg Lecithin

Phosphatidylcholine

Soy Lecithin

Acylglycerols:

Behenate

Glycerol

Glycerol

Glycerol

Monostearate

Palmitostearate

Ethylene Oxide/Propylene Oxide

Copolymers:

Poloxamer 188

Poloxamer 182

Poloxamer 407

Poloxamine 908

Fatty Acids:

Behenic Acid

Decanoic Acid

Palmitic Acid

Stearic Acid

Sorbitan Ethylene Oxide/Propylene

Oxide Copolymers:

Polysorbate 20

Polysorbate 60

Polysorbate 80

Waxes:

Cetyl Palmitate

Alkylaryl Polyether Alcohol

Polymers:

Tyloxapol

Cyclic Complexes:

Cyclodextrin

Bile Salts:

Sodium Cholate

Sodium Glycocholate

Sodium Taurocholate

Sodium Taurodeoxycholate

Hard Fat Types:

Witepsol W 35

Witepsol H 35

Alcohols:

Ethanol

Butanol


18

The time of addition of the cryoprotectant affects the quality of the

lyophilizates. Addition of cryoprotectant prior to homogenization helps in reducing

the increase in the particle size. Better particle size results are obtained when SLN

lyophilizates are redispersed using a bath sonicator as opposed to simple hand

shaking. The removal of water and increase in particle concentration during

lyophilization compromises the protective effect of the surfactant and hence favors

particle aggregation. Mehnert et al. recommends a sugar/ lipid weight ratio of 2.6-3.9.

Extensive research has been done in optimizing the lyophilization procedure of SLN

dispersions [144]. Results on the rate of freezing (Slow freezing in a deep freeze at -

70°C, rapid freezing in liquid nitrogen) are ambiguous and hence the procedure has to

be optimized on a case-by-case basis. Thermal treatment (2 h at -22°C followed by 2

h at -40°C) of the frozen SLN dispersion has also been reported to improve the results

[144]. Rapid cooling helps to decrease freezing out effects by forming small and

heterogeneous crystals.

1.10 Stability of SLNs and Spray Drying

Although rarely used, spray drying is another technique that can be used to

transform an aqueous SLN dispersion into a dry product. The production cost is lower

with spray drying when compared to lyophilisation [145]. Spray dryers utilize hot

gases and atomizers or spray nozzles to disperse the SLN dispersion and hence cause

aggregation and partial melting of the SLN particles. Freitas suggests the use of high

melting point lipids (>70°C), low lipid content in the dispersion, ethanol-water

mixtures (10/90 v/v) as the dispersion medium, addition of about 20-30 %

carbohydrates such as trehalose to control particle aggregation during spray drying

[146].


19

1.11 Applications of SLNs in Drug Delivery System

SLNs are composed of physiological lipids and hence the pathways for lipid

transportation and metabolism already present in the body determine the in vivo fate

of the carrier[147] . Enzyme lipases are most important for SLNs. SLNs are stable for

a long period of time and easy to scale up when compared to other colloidal systems

and thus may be important for many modes of targeting [148]. Anticancer agents are

usually delivered systemically degradation [109]. SLNs can be administered

intravenously owing to their small size [149]. They have been reported to be useful as

drug carriers to treat tumors. They provide a novel and a unique drug delivery system

to prevent rapid clearance by the immune system. Stealth nanoparticles can be used to

target specific tissues in accessible cells. Fluorescent SLNs prepared using fluorescent

markers and drugs have been successfully tested in animal models[149, 150]. Tumor

targeting has been reported with SLNs loaded with methotrexate and camptothecin

[151-153]. Longer circulation times have been reported to be achieved with paclitaxel

[154].

SLNs can penetrate the Blood Brain Barrier (BBB) due to adsorption of blood

proteins such as apolipoproteins on lipid nanoparticles surface which in turn may lead

to interactions with endothelial cells that facilitate crossing the BBB [155, 156]. Such

properties have been reported for the drugs such as tobramycin, doxorubicin and

idarubicin [157-160].

SLNs can be used in the formulation for delivery of gene vector [161]. DNA

degradation can be avoided and target specific delivery can be achieved by its

incorporation in the SLN. Increase in the bioavailability and decrease in the dosing

frequency has been reported to be achieved by incorporating antitubercular drugs such

as rifampicin, isoniazid, and pyrazinamide in the SLNs [162-164].


20

SLNs have been used for topical application of various drugs as it give the

potential advantage of delivering the drug directly to the site of action [165]. Research

has been done for the incorporation of active ingredients such as anticancer drugs,

imidazole antifungals [166], DNA [167, 168], flurbiprofen [169], glucocorticoids

[170], isotretinoin[171], triptolide [172, 173], and Vitamin A [174-176] into the

SLNs. SLNs are known to be suitable as carriers for UV-blockers due to their

particulate character and adhesive properties [177]. SLNs aid in achieving better

localization, occlusiveness, controlled release and increased skin hydration in topical

formulations [178, 179].

Figure 6. Metabolism of lipid in body

1.12 Limitations of Solid Lipid Nanoparticles

SLNs have the potential to overcome many of the disadvantages associated with

other nanoparticle drug delivery systems. However, they too may have limitations,

most of which are associated with the lipid crystallinity of the particles. On one end of

the spectrum, SLNs that exhibit high crystallinity may be limited by low drug loading

[180]. There may simply be little room for the drug to be inserted among the tightly

packed lipid molecules [181]. On the other end of the spectrum, working with less


21

crystalline lipids (or reducing the crystallinity of the lipid through the addition of

other lipids and/or surfactants) may lead to stability issues [182, 183]. If the lipid

crystallizes over time, it may lead to drug expulsion [184], particle size growth or

gelation[135, 185, 186]. However, by characterizing the physicochemical properties

of the SLNs, these issues may be anticipated and avoided through changes to the

composition (e.g., lipids, surfactants), preparation procedure, or storage conditions

[147].

1.13 Niclosamide

Niclosamide (NIC) is oral anthelminthic drug having chemical name 5-chloro–

N–(2–chloro–4–nitrophenyl)–2–hydroxybenzamide [187]. NIC is yellowish or

yellowish–white, fine crystals, practically insoluble in water and slightly soluble in

ethanol [188]. NIC is used from five decades against tapeworm infections like Taenia

saginata, Diphyllobothrium latum, Taenia solium and Hymenolepis nana infections

[189]. NIC acts by inhibiting oxidative phosphorylation in mitochondria and

anaerobic ATP production [190]. Modern studies shows that NIC is very effective

against cancerous cell [191]. It has shown anti-proliferative activity but the exact

mechanism against cancer cells is unknown [192]. Besides, NIC gave striking anti-

tumor activity in animal models [187].

Niclosamide belongs to BCS-II drugs having poor water solubility i.e. 0.23

μg/ml [193]. One of the modern approaches to enhance NIC solubility is to modify it

chemically and several water-soluble NIC derivatives have been created [194].


22

Figure 7. Chemical Structure of Niclosamide

Table 3. Properties of Niclosamide

1.14 Sulfasalazine

Sulfasalazine (SZN)- a sulfa group containing drug & the derivative of mesalazine,

formed by an azo bond between salicylate and sulfapyridine. Sulfasalazine is a kind of

drug known as Disease-Modifying Anti-Rheumatic Drug (DMARD). DMARD have

the effect of dulling down the underlying disease course, rather than only curing

symptoms. SZN is used to treatment rheumatoid arthritis and also for the treatment of

other kind of arthritis related with ankylosing spondylitis and inflammatory bowel

disease [195].

PROPERTIES OF NICLOSAMIDE

Formula C13H8Cl2N2O4

Molar mass 327.119 g/mol

Melting point 225 to 230 °C

Bioavailability <10%

Biological half-life 10-12 hours

Supplier Shaigan Pharmaceuticals, Rawalpindi-Pakistan


23

Figure 8. Chemical Structure of Sulfasalazine

Table 4. Properties of Sulfasalazine

1.15 Background of the Study

Two classes of BCS i.e. II and IV of poor water solubility offer many

challenges for researchers working on drug delivery system [196]. Various

approaches have been used to decrease the particle size in order to enhance water

solubility & permeability of these drugs [197].

Both lipid-based technologies and nano-technology approaches are used to

augment solubility/bioavailability of poor water-soluble drugs [51]. In Nano

technological-based approaches, the solubility and dissolution rate of compounds can

PROPERTIES OF SULFASALAZINE

Formula C18H14N4O5S

Molar mass 398.394 g/mol

Melting point 245 °C

Bioavailability <15%

Biological half-life 5-10 hours

Supplier Ferozsons Lab. Ltd. Nowshera-Pakistan


24

be improved by decreasing the particle size or increasing the surface area. Ultrafine

particles of drugs can be shaped either top-down or bottom-up techniques. On other

hand, lipids have also twisted further profitable & academic consideration as a

favourable approach to enhance bioavailability of lipophilic drugs [52]. SLNs concept

actually comes from o/w emulsion [198]. SLNs are actually colloidal system that have

attained importance as substitute of polymer based nano-particles along with lipo-

somes, nano-capsules (lipid-based) & nano-emulsions [43].

In this study, drugs such as Niclosamide and Sulfasalazine were successfully

loaded in SLNs containing stearic acid as solid lipid. Both in-vitro & in-vivo released

data of drug loaded-SLNs confirmed that SLN system is most appropriate to improve

the oral drug delivery of NIC and SZN with improved water solubility, permeability

and lastly bioavailability.

CHAPTER 2 LITERATURE REVIEW

25

Chapter 2 LITERATURE REVIEW

Solid Lipid Nanoparticles as drug carriers for topical glucocorticoids, in which they

studied skin atrophy systemic side effects which occurred after applying conventional

prednicarbate cream which could be avoided when this drug was formulated as SLN,

prednicarbate uptake was enhanced and it was accumulated in the epidermis with low

concentration in the dermis [199].

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic

and dermatological preparations, in it they introduced Solid Lipid Nanoparticles for

topical drug application, it showed low toxicity, due to its small size close contact with

skin increases the amount of drug penetration in the skin, increased skin hydration and

also enhanced the chemical stability of compounds sensitive to light, oxidation and

hydrolysis [180].

The influence of the crystallinity of lipid nanoparticles on their occlusive properties,

had investigated the occlusive properties of Solid Lipid Nanoparticles. The formation of

lipid film on skin followed by occlusion was described for lipid nano-particles [200].

Composition, quality control and antimicrobial activity of the essential oil of long-

time stored dill (Anethum graveolens L.) seeds from Bulgaria, studied the antimicrobial

activity of Essential oil of dill seed which was stored for more than 35 years and resulted

into high activity of the essential oil of A. graveolens against Asperigillus niger and yeast

Saccharomyces cerevisiae and Candida albicans [201].


26

Solid lipid nanoparticle and microemulsion for topical delivery of triptolide,

developed solid lipid nanoparticles and microemulsion for topical delivery of triptolide.

Triptolide loaded SLNs showed more and also anti-inflammatory activity [172].

The influence of solid lipid nanoparticles on skin hydration and viscoelasticity – in

vivo study, studied the influence of solid lipid nanoparticles on the skin hydration,

viscoelasticity and vivo study. The reduction in trans-epidermal loss of water produced by

occlusion followed by increase in skin hydration when SLNs were applied dermally [202].

Podophyllotoxin-loaded solid lipid nanoparticles for epidermal targeting, compared

podophyllotoxin-SLNs with podophyllotoxin-tincture with regard to permeation of skin

and targeting effect, localization effect in the epidermis was suggested by them and

reduction in systemic side effect is expected after application of podophyllotoxin using a

formulation containing solid lipid nanoparticles [203].

Artemisia arborescens L. Essential Oil-Loaded Solid Lipid Nanoparticles for

Potential Agricultural Application: Preparation and Characterization, formulated the

essential oil loaded Solid Lipid Nanoparticles and concluded that the formulations

demonstrated a high physical stability and a good capability to reduce the essential oil

evaporation of Artemisia arborescens for agricultural application [204].

SLNs were fabricated containing Vitamin A. In vitro studies of vitamin A palmitate

from nanoparticulate dispersion and it gel showed prolonged drug release upto 24 hrs. and

penetration studies also showed 2 times higher drug concentration in the skin when

compared with conventional gel. In vivo studies showed increase in thickness of sratum

corneum with improved skin hydration and no skin irritation [174].


27

Nanostructured lipid carriers (NLC) in cosmetic dermal products, developed Solid

Lipid Nanoparticles at the beginning of 1990 and introduced it as alternate carrier system

to emulsion, liposomes and polymeric nanopacticles. They incorporated many drugs into

solid lipid nanoparticles & nanostructured lipid carriers for various routes of drug

administration and resulted in, to provide sustained drug release & increase in stability and

inert drug carriers [205].

Solid lipid nanoparticles (SLN) of tretinoin: Potential in topical delivery, Studied

tretinoin loaded solid lipid nanoparticles formulation gel release nanoparticles-based

tretinoin-gel resulted in remarkably fewer erythremic episodes in comparison with the

marketed product [206].

Cyproterone acetate loading to lipid nanoparticles for topical acne treatment:

particle characterization and skin uptake, developed cyproterone acetate loaded lipid

nanopartocles for topical acne treatment. Application of antiandrogen drug cyproterone

acetate loaded solid lipid nanoparticles increased the skin penetration at least four folds

over the uptake from the conventional cream and emulsion [207].

Lipid nanoparticles for prolonged topical delivery: An in vitro and in vivo

investigation, formulated ketoprofen and naproxen loaded nanoparticles using hot high

pressure homogenization and ultrasonication technique, nanoparticle behavior on human

skin showed increase in drug penetration and permeation due to reduced particle size. A

prolonged anti inflammatory effect was also observed and reported usefulness of lipid

nanoparticles as carriers for topical administration [208].

Characteristics of Anethum graveolens (Umbelliferae) Seed Oil: Extraction,

Composition and Antimicrobial Activity, studied the extraction, composition and


28

antimicrobial activity of Anethum graveolens. They extracted the dill oil by adopting

steam distillation process and separated the components using gas chromatography. The

antimicrobial activity was studied against microorganisms and concluded that dill seed oil

has antimicrobial activity against Escheria coli, Styplococuss aureus, Bacillus subtilis

[209].

Development of SLNs from natural lipids: Application to topical delivery of

Tretinoin, developed SLN of tretinoin by emulsification –solvent diffusion teery of

technique and evaluated SLN based gel for topical delivery of tretinoin.

They demonstrated the investigation by improvement in photostability when

compared with methanolic TRE and also prevented isomerization. They stated

improvement in skin tolerability and has more permeation profile compared to its

marketed cream [210].

Formation and stabilization of Ibuprofen nanoparticles in supercritical fluid

processing, developed SLN and NLC for improved dermal delivery of lidocain and it was

formulated into hydrogel for topical application. They concluded that SLN formulation

was stable with respect to particle size, polydispersity and entrapment efficiency for 6

months at 40_C/75% relative humidity. Both SLN & NLC resulted in five fold and six

fold increase in duration of anesthesia respectively, when compared with xylocain Rx gel

[211].

Studies concerning the entrapment of Anethum graveolens essential oil in liposomes,

prepared the liposomes (MLV and SUV) and studied the entrapment efficiency of

essential oil of dill seed which is influenced by liposomal composition, size and its

lamellarity [212].


29

Applications of novel drug delivery system for herbal formulations, reviewed

standardized plant extracts or mainly polar phyto constituents like flavnoids, terpenoids,

tannins, xanthenes when administered through novel drug delivery system show much

better absorption profile which enables them to cross the biological membrane resulting in

enhanced bioavailability [213].

Characterization of Nigella Sativa L. Essential Oil-Loaded Solid Lipid Nanoparticle,

prepared Solid Lipid Nanoparticles loaded with Nigella sativa essential oil by adapting

pressure homogenization technique using hydrogenated palm oil, softisan 154 and Nigella

sativa essential oil as lipid matrix and sorbitol and water as surfactant and concluded that

results obtained showed that solid lipid nanoparticles formulations are suitable carriers in

pharmaceutical and cosmetic fields [214].

Importance of novel drug delivery systems in herbal medicines, summarized various

drug delivery technologies which could be used for herbal actives for better therapeutic

effect [215].

Transdermal Drug Delivery Enhancement by Compounds of Natural Origin,

reviewed transdermal drug delivery enhancement by compounds of natural origin in which

he studied different compounds from natural origin and found out that many chemical

compounds extracted from natural sources showed potential as skin penetration enhancing

agents and also observed that the effectiveness of the penetration enhancer depends not

only on their concentration in the formulation but also on the physio-chemical

characteristics of the drug to be transported through / into the skin layers [216].

Giyoong Tae, et al, In-vivo tumor targeting of pluronic-based nano-carriers, studied

and demonstrated that chitosan conjugated pluronic based nano carrier can be used as


30

novel platform for transcutaneous delivery of hydrophilic macromolecules and other drug

delivery applications [217].

Heamalatha S.,et al, Pharmacognostical, Pharmacological, Investigation on Anethum

graveolens Linn, concluded in the article as Anethum graveolnes has been recognized in

different system of traditional medicines for the treatment of different diseases in human

beings and the review support all updated information on its pharmacognosy,

pharmacological activities and traditional use [218].

Phytochemical analysis and antibacterial efficacy of dill seed oil against multi-drug

resistant clinical isolates, evaluated dill seed-oil for phyto-chemical ingredients and anti-

bacterial activity & TLC bio-autography. Phyto-chemical analysis disclosed the presence

several chemical constituents and antibacterial

activity was carried out on 8 multi drug resistant different strains both gram +ve and

gram -ve bacteria and two standard strains and resulted that it shows broad antibacterial

activity against both gram +ve and gram -ve bacteria [219].

Donsib Francesco, et al, Design of nanoemulsion-based delivery systems of natural

antimicrobials: Effect of the emulsifier, investigated the effect of nanoemulsion delivery

system on the antimicrobial activity of different essential oil components which was

significantly affected by the formulation of the nanoemulsion where different bioactive

compounds were encapsulated [220].

Preparation and characterization of solid lipid nanoparticles loaded with

frankincense and myrrh oil, prepared and characterized Solid lipid nanoparticles for oral

delivery of frankincense and myrrh essential oils. Solid lipid nanoparticles were prepared

by high pressure homogenization by using compritol 888 ATO and soybean lecithin and


31

tween 80 which were characterized for standard parameters of Solid lipid nanoparticles

and they presented the report that it can be used as drug carriers for hydrophobic oil drug

extracted from traditional Chinese medicines [221].

Chemical Constituents of Essential Oil from Anethum Sowa Kurz. Seed, in her

research article mentioned the major constituents of dill seed oil obtained after its

hydrodistillation and the analysis of which is done by GCMS [222].

Antidepressant Activity of Curcumin Loaded Solid Lipid Nanoparticles (C-SLNs)

In Mice, formulated Solid lipid nanoparticles of Curcumin obtained from the rhizome of

the herb Curcuma longa L. and studied its antidepressant effects on rodent models. It

showed that Curcumin loaded solid lipid nanoparticles with improved bioavailability and

permeability which possess higher antidepressant potential on administration of single and

much lower dose when compared to free Curcumin [223].

Thapa Raj kumar, et al, Herbal Medicines Incorporated Nanoparticles:

Advancements In Herbal Treatment, reviewed the use of nanotechnology to overcome

several problems for herbal medicine to formulate it [224].

Formulation and Evaluation of Herbal Antidandruff Shampoo Containing Garlic

Loaded Solid Lipid Nanoparticles, prepared and evaluated herbal antidandruff shampoo

containing Garlic loaded Solid Lipid Nanoparticles which was formulated by hot

homogenization method and evaluated for zeta potential, particle size, polydispersity

index, scanning electron microscopy and drug release and concluded that it is more

effective for the treatment of dandruff on scalp and hair with no side effects [225].

Solid lipid nanoparticles (SLNs) gel for topical delivery of aceclofenac invitro and

invivo evaluation, prepared a gel of acelofenac for topical delivery and evaluated for in


32

vitro and in vivo and concluded that and drug release of solid lipid nanoparticle gel

formulation was better controlled as compared to SLN dispersions and in vivo anti

inflammatory study showed that action of acelofenac was enhanced for SLN dispersion

and gel formulation [226].

Role of nanoparticles for production of smart herbal drug−An overview, reviewed

and summarized role of nanoparticles for production of smart herbal drug. In this article

fifteen herbal plant/plant parts were reviewed along with information regarding botanical

identification, its active ingredients, pharmacological activities drawbacks related with

traditional dose, method of their action of nano-carrier and their efficacy with positive

results on above matter. They concluded that implementation of these approaches on a

large scale that includes more plant with high therapeutic properties [227].

Vijayan V., et al, formulated and characterized SLN loaded Neem oil for topical

treatment of acne which was prepared by double emulsification method using different

concentration of lecthin and Tween 80 the result concluded that neem oil loaded Solid

Lipid Nanoparticles with more lecithin content in their colloid shows sustained effect

which satisfactorily produced antibacterial action on acne microbes so it could be used

successfully for prolonged treatment of acne [228].

Applications of Nanotechnology Based Dosage Forms for Delivery of Herbal Drugs,

reviewed herbal medicines are globally accepted as alternative system of therapy in

pharmaceuticals but the drug delivery system for herbal drug is quite traditional and out of

date. As number of plant constituents like flavanoids, tannis, terpenoids show enhanced

therapeutic effect at similar or less dose when incorporated into novel drug delivery

system as compared to conventional system [229].


33

Antibacterial and Antioxidant Activities of Anethum graveolens L. Dried Fruit

Extracts, studied the antioxidant and antibacterial activity of Anethum graveolens L. dried

fruits extract against five pathogenic bacteria and found that

essential oil fraction exhibited antibacterial activity against it [230].

An effective tool for enhancing bioavailability and bioactivity of phytomedicine,

studied application of nanotechnology which leads to increase in bioavailability and

bioactivity of phytomedicine by reducing the size of the particles, surface modification,

entrapping the phytomedicine with different polymers of micro and nano material. Nano

material aids the targeted and sustained delivery and improved the pharmacokinetic

profile, diffusion of drug into various organs by crossing the barriers [231].

Application of nano and micro particles on the topical therapy of skin related

immune disorders, studied the comparison of normal and pathological skin structure,

penetration route of nano and micro drug particles, in-vivo and in-vitro evaluation methods

for topical therapy and also highlighted applications of particles from herbal medicines for

skin immune disorder [232].

2.1 Solid Lipid Nanoparticle Production Techniques

At the beginning of 1990s, the advantages of solid particles, emulsions and

liposomes were combined by the development of the ‘solid lipid nanoparticles’, as a

carrier system for pharmaceuticals and cosmetics. The use of lipids in solid form for drug

delivery is identified for many years in the form of lipid pellets. Mostly, biocompatible

and biodegradable lipids are used. Similar to emulsions and liposomes, the SLN consist of

toxicologically acceptable excipients.


34

There are two basic production methods for SLN, the high pressure homogenization

technique developed by Muller and Lucks, 1996 and the micro emulsions technique

invented by Gasco in Turin [66]. In the beginning of SLN research, there were only three

research groups working on this topic, the groups of Muller, Gasco, and Westesen [233].

Subsequently more attention was paid to this important area which is evident from the

increase of research groups working in the area and the number of published papers. In the

beginning, SLN were developed primarily for intravenous administration and later, they

were exploited for oral drug delivery, e.g., that of cyclosporine [234].

2.2 Solvent Emulsification Diffusion Technique

Hu et al. used the concept of emulsion solvent diffosion to produce SLN (Hu et al.,

2002). Model drug mifepristone and monostearin were dissolved completely in a mixture

of acetone and ethanol in water bath at 50°C and the resultant organic solution was poured

into an acidic aqueous phase under agitation at 25 °C followed by centrifugation to get

SLNs. The aim of this investigation was to assess a prolonged release with this method

and the results exhibited a biphasic drug release pattern with an initial burst and prolonged

release over 4 days. He reused the solvent-diffusion technique in aqueous phase to

establish a novel preparation method for peptide-loaded SLN [235].

The model peptide gonadotropin was incorporated to study the entrapment

efficiency, size, zeta potential (charge) and drag delivery characterization. The results

demonstrated the principle suitability of SLN as a prolonged release formulation for

hydrophilic peptide drugs.

The emulsification-diffusion method traditionally used to prepare polymeric

nanoparticles was adapted to obtain lipidic nano-spheres (LN) using four model


35

lipids[236]. The results showed that particle size could be reduced by rising process

temperature, stirring rate/time, the concentration of surfactants, and by decreasing the lipid

concentration. It was observed that the influence these parameters was allied with a

mechanism which is based on physico-chemical instability. In this way, it was proposed

that the fast solvent-diffusion made areas of local saturation near interface, and LN are

fabricated because of resultant interfacial phase-transformations and aggregation of lipid

[236].

2.3 Micro Emulsion Method

Micro emulsions, or swollen micelles, represent an intuitively interesting approach

for producing solid lipid nanoparticles. As elaborated by Moulik and Paul, micro

emulsions are thermodynamically stable, isotropic, and clear systems comprised of water,

lipids, and surfactants [237]. Given appropriate conditions, the lipid/surfactant

constituents of micro emulsions self-assemble into spherical particles typically ranging

from 5-100 nm. These particles are polydisperse in nature, but polydispersity decreases

with decreasing particle size. Gasco optimized the synthesis of producing solid lipid

nanoparticles from micro emulsions [66]. Micro emulsions using stearic acid and

surfactants were formed at 65-70 °C, and then were dispersed into near freezing water at

ratios of 1:25-1:50 hot micro-emulsion to cold water.

Cavalli et al. reported, stearic acid when stabilized by ionic and nonionic surfactants,

respectively [238]. The minimal energy requirement for micro emulsion formation is a

significant advantage. The theoretical stability is quite attractive for long-term retardation

of phase separation phenomena which gives rise to storage instabilities, i.e. particle size


36

growth. Thus, lipid nanoparticles formed by micro emulsions are amenable to sensitive

biomolecules.

Cavalli et al. prepared paclitaxel loaded SLN and tobramycin loaded SLN from

micro emulsion technique [238]. Zara et al. prepared SLN using the same technique for

pharmacokinetics and tissue distribution study of doxorubicin loaded [239]. Heydenreich

et. al in 2003 later prepared cationic SLN by the micro emulsion technique with

polysorbate 80 (Tween 80) and butanol as surfactants. The SLN consisted mainly of

stearyl amine and different triglycerides with diameter range of 100--500 nm and zeta

potential around +15 inV. Three different purification methods, ultrafiltration,

ultracentrifugation and dialysis, were investigated and compared with the cellular toxicity

and physical stability of the dispersions.

Curcuininoids loaded SLN were fabricated by using a micro emulsion technique at

75°C [240]. It was observed that change in concentration of excipients had significant

effects on loading capacity of curcuminoid, the average particle size, and size-distribution.

The results revealed that after storage in the absence of sunlight for 6 months, the

percentages of the remaining curcumin, bisdemethoxycurcumin and demethoxycurcumin

were 91, 96 and 8 8 , respectively. For micro emulsion technique, the capital and operating

expenses would be lower than high shear homogenization and high pressure

homogenization techniques but the major disadvantage of the micro emulsion approach

was the sensitivity of micro emulsion systems to minor changes in composition or

thermodynamic variables, which can cause significant phase transitions. A process

optimized for a particular system may no longer work if the composition was modified

only slightly. This lack of robustness leads to unacceptably high development costs.


37

Additionally, the solidification process shifted the system to a thermodynamically unstable

state, undermining the very advantage of a micro emulsion approach.

Table 5. Emulsifiers used for the production of lipid nanoparticles

EMULSIFIERS/CO-EMULSIFIERS HLB VALUE

Solutol HS 15 15

Polysorbate 80 15

Polysorbate 65 10.5

Polysorbate 20 16.7

Poloxamer 407 21.5

Poloxamer 188 29

Lecithin 4-9

Cremophor EL 12-14


38

Table 6. Types of Lipids use in preparation of Lipid based nanoparticles

2.4 Stability of SLNs

The stability of SLNs are generally considered from two angles, (1) distribution of

particle-size and (2) crystalline form of lipid. The degree of polydispersity(PDI) can

influence the growth of particle size through Ostwald-ripening and can affect the overall

kinetics of drug release. Similarly the crystalline state of lipid can be strongly correlated

with drug encapsulation, drug-release, and the particle-geometry [44]. The droplet of

S.NO TYPE OF LIPID

1 Beeswax

2 Behenic acid

3 Carnauba wax

4 Cetyl palmitate

5 Glyceryl behenate

6 Glyceryl monostearate

7 Glyceryl palmitostearate

8 Goat fat

9 Palmitic acid

10 Softisan 142 and Softisan 154

11 Stearic acid

12 Theobroma oil

13 Trilaurin

14 Trimyristin (Dynasan 114)

15 Tripalmitin (Dynasan 116)

16 Tristearin (Dynasan 118)

17 Waxes

18 Witepsol bases


39

emulsion have the ability to keep a discrete droplet of uniform size depends on the

dispersing ability of surfactants. The emulsion droplet will be stable for longer time, if the

dispersing ability of surfactant is enough. But whe surfactants’ dispersing ability is not

enough for emulsion-system, then characteristic phase-separation process will start

quickly like Ostwald ripening, creaming, coalescence and flocculation. Electrostatic

repulsion results from the formation of an electrical double layer at the lipid-water

interface. Ionic surfactants, such as negatively charged lecithin adsorbed at the interface

attract solution counter ions, cations in the case of lecithin, into the interfacial region. The

counter ions effectively adsorb onto the oppositely charged interface. The net charge at the

interface affects the ion distribution in the nearby region, increasing the concentration of

counter ions close to the interface. Thus, an electrical double layer is formed in the

interfacial region. Zeta potential is a function of the surface charge of the particle, any

adsorbed layer at the interface, and the nature and composition of the surrounding

environment. Particles are prevented by steric-stabilization to small distances which is

necessary for coalescence and flocculation. Non-ionic surfactant operates by steric-

stabilization, and ethylene-oxide or propylene oxide co-surfactant are normally employed

for their steric-stabilization abilities. The polyoxypropylene chain adsorbs onto the

hydrophobic interface, and the polyoxyethylene chain extends into the aqueous phase in a

coil configuration. Given sufficient surfactant concentration and hydrophilic chain length,

often > 2 0 ethylene oxide units, the hydrophilic coils extending outward from the surface

maintain other particles at distances required for stability. Unlike systems stabilized by

ionic surfactants, those stabilized by nonionic surfactants are independent of bulk

electrolyte concentration. However, non-ionics are affected by temperature.


40

Hydrophilicity of the polyoxyethylene chain decreases with increasing temperature as

chain dehydration occurs. As dehydration increases, the polyoxyethylene chain adsorbs

more strongly on the hydrophobic surface, reducing the steric boundary around the

particle. Above a critical temperature, known as the critical flocculation temperature,

flocculation occurs as the steric hindrance no longer exceeds the van der Waals attraction

between particles. In emulsion systems, the temperature dependency of nonionic

surfactants can give rise to a phase inversion, i.e. from oil-in-water to water-in-oil. This

temperature is referred to as the phase inversion temperature. Regularly, the finest

stabilization approach is to invoke both these approaches (electrostatic approach and steric

approach). This approach has been commonly used in liposome drug delivery system

[241]. However, one must remain cognizant of the effect of steric stabilization on the zeta

potential. Adsorption of the steric stabilizer shifts the shear plane outward, reducing the

zeta potential. A proper balance between electrostatic repulsion and steric stabilization

must be obtained for long-term stability, if stability depends on both mechanisms.

Lipid crystallinity is another dimension of lipid nanoparticle stability significantly

impacting lipid nanoparticle drug incorporation and release characteristics. Crystallization

is a balance between attractive intermolecular forces and entropic factors. In lipids, van

der Waals forces drive non-polar molecules closer to one another. Entropy favors

increased molecular disorder, driving molecules farther apart. As intermolecular attraction

increases, or entropy decreases, liquids crystallize more readily. As temperature decreases,

entropy decreases and the intermolecular distance decreases. Therefore, intermolecular

attraction outweighs entropy, and crystallization will commence at a site of nucleation.


41

Historically, four states of crystallinity are associated with lipids, but more recent research

has revealed numerous varieties of crystalline structures in lipids [242].

Lipid mixtures, surfactant mixtures, and rapid cooling techniques promote the

crystal structure. Using lipids of dissimilar geometries inhibits closely packed, highly

ordered crystal structures. For example, introducing oleic acid, cw-9-octadecanoate, into

tripalmitin inhibits close, ordered acyl chain packing due to the cis double bond of oleic

acid. Likewise, introducing surfactants whose hydrophobic tails are geometrically

dissimilar to the core lipid inhibits highly ordered crystal formation. Sterols, such as the

bile salts like sodium taurocholate, possess a bulky, five ring hydrophobic region which

does not permit close highly -ordered crystal formation, at least near the interface. As

noted before, rapid cooling does not provide adequate time for the crystallization process

to form the more highly ordered p crystal. These techniques provide researchers with

opportunities to produce solid lipid nanoparticles in the a crystal form. Despite the

stability challenges, optimized solid lipid nanoparticle dispersions can be stable for more

than one year [110]. By photon correlation spectroscopy (PCS) analysis, Muller et al.

demonstrated that glycerol palmitostearate and tribehenate nanoparticles were stable for 3

years [135]. To evade in-stability problems in aqueous-dispersions, scientists have

established lyophilization and spray drying techniques which is very successful for long

term SLNs stability [135]. Historically, four states of crystallinity are associated with

lipids, but more recent research has revealed numerous varieties of crystalline structures in

lipids[243].


42

2.5 Drug Loading and Release from Lipid Nanoparticles

A variety of drugs, including agents for treating cancer, AIDS, fungal infections,

high blood pressure, mental illness, skin disease, and imaging have been loaded into solid

lipid nanoparticles. For efficiency and efficacy reasons, the amount of drug that can be

loaded is very important. Calculated as the ratio of drug weight to the sum of drug and

lipid weight, loading capacity typically ranges from 1-5% [108]. For HPH, Muller

suggests that capacity is determined by the drug solubility in the melted lipid, the

miscibility of the melted drag and melted lipid, and the physiochemical structure of the

solid lipid.

2.6 Enhanced Sustained Drug Release

Obtaining controlled drug release from lipid nanoparticles remained elusive as, more

often than not, burst release kinetics had been observed [108]. Muhlen reported the first

controlled release of a drug, prednisolone, from HPH produced solid lipid nanoparticles.

In vitro drug release was obtained for 7 weeks. Release kinetics were dependent on the

lipid matrix, surfactant concentration, and HPH production parameters, but were

independent of particle size. The size independence suggested the mass transfer was not

diffusion limited. Burst release increased with increasing processing temperature and

increasing surfactant concentration, leading Muller to suggest that drug partitioning into

the aqueous phase during homogenization negatively affected sustained release. As

temperature and surfactant levels increased, drug solubility in water increased. As

temperature decreased, Muller rationalized that the lipid crystallized initially in the center,

and the drug repartitioned into the lipid; however, because the lipid core had already


43

crystallized, the core was unavailable to the drug. As the system continued to cool, the

drug solubility in the water diminished, and the drug was concentrated in the lipid ‘shell’

region. This enriched shell profile then promoted drug burst release [108, 154, 244-247].

To obtain sustained drug release, Muller suggested a diffusion controlled release

mechanism and a uniform drug profile throughout the lipid shell or an enriched ‘core’

[108]. If a delayed release profile was desirable, the proposed drug enriched core/lipid

shell model represents an interested option. Existing synthesis techniques do not provide

precise control for constructing prescribed desired drug profiles. The process remains a

trial-and-error based approach in which changing drugs, lipids, surfactants, concentrations,

and process parameters lead to unpredictable results. An improved production technology

is required to provide this level of control.

2.7 Mechanism of Action of Lipid-Based Delivery Systems

A wide variety of hypotheses have been put forth to explain the mechanism by

which lipid-based systems enhance the oral bioavailability of compounds. In general,

these hypotheses can be divided into two Categories: physical-chemical (e.g., wetting

effects or enhanced solubility) and biochemical (inhibition of efflux transporters). While

the literature is full of examples of successful enhancement of bioavailability by lipid

systems, a mechanistic understanding of the enhancement process has not been fully

developed. It is quite possible that several mechanisms are in operation simultaneously. A

better understanding of the mechanism(s) associated with enhancement of absorption

would help formulators make rational, science-based decisions as to the suitability of

lipid-based systems for a particular application. With respect to biological effects, there is

new interest in the chemo protective effect of multidrug-resistant transporters for in vivo


44

cancer cells. Data suggest that it may also be possible for lipid systems to avoid p-

glycoprotein-based drug efflux in enterocytes [248]. Some evidence suggests that

polymeric surfactants typically employed in lipid formulations may be critical as ATP-

depletion agents [249]. Restricting the supply of ATP might slow the activity of active

transporters. The growing appreciation of MDR (multidrug resistance) transporter(s) in

cancer chemotherapy makes this an important field awaiting further mechanistic studies

[250]. Perhaps or even more potential for widespread use, SLN have been studied for their

ability to target drug delivery to specific regions of the gastrointestinal tract[108]. No

matter what the mechanism by which lipid-based systems operate in the gastrointestinal

tract, physical chemical processes at work in the lipid aggregate play a decisive role in the

successful application of formulations. A better mechanistic understanding of the

physical/chemical aspects of the aggregates will aid the rapid and rational application of

lipid-based formulations.

2.8 Enhancement of Bioavailability by Lipid Nanoparticles

First of all, identical to the drug nanocrystals the SLN possess adhesive properties.

They adhere to the gut wall and release the drug exactly where it should be absorbed. In

addition the lipids are known to have absorption promoting properties, not only for

lipophilic drugs such as Vitamin E but also drugs in general [251]. There are even

differences in the lipid absorption enhancement depending on the structure of the lipids,

for example MCT (medium chain triglyceride) lipids are more effective than LCT (long

chain triglyceride) [252]. Basically, the body is taking up the lipid and the solubilized drug

at the same time. It can be considered as a kind of “Trojan horse” effect.


45

Possible Mechanisms of Oral Bioavailability Enhancement In Vivo

a. Enhanced wetting of hydrophobic solids with formulation components

resulting in enhanced rate of dissolution

b. Increased rate of dissolution into the aqueous environment from oil droplets

of high surface area

c. Enhanced thermodynamic activity via supersaturation of the aqueous

environment of the GI tract

d. Promotion of absorption via intrinsic lipid pathways

e. Targeting of small hydrophobic particles toward Peyer’s patches

f. Short circuiting of the aqueous boundary layer

g. Inhibition of active drag efflux from enterocytes.

CHAPTER 3 MATERIALS AND METHODS

46

Chapter 3 MATERIALS AND METHODS

3.1 Materials

3.1.1 Chemicals

1. Niclosamide (NIC) was a generous gift from Shaigan Pharmaceuticals Pvt. Ltd,

Pakistan

2. Sulfasalazine was a generous gift from Ferozsons Labs Nowshera, Pakistan.

3. Stearic Acid Acros Organics Thermo Fisher Scientific (USA),

4. Tween-80 Acros Organics Thermo Fisher Scientific (USA)

5. Poly Ethylene Glycol-400Thermo Fisher Scientific (USA).

6. Hydroxy propyl methyl cellulose (Colorcon Ltd, Dartford Kent, UK)

7. Cross linked poly vinyl pyrrolidone (BASF AG, Ludwigshafen, Germany)

8. Micro crystalline cellulose (FMC BioPolymers, Philadelphia, PA, USA)

9. Calcium hydrogen phosphate dihydrate (JRS PHARMA GmbH & Co. KG,

Rosenberg, Germany)

10. Talc (Luzenac® pharma; Luzenac Europe, Toulouse, France)

3.1.2 Instrumentations

a. Dynamic Light Scattering

Particle size measurements, Poly dispersity index and Zeta-Potential analyses (ζ)

were carried out through Photon Correlation Spectroscopy (Zeta-sizer Nano ZS-90,

Malvern Instruments-UK) at 90° scattering angle and 25°C. [Figure 9].


47

Figure 9. ZS-90, Malvern Instruments

b. Scanning Electron Microscope

For morphological studies, Scanning electron microscopy (SEM) was performed at

magnification of 30,000X and acceleration voltage of 20KV using Scanning Electron

Microscope JSM-910, JEOL (Japan). [Figure 10].

Figure 10. Scanning Electron Microscope


48

c. Differential Scanning Calorimeter

Differential Scanning Calorimeter Perkin Elmer (USA) compared thermal

properties of the unprocessed drugs and lyophilized formulations. Samples were heated

from 40 ºC to 300ºCat heating rate of 10ºC/min. [Figure 11].

Figure 11. Differential Scanning Calorimeter

d. Powder-X-ray Diffraction (P-XRD)

Powder X-ray Diffraction JEOL (Japan) technique was applied to analyse crystalline

nature of unprocessed drugs and lyophilized formulations. P-XRD analysis were carried

out by CuKa radiation and scanned up to 80º, step size 2Ø-0.05º and step time 1.0 second,

divergence slit 1º, receiving slit 0.2mm and scattering slit 1º (12).

e. Fourier Transform Infrared Radiation (FT-IR) Measurements

Fourier transform Infrared Spectrometer Prestige-21 Shimadzu (Japan) study was

carried out to confirm the compatibility among different formulation’s components.

Spectra were taken in the range of 2000 to 400cm−1. For compatibility between drug and


49

excipients, the peaks and patterns shaped were compared. The spectra were recorded at

room temperature. [Figure 12].

Figure 12. Infra-Red Spectroscope

f. Freeze Dryer

Freeze dryer (Heto PowerDry LL1500- Thermo Electron Corporation-USA).

g. Nano-Drop-Spectrophotometer (Thermo scientific 2000c/2000 UV-VIS

Spectrophotometer)

h. High Performance Liquid Chromatography with reversed phase column

(Supelco C18, 25cm in length, 4.6 mm width and 5µm particle size) protected

with a pre-column (Supelco Cl8) was used.

3.2 Methods

3.2.1 Fabrication of Blank SLNs by Microemulsion Technique

Micro-emulsion technique was optimized to fabricate unloaded SLNs through four

variables i.e. different concentrations of Stearic acid (SA), Tween-80, Polyethylene Glycol

(PEG) and stirring time. Twelve different SLN formulations (BME-1 to BME-12) were

prepared [Table 7]. Stearic acid was melted above its melting point (at least 5°C);


50

surfactant (Tween-80) & PEG were dissolved in de-ionized water and heat up to 75 ⁰C

with continuous magnetic stirring at 1200 rpm. Both oily phase and aqueous phase were

mixed and continued stirring for different time intervals to obtain micro emulsion

followed by addition of cold water (1:25) under magnetic stirring to get SLNs dispersion

[69]. The SLNs dispersion was centrifuged at 30,000 rpm for 15 minutes at room

temperature [51].

Table 7. Blank SLNs formulations for Micro-emulsion Technique.

FORMULATION STEARIC ACID

(g)

TWEEN-80

(g)

PEG-400

(g)

STIRRING TIME

(min)

BME-1 2 1 00 5

BME-2 1 1 00 5

BME-3 2 3 00 5

BME-4 1 2 00 5

BME-5 1 1.9 0.1 5

BME-6 1 1.8 0.2 5

BME-7 1 1.7 0.3 5

BME-8 1 1.6 0.4 5

BME-9 1 1.5 0.5 5

BME-10 1 1.6 0.4 10

BME-11 1 1.6 0.4 15

BME-12 1 1.6 0.4 20


51

Figure 13. Schematic diagram of Micro-Emulsion Method

3.2.2 Fabrication of Blank SLNs by Solvent Emulsification Diffusion Technique

Solvent emulsification-diffusion (SED) technique was optimized for unloaded SLNs

through different formulation variables and process parameters i.e. stearic acid, Tween-80,

Polyvinylpyrrolidone (PVP) concentrations and stirring time. Twelve different unloaded

SLN formulations (BSE-1 to BSE-12) were prepared [Table 8]. Specified quantity of

stearic acid was dissolved in water miscible organic solvent (ethanol) [253] Tween-80 and

Polyvinylpyrrolidone (PVP) in specified quantity were dissolved in deionized water.

Organic and aqueous phases were mixed on magnetic stirrer at 1200 rmp for specific time

intervals to obtain lipid matrix dispersion followed by dilution with excess amount of

water. This dilution lead to the diffusion from inner phase into outer phase resulting in


52

lipid aggregation in the form of SLNs [236]. Organic solvent was removed using rotary

evaporator (Julabo-US) at reduced pressure of 60 mbar.

Table 8. Blank SLNs for Solvent Emulsification Diffusion method

3.2.3 Fabrication of Niclosamide loaded SLNs by Microemulsion Technique

By using the optimized conditions of BME-11, five different formulations (NME-1

to NME-5) of NIC loaded SLNs were prepared on basis of lipid drug ratio [Table 9]. NIC

was dissolved in melted stearic acid, the rest of protocol followed was same as used for

unloaded SLNs.

FORMULATION STEARIC ACID

(g)

TWEEN-80

(g)

PEG-400

(g)

STIRRING TIME

(min)

BSE-1 2 1 00 5

BSE -2 1 1 00 5

BSE -3 2 3 00 5

BSE -4 1 2 00 5

BSE -5 1 1.9 0.1 5

BSE -6 1 1.8 0.2 5

BSE -7 1 1.7 0.3 5

BSE -8 1 1.6 0.4 5

BSE -9 1 1.5 0.5 5

BSE -10 1 1.6 0.4 10

BSE -11 1 1.6 0.4 15

BSE -12 1 1.6 0.4 20


53

Table 9. Different NIC loaded SLNs formulations by Microemulsion Method

3.2.4 Fabrication of NIC-SLNs by Solvent Emulsification Diffusion Technique

Using optimized conditions of BSE-11, different formulations (NSE-1 to NSE-5) of

NIC loaded SLNs were prepared based on lipid drug ratio [Table 10]. Specified quantity

of NIC was dissolved in ethanol along with stearic acid, the rest of protocol followed was

same as used for unloaded SLNs [Figure 14].

Table 10. NIC loaded SLNs by Solvent Emulsification Diffusion Method

FORMULATION NICLOSAMIDE

(mg)

SA

(gm)

TWEEN-80

(g)

PEG-400

(g)

STIRRING

TIME

(min)

NME-1 200 1 1.6 0.4 15

NME-2 100 1 1.6 0.4 15

NME-3 66.6 1 1.6 0.4 15

NME-4 50.0 1 1.6 0.4 15

NME-5 40.0 1 1.6 0.4 15

FORMULATION NICLOSAMIDE

(mg)

SA

(gm)

TWEEN-80

(g)

PVP-29000

(g)

STIRRING

TIME (min)

NSE-1 200 1 1.6 0.4 15

NSE-2 100 1 1.6 0.4 15

NSE-2 66.6 1 1.6 0.4 15

NSE-4 50.0 1 1.6 0.4 15

NSE-5 40.0 1 1.6 0.4 15


54

Figure 14. Schematic diagram of Solvent-Emulsification-Diffusion

3.2.5 Fabrication of Sulfasalazine Loaded SLNs by Microemulsion Technique

Using optimized conditions of BME-11, five different formulations (SME-1 to

SME-5) of SZN-SLNs were prepared on basis of lipid drug ratio [Table 11]. Specified

amount of SZN was dissolved in melted stearic acid (75⁰C). The rest of protocol followed

was the same as adopted for unloaded SLNs.

3.2.6 Fabrication of SZN-SLNs by Solvent Emulsification Diffusion Technique

Using optimized conditions of BSE-11, different formulations (SSE-1 to SSE-5) of

SZN loaded SLNs were prepared based on lipid drug ratio. Specified quantity of SZN was

dissolved in ethanol along with stearic acid; the rest of protocol followed was same as

used for unloaded SLNs.


55

Table 11. SZN loaded SLNs formulations by Microemulsion Method

Table 12. SZN loaded SLNs by Solvent Emulsification Diffusion Method

FORMULATION SULFASALAZINE

(mg)

SA

(gm)

TWEEN-80

(g)

PEG-400

(g)

STIRRING

TIME

(min)

SME-1 200 1 1.6 0.4 15

SME-2 100 1 1.6 0.4 15

SME-3 66.6 1 1.6 0.4 15

SME-4 50.0 1 1.6 0.4 15

SME-5 40.0 1 1.6 0.4 15

FORMULATION SULFASALAZINE

(mg)

SA

(gm)

TWEEN-80

(g)

PVP-

29000 (g)

STIRRING

TIME

(min)

SSE-1 200 1 1.6 0.4 15

SSE-2 100 1 1.6 0.4 15

SSE-2 66.6 1 1.6 0.4 15

SSE-4 50.0 1 1.6 0.4 15

SSE-5 40.0 1 1.6 0.4 15


56

3.2.7 Lyophilization

The SLNs were lyophilized by means of freeze dryer (Heto PowerDry LL1500-

Thermo Electron Corporation-USA). Fructose solution (5%) was used as cryoprotectant.

[254] After overnight storage at -20⁰C, SLNs were transferred to freeze dryer (-75⁰ C) and

lyophilized for 48 hrs [255].

3.2.8 Calibration curve of Niclosamide

Specific amount of Niclosamide (NIC) was transferred into 100ml of volumetric

flask, add 25ml of methanol, and sonicated by using bath sonicator (Elma E30H) for 30

min make up the volume 100ml with methanol. Transferred 5ml from this solution into

50ml volumetric flask and dilute the volume with methanol. By changing the amount of

NIC, solutions of different concentration were prepared. Nano Drop Spectrophotometer

(Thermo-scientific 2000c/2000 UV-Vis Spectrophotometers) was used to measure the

absorbance at λmax of 332nm. Calibration curve was plotted between concentration and

absorbance [256].

3.2.9 Calibration curve of Sulfasalazine

Transfer SZN working standard accurately weighted to 100 ml volumetric flask

Dissolved in 0.1N NaOH solution and make the volume 100 ml. Took 5 ml from this

solution and transferred to 1000 ml volumetric flask containing 750 ml of water, mixed

well and added 20 ml of 0.1 N acetic acid. Made final volume 1000 ml with water. By

changing the amount of SZN different concentration solutions were prepared. Absorbance

was measured by Nano Drop Spectrophotometer (Thermo-scientific 2000c/2000 UV-Vis


57

Spectrophotometers) at λmax of 359nm. Calibration curve was obtained by plotting

concentration against absorbance.

3.2.10 Entrapment efficiency

The loaded nanoparticles were centrifuged at a high speed of 30,000 rpm for 15 min

at 25 °C and the supernatant was assayed for unloaded drug concentration by Nano Drop

Spectrophotometer [257]. Percent Entrapment efficiency was then calculated as follows:

3.2.11 Drug Loading Capacity

The percent drug loading capacity was calculated using the following equation

[258];

3.3 Characterization

3.3.1 Particle size and PDI

Particle size and Polydispersity Index (PDI) were measured through Photon

Correlation Spectroscopy (PCS) also known as Dynamic Light Scattering by using a

Zetasizer Nano (ZS-90, Malvern Instruments, Malvern, UK). The PDI is a measure of

nano dispersion homogeneity and ranges from 0 to 1[259]. All samples were diluted with

deionized water & measured at scattering angle of 90° & room temperature [260].


58

3.3.2 Zeta Potential

Nanoparticles were characterized with Zetapotential (ζ) by using Zeta-sizer (ZS-90

Malvern-UK) to study the ability of dispersion in account of stability. The measurements

were carried out using an aqueous dip cell in an automatic mode by placing diluted

samples (with deionized water) in the capillary measurement cell [260].

3.3.3 Scanning Electron Microscopy

The stability and behaviour of nanoparticles can be determined by the arrangement

of components and molecules orientation within nanoparticles [261]. For this purpose,

scanning electron microscopy (SEM) was performed by using Scanning Electron

Microscope (JSM910, JEOL Japan) at different acceleration voltages magnifications.

[262].

3.3.4 Differential Scanning Calorimeter

Thermal properties of the pure drugs, and lyophilized formulations were analysed

using Differential Scanning Calorimeter (DSC). All samples were studied using crimped

Al-pans and over temperature range of 40 to 300 ºC with heating rate of 10 ºC/min [94].

3.3.5 Powder X-ray Diffractometry

The crystalline behaviour of unprocessed drugs and SLNs (lyophilized) formulation

were studies using JDX-3532, JEOL Japan diffractometer by exposing them to Cu-Ka

radiation (40kV & 30mA) and scanned from 5º- 80º, 2Ø at a step-size 0.05º and step-time

of 1.0 sec with divergence slit 1° [263].


59

3.3.6 FT-IR Studies

FT-IR analysis of all samples (unprocessed drugs & drug-loaded SLNs) were

performed using IR Prestige-21 Shimadzu, Japan spectrometer. Pellet method was used to

prepare samples using Potassium Bromide (KBr) at 5 x 106 Pa pressure in order to

produce a clean and transparent disc of 0.2cm in thickness and 2cm in diameter. All

spectra were recorded at 25 °C from 2000 cm-1 to 400 cm-1[264].

3.3.7 In-vitro drug release

In-vitro release studies was performed using dialysis bag method [265, 266]. Dialysis bags

of molecular weight cut off 12 K-14 K (Spectrum Lab, Canada) were soaked in deionized

water for about 12 hours before use. SLNs dispersion (1 ml) from each formulation was

transferred to dialysis bag and fixed both ends of dialysis bag with thread. Placed each

sample into 250 ml phosphate-buffer solution (pH 7.4) at 50 rpm. Took samples for

analysis after specific interval of time (1-12 hour) while same volume of phosphate-buffer

solution was replaced. Amount of drug release were determined by using UV-

spectrophotometer against blank phosphate buffer solution [256]. Numerous kinetic

models were applied to find out in-vitro release rate and mechanism [244].

3.3.8 Drug release Mechanism

Data obtained from in-vitro release were studied through four kinetic models like;

First- order, Zero-order, Higuchi model & Korsmeyer-Peppas equations to obtain release

rate and also to find out drug release mechanism.


60

3.3.9 Stability

Freshly prepared nanoparticles were stored refrigerated temperature (5±3 ⁰C) and

room temperature (25⁰C ±2) and 60% ±5 RH for period of 3 months [267]. Samples were

kept for 90 days for stability analysis. At different time intervals of the days on 1, 15, 30,

45, 60 and 90, the average size and PDI were measured for both samples. Statistical

analysis of the data was performed using the two-tailed t-test. A probability of less than

0.05 (P<0.05) was considered significant in this study [267].

3.4 In-Vivo Study

3.4.1 Dose Administration

Prior approval was taken from Research Ethics Committee (vide letter

No.DREC/20160503-14). Rabbits (2±0.2 kg) were kept fasted for 12 hours before oral

administration but allowed for water. Two groups of rabbits were made each having 6

animals drug loaded SLNs were administered to Group-I while marketed drug to Group-II

(100 mg.kg-1). Blood (0.5 ml) were taken from marginal ear vein at various time intervals

(0−24 hour). Blood was heparinized followed by centrifugation to separate plasma, which

was stored at -20°C.

3.4.2 Quantification of Niclosamide by HPLC

Plasma was quantified for NIC by using HPLC technique [265]. Acetonitrile:

Potassium Dihydrogen Phosphate (40:60 v/v) were used as mobile phase. Reversed phase

column (Supelco C18, 25 cm in length, 4.6 mm width and 5 µm particle size) protected

with a precolumn (Supelco Cl8) was used. The column was kept at 37oC, flow rate l

ml/min and retention−time 4.5 minutes. Plasma sample was mixed with two volumes of


61

acetonitrile and placed at -20oC for 10 minutes followed by centrifugation to precipitate

protein. The supernatant (20 µl) was injected for the detection of NIC concentration using

UV detector (λmax 290 nm) [268]. The concentration was quantified using calibration

curve.

3.4.3 Quantification of Sulfasalazine by HPLC

Plasma was quantified for SZN by using HPLC technique [265]. Methanol was used

as mobile phase. Reversed phase column (Supelco C18, 25cm in length, 4.6 mm width and

5µm particle size) protected with a precolumn (Supelco Cl8) was used. The column was

kept at 37oC, flow rate l ml/min and retention−time 3 minutes. Plasma samples were mixed

with two volumes of methanol and placed at -20oC for 10 minutes followed by

centrifugation to precipitate protein. The supernatant (20µl) was injected or the detection

of SZN concentration using UV detector (λmax 358nm).The concentration was quantified

from area of chromatographic peak by using calibration curve.

3.4.4 Analysis of data

Based on non-compartmental model, pharmacokinetic limits were measured. Area

under curve from 0 to t (AUC0→t) was calculated from concentration−time curve by

trapezoidal rule [269]. Peak plasma concentration (Cmax) and peak plasma concentration

time (Tmax) were obtained directly from plasma concentration−time curve. Area under total

plasma concentration−time curve from time 0 to 24 hours was measured using equation:


62

Ct is drug concentration observed after 24 hours while Ke is the elimination rate

constant. Relative bioavailability (Fr) at 24th hour for same dose was measured using

equation:

Data from pharmacokinetic parameters were analyzed statistically using one−way

analysis of variance and t−test (p<0.05).

3.5 Granulation

3.5.1 Static Bed Drying

Static bed drying was performed. Trays were moved slowly under forced air oven. This

drying is suitable to dry large batches of SLNs.

3.5.2 Comparatives study of Lyophilized and Static dried SLNs

Scanning Electron Microscopy analysis of lyophilized and static bed dried SLNs were

compared to observe the change by changing the drying method.

3.5.3 Wet-Granulation

During wet-granulation, binder solution was added to the dried SLNs followed by passing

through desired mesh size and again dried. The dried granules were again passed through

a smaller mesh for reducing the granules size even further. Excipients used for granulation

purpose are enlisted below;


63

Table 13. Excipients used during wet granulation method

S. NO MATERIAL

1 Lactose monohydrate

2 Starch

3 Polyvinylpyrrolidone (K-30)

4 Isopropyl alcohal (IPA)

5 Primogel

6 Magnesium stearate

7 Aerocil (200)

In step-1, lactose monohydrate, starch and aerocil (200) as well as dried powder of

SLNs formulation were passed via mesh (size 16) to obtain particles of uniform size. After

adding dried powder of SLNs formulation with lactose monohydrate, starch and aerocil

(200), performed geometrical mixing for 15 minutes. Gloves and mask were used through-

out the process to ensure evade of any chance of contamination.

In step-2, polyvinylpyrrolidone (K-30) was dissolved in isopropyl alcohal (IPA)

and thoroughly mixed to form a clear solution followed by mixing with the resultant

product of step-1. After mixing, it was placed in tray drier for drying purpose followed by

passing via mesh (size 12) to obtain granules of uniform size. Finally, magnesium stearate

and aerocil (200) were mixed with the granules for 10 minutes to ensure its high flow

property. The resultant granules were further coated to get single-layered and double-

layered granules.

3.5.4 Coating of Granules

The resultant granules were further coated to get single-layered granules. Materials

employed for single layer coated are enlisted below;


64

Table 14. Excipients used during Coating

In step-I, methocil was dissolved in isopropyl alcohol (IPA), then methylene

chloride was add to it followed by mixing properly. In step-II, titanium dioxide (TiO2) was

dissolved in isopropyl alcohol (IPA), then added to the resultant mixture of step-I

followed by thoroughly mixing to prepare the desired white solution (spraying solution).

The white solution (spraying solution) was spray via spray gun on the already

prepared granules followed by drying to obtain the coated granules.

3.5.5 Capsule Shells Filling

Granules were fill manually in hard gelatine capsule shells (size 5; Capsugel, North

Peapack, NJ, USA) following standard operating procedure (SOP).

3.6 Similarity Study

In-vitro drug release profile of marketed tablets was obtained under same conditions

as applied for our prepared formulations and similarity factor was determined from release

data. This data was analysed by using the following equation;

S.NO MATERIAL

1 Methocil (E5)

2 Titanium dioxide (TiO2)

3 Methylene chloride

4 Isopropyl alcohal (IPA)


65

N is the integer of time, dissolution of reference sample = Ri in time i and

dissolution of test product = Bi in time i. if the value of f2 is greater than 50 then the two

products are consider to have similar release characteristics.

3.7 Statistical Analysis

Results of the current study were calculated as mean ± standard error of the mean.

Each parameter of experimental groups were compared by using t-test with the help of

SPSS (version 16). The difference between means of two groups was considered

significant, if the value of "P" was less than 0.05.

CHAPTER 4 RESULTS AND DISCUSSION

66

Chapter 4 RESULTS AND DISCUSSION

4.1 Micro Emulsion Technique

4.1.1 Particle size and PDI of Blank SLNs by Micro emulsion Technique

Blank SLNs were optimized via different variables including concentrations of

surfactant, concentrations of co-surfactant and stirring time. Significant changes were

observed by changing these variables as given in Table 15. Maximum particle size

was observed for BME-1 i.e. 855.2±2.5 and lowest for BME-12 i.e. 203.2±2.5.

Similarly, the highest PDI was shown by the BME-3 (0.650±0.002) and lowest by

BME-11 (0.388±0.008) formulation. Optimized blank formulation (BME-11) with

average particle size 205.5±2.9 nm and PDI 0.388±0.008 was designated for drug

loading.

Different SLNs formulations fabricated on basis of lipid and surfactant ratio

showed; as the concentration of surfactant increases, particle size reduces while there

is no significant change in PDI. Various studies reported that increase in lipid

concentration results in larger particle size and broader particle size distribution [270].

The formulation BME-4 with Particle size 239.3±1.4nm and PDI 0.537±0.004 was

selected for further optimization on basis of surfactant and co-surfactant ratio.

Blank SLNs formulations prepared on basis of surfactant and co-surfactant ratio

showed that increase in concentration of co-surfactant reduce the particle size up to

BME-8 while further increase in co-surfactant concentration led to increase in particle

size. Therefore, the BME-8 with particle size 214.1±1.4nm was selected for further

optimization on basis of stirring time.


67

Literature shows that stirring time effects PDI while having almost no effect on

particle size [271]. During further optimization process, PDI decreased with increase

in stirring time and after 15 min of stirring, the optimum size (205.5±2.9) and PDI

(0.388±0.008) were observed for BME-11 formulation which was selected for drug

loading.

Table 15. Size and PDI of blank SLNs formulations Mean±SD (n=3)

FORMULATION SIZE (nm) PDI

BME-1 855.2 ± 2.5 0.565 ± 0.003

BME-2 544.5 ± 1.3 0.551 ± 0.008

BME-3 273.3 ± 2.9 0.650 ± 0.002

BME-4 238.9 ± 2.6 0.537 ± 0.004

BME-5 236.8 ± 5.1 0.514 ± 0.003

BME-6 233.9 ± 2.5 0.504 ± 0.003

BME-7 217.8 ± 1.5 0.506 ± 0.002

BME-8 214.1 ± 1.4 0.608 ± 0.002

BME-9 215.6 ± 2.6 0.566 ± 0.003

BME-10 212.8 ± 1.8 0.551 ± 0.001

BME-11 205.5 ± 2.9 0.388 ± 0.008

BME-12 203.2 ± 2.5 0.50 ± 0.0010

4.2 Fabrication of Niclosamide by Micro Emulsion Technique

4.2.1 Particle size, PDI and Zeta Potential of Niclosamide SLNs

Five different formulations of Niclosamide loaded SLNs (NME−1 to NME−5)

based on drug lipid ratio were fabricated. The optimized NME-3 nanoformulation

gave average particle size 204.2±3.2 nm, PDI 0.328±0.02 and Zeta potential -33.16±2

mV [Figure 15 & 16]. Results showed that particle size reduced after drug loading,

which is due to decreased free lipid content [272]. In addition, PDI was controlled and

reduced by increasing stirring time [273]. The PDI <0.5 and zeta potential in the


68

range of ±30 demonstrated that the produced nanoformulation would be stable in

nature [274]. In the prepared NIC-SLNs, these values were within the range, which

exhibit electrostatic stabilization. Consequently there would be no aggregation, which

can potentially led to prevent Ostwald ripening and particles growth [171].

Figure 15. Average Particle size of NME−3 formulation

Figure 16. Zeta Potential of NME−3 formulation


69

4.2.2 Entrapment Efficiency of Niclosamide Loaded SLNs

To check the entrapment efficiency of SLNs loaded with NIC, it was observed

that maximum percent entrapment efficiency was for NME-3 i.e. 89.1±0.03

nanoformulation whereas for NME-1 nanoformulation percent entrapment efficiency

was 38.2 ±0.04 [Table 16]. As the concentration of lipid decreased the entrapment

efficiency reduced. Drug lipid ratio (15:1) showed maximum entrapment efficiency

whereas further increase in lipid ratio led to reduce entrapment efficiency. Moreover,

concentration of the chosen excipients including stearic acid (1.0gm), Tween80

(1.6gm) and PEG (0.4gm) were found the effective combination to demonstrate

maximum encapsulation of the drug with higher loading efficiency [275].

4.2.3 Drug Loading Capacity of Niclosamide Loaded SLNs

Drug Loading Capacity (DLC) of NIC loaded SLNs are given in Table 16.

Among the drug-loaded formulations, highest loading capacity (3.01±0.04) was

observed for NME-3 whereas the formulation NME-5 showed the lowest loading

capacity (2.06±0.03). As the concentration of lipid decreased the loading-capacity

reduced. Drug lipid ratio (15:1) showed maximum loading capacity whereas further

increase in lipid ratio led to reduce loading capacity.

There has been previously reported that in lipid based nanoparticles, the binding

energy of the APIs with the lipids play a key role to effectively encapsulate the drug

in the lipid layers [276]. In this case, it might be attributed to the high binding energy

of the NIC with stearic acid that results in maximum drug loading.


70

Table 16. EE% and DLC% of Niclosamide Loaded SLNs Formulation

FORMULATION EE% DLC%

NME-1 38.2 ± 0.04 2.43 ± 0.02

NME-2 55.4 ± 0.02 2.58 ± 0.03

NME-3 89.1 ± 0.03 3.01 ± 0.04

NME-4 66.3 ± 0.02 2.36 ± 0.04

NME-5 51.1 ± 0.01 2.06 ± 0.03

4.2.4 Scanning Electron Microscopy of optimized Niclosamide Loaded SLNs

SEM micrograph of NIC loaded SLNs evidently denoted that the prepared solid

lipid nanoparticles of Niclosamide were spherical in shape and had smooth surface.

Micrograph showed that the size of the nanoparticles were below 210 nm with

homogeneous distribution of the produced nanoparticles [Figure 17].

Figure 17. SEM image of Niclosamide Loaded SLNs (NME-3)


71

4.2.5 DSC thermogram of Niclosamide SLNs optimized

DSC thermograms of Niclosamide (free drug), and NME-3 nanoformulation

were recorded separately. Endothermic peak was observe for pure NIC at 229°C

while for NME-3 nanoformulation at 188.5oC [Figure 18]. Tiny peak was observed at

188.5 °C for NME-3 nanoformulation, which is because of reduced particle size,

enlarged surface area and closed contact between stearic acid and drug. The melting

point of the drug shifted to lower scale with disappearance of components peaks is

the indication of the complete dispersion of the drug in lipid layers. The shifting of the

melting point peak of active pharmaceutical ingredients (API) in SLNs using stearic

acid has previously been reported [277].

Figure 18. Thermograms of Pure Niclosamide and NME-3

4.2.6 PXRD of optimized Niclosamide loaded SLNs

Powered X-ray Diffraction (P-XRD) pattern showed larger peak counts at

highest for NIC (pure drug) compared NME-3 nanoformulation [Figure 19]. This

study helped to explore the drug nature after encapsulation. It was observed that drug


72

entrapped in the SLNs was in the disordered-crystalline or amorphous state. The

reduction in intensities and also disappearance of minute peaks of NME-3

nanoformulation are indicative for reduction in crystalline nature [274, 278]. This

phase further facilitates sustained drug release from SLNs [279].

Figure 19. P-XRD Spectra of Pure Niclosamide and NME-3

4.2.7 FT-IR Study of optimized Niclosamide loaded SLNs

Major peaks of Pure Niclosamide (NIC) appeared at wave numbers 1572, 1515,

1613, 1285, 1650, and 1218 cm-1 which were also found in NME-3 nanoformulation

[280]. On the basis of the calculated and experimental results; assignment of the

fundamental frequencies were examined. The difference between the observed and

scaled wavenumber values of most of the fundamentals is very small. Overall, there is

no significant change in FT-IR spectra of NIC free drug and NME-3 nanoformulation

which revealed no interaction between drug and other formulation components

[Figure 20].


73

Figure 20. FT-IR Spectra of Pure Niclosamide (A) and NME-3 (B)

4.2.8 In-vitro drug release of optimized Niclosamide loaded SLNs

During in-vitro study, cumulative percent drug release from NME-1 to NME-5

formulations were 100%, 100%, 97.31%, 57.35% and 55.15% respectively [Table.

17] & [Figure 21]. When the quantity of drug payload reduced from 200mg to 40mg,

the cumulative percent drug release decreased from 100% to 55.15% only during the

12 hrs in-vitro study. Hence, it is concluded that increased drug payload resulted in

improved sustained release profile.

Kinetic models study showed that NIC-SLNs followed zero order kinetics.

Release exponent (n) was greater than 0.89, regarded as Super case II diffusion

mechanism [281, 282].

4.2.9 Drug release Mechanism of optimized Niclosamide loaded SLNs

By putting the drug release data in different kinetic models, it was observed that

NIC-SLNs followed zero order kinetics. Release exponent (n) was greater than 0.89,

regarded as Super case II diffusion mechanism [281]. R2 value and n values are given

in Table 18.


74

Figure 21. Percent Drug Release from NIC-SLNs Formulations

Table 17. Cumulative Percent Drug Release from NIC-SLNs Formulations

TIME (HR) CUMULATIVE PERCENT RELEASE

NME-1 NME-2 NME-3 NME-4 NME-5

0 0 0 0 0 0

1 17.46 17.41 21.46 9.11 7.33

2 32.56 34.78 30.26 11.78 10.78

3 43.55 43.51 39.14 23.51 22.41

4 55.19 55.62 48.19 31.33 27.53

5 67.48 68.88 56.48 34.88 33.18

6 76.21 78.21 64.21 39.21 37.21

7 85.63 93.63 72.63 43.63 41.73

8 91.22 100 79.22 47.42 43.21

9 96.56 100 82.23 49.65 45.54

10 100 100 85.16 52.55 48.51

12 100 100 97.31 57.35 55.15


75

Table 18. Kinetic Models for Niclosamide-SLNs

4.2.10 Stability Study of optimized Niclosamide loaded SLNs

No significant change was observe in size and PDI of NME-3 nanoformulation

stored at refrigerated temperature. However, for the initial four weeks some growth

was observed at room temperature followed by stabilization for rest of the period.

This is also because of the amorphous nature of the NME-3 nanoformulation and

might be degradation of both drug and lipid having low glass-transition temperature.

It is also common that particles in amorphous solid deposit on the surface of the

larger ones [274, 278]. Statistically data was analysed by two-tailed t-test, which

showed p-value 0.03 for particle size and 0.05 for PDI [Table 19].

FORMULATIONS ZERO

ORDER

(R2)

FIRST

ORDER

(R2)

HIGUCHI

MODEL

(R2)

KORSMEYER-

PEPPAS MODEL

RELEASE

EXPONENT

(R2) (n)

NME-1 0.9574 0.9808 0.9754 0.80632337 0.9415

NME-2 0.9741 0.9656 0.9541 0.91632848 0.963

NME-3 0.9812 0.9772 0.9573 0.89311668 0.962

NME-4 0.9943 0.9877 0.9455 0.93456989 0.943

NME-5 0.9962 0.9882 0.9417 0.976809871 0.935


76

Table 19. Stability study of NIC-SLNs (NME-3)

4.2.11 In-Vivo Study of optimized Niclosamide loaded SLNs

After oral dose, plasma concentration−time curves of NME-3 nanoformulation

and marketed drug i.e. Mesan® in rabbits are shown in Figure 22. Pharmacokinetic

parameters are measured [Table 20]. It was observed that at all time points, the

Niclosamide plasma concentrations were significantly higher in rabbits treated with

SME-3 nanoformulation than for those treated with Mesan®.

Peak plasma concentration (Cmax) for Mesan® and NME-3 formulation was

1.84±0.24 μgml-1 and 3.97±0.24 μgml-1 respectively. AUC0→24 for Mesan® was 1.51

μg.hr.ml-1 whereas, for NME-3 was 16.74 μg.hr.ml-1. NME-3 nanoformulation

showed 2.15−fold increase in Cmax and 11.06−fold increase in AUC0→24 compared to

Mesan® [Table 20].

From these results, it could be conclude that NIC absorption was significantly

improved by using SLNs formulation compared to conventional dosage form

(Mesan®).

WEEK SIZE (nm) (5±3⁰C) SIZE (nm) (25±2⁰C) PDI (5±3⁰C) PDI (25±2⁰C)

Zero Week 204.2 204.2 0.328 0.328

1st Week 204.5 215.4 0.331 0.341

2nd Week 205.9 218.3 0.333 0.400

6th Week 207.1 231.2 0.341 0.503

8th Week 212.1 235.5 0.341 0.506

12th Week 213.5 245.5 0.343 0.557

Mean 207.8 256 0.33 0.45

±SD 3.97 225 0.006 0.117

p-Value 0.03 0.05


77

The results obtained from pharmacokinetic study revealed that NIC absorption

was significantly enhanced by taking up SLNs as drug delivery system. The small

particle size of SLNs may have bio−adhesion with gastrointestinal wall or go through

the inter−villar spaces results in rising duration of residence in GIT [283, 284]. This

long term sticking with GIT will result in boosted bioavailability. Tween−80 may

have paid to increase in affinity between stearic acid and intestine which leads to

enhance permeability [285, 286].

Figure 22. Comparative in−vivo drug release from NME−3 & marketed drug

Table 20. Pharmacokinetic parameters of NIC-SLNs (NME-3) & Mesan®

(n=6, x¯±SD)

PARAMETERS NIC-SLNS (NME-3) MARKETED DRUG

Cmax (μg mL−1) 3.97±0.24 1.84 ±0.24

Tmax (h) 12±0.1 6±.03

AUC (μg h mL−1) 16.74 1.51

Fr 11.08


78

4.3 Fabrication of Sulfasalazine by Micro Emulsion Technique

4.3.1 Particle size, PDI and Zeta Potential of Loaded Sulfasalazine SLNs

Five different formulations of Sulfasalazine loaded SLNs (SME−1 to SME−5)

based on drug lipid ratio were fabricated. The optimized SME-3 nanoformulation

showed average particle size 217.2±3.2nm, PDI 0.373±0.02 and zeta potential -

35.26±2mV [Figure 23 & 24]. Results showed that particle size reduced after drug

loading, which is due to decreased free lipid content [272]. In addition, PDI was

controlled and reduced by increasing stirring time [273]. The PDI <0.5 and zeta

potential in the range of ±30 demonstrated that the produced nanoformulation would

be stable in nature [274]. In the prepared SZN-SLNs, these values were within the

range, which exhibit electrostatic stabilization. Consequently there would be no

aggregation, which can potentially led to prevent Ostwald ripening and particles

growth [171].

Figure 23. Particle size of SME-3 Formulation


79

Figure 24. Zeta Potential of SME-3 Formulation

4.3.2 Entrapment Efficiency of Sulfasalazine Loaded SLNs

In order to check the entrapment efficiency of SLNs loaded with SZN, it was

observed that maximum percent entrapment efficiency was for SME-3

nanoformulation whereas for SME-1 nanoformulation percent entrapment efficiency

was 28.2 ±0.05 [Table 21]. As the concentration of lipid decreased the entrapment




(1.6gm) and PEG (0.4gm) were found the effective combination to demonstrate


4.3.3 Drug Loading Capacity of Sulfasalazine Loaded SLNs

Drug Loading Capacity (DLC) of SZN loaded SLNs are given in Table 21.


observed for SME-3 whereas the formulation SME-5 showed the lowest loading



80







Table 21. EE & DLC of SZN loaded SLNs formulations


SME-1 28.3 ± 0.05 2.57 ± 0.02

SME-2 56.4 ± 0.07 2.68 ± 0.04

SME-3 89.1 ± 0.03 2.87 ± 0.05

SME-4 64.3 ± 0.03 1.56 ± 0.04

SME-5 54.1 ± 0.07 1.06 ± 0.06

4.3.4 Scanning Electron Microscopy of optimized Sulfasalazine Loaded SLNs

SEM micrograph of SZN loaded SLNs evidently denoted that the prepared solid lipid

nanoparticles of Niclosamide were spherical in shape and had smooth surface.




81

Figure 25. SEM image of Sulfasalazine Loaded SLNs (SME-3)

4.3.5 DSC thermogram of Sulfasalazine SLNs

DSC thermograms of SZN (free drug), and SME-3 nanoformulation were

recorded separately. Endothermic peak was observe for pure SZN at 245°C while for

SME-3 nanoformulation at 238.5oC [Figure 26]. Tiny peak was observed at 238.5 °C

for SME-3 nanoformulation, which is because of reduced particle size, enlarged

surface area and closed contact between stearic acid and drug. The melting point of

the drug shifted to lower scale with disappearance of components peaks is the

indication of the complete dispersion of the drug in lipid layers. The shifting of the




82

Figure 26. DSC thermogram of Pure Sulfasalazine and SME-3 formulation

4.3.6 PXRD of optimized Sulfasalazine loaded SLNs


highest for SZN (pure drug) compared SME-3 nanoformulation [Figure 27]. This

study facilitated to discover nature of drug after encapsulation. It was observed that

drug entrapped in the SLNs was in the disordered-crystalline or amorphous state. The

reduction in intensities and also disappearance of minute peaks of SME-3

nanoformulation are revealing for decrease in crystalline nature [274, 278]. This

phase further assists sustained drug release from SLNs [279].

4.3.7 FT-IR Study of optimized Sulfasalazine loaded SLNs

FT−IR spectra of the pure SZN and SME-3 nanoformulation were obtained

between 400–2000 cm-1 [Figure 28]. C−O in phenol at 1281 cm-1 of the SZN & SME-

3 nanoformulation remained the same. Similarly, OH−group of free SZN at 1394 cm-

1remained same in SME-3 nanoformulation [287]. C=C vibration of the benzene ring

at 1700cm-1 remained same in free SZN and its SLNs. The spectrum of SZN (free

drug) and SME-3 formulation showed sharp peaks at 1427 & 1618cm-1 allocated to

symmetric and asymmetric stretching of the carboxylate group. Based on the


83

calculated and experimental results; assignment of the fundamental frequencies were

examined. The difference between the observed and scaled wavenumber values of

most of the fundamentals is very small. Overall, there is no significant change in FT-

IR spectra of SZN free drug and SME-3 nanoformulation, which revealed no

interaction between drug and other formulation components.

Figure 27. P-XRD Spectra of Pure Sulfasalazine and SZZ−SLNs (SME-3)


FT−IR spectra of the pure SZN and SME-3 nanoformulation were obtained

between 400–2000 cm-1 [Figure 28]. C−O in phenol at 1281 cm-1 of the SZN & SME-

3 nanoformulation remained the same. Similarly, OH−group of free SZN at 1394 cm-

1remained same in SME-3 nanoformulation [287]. C=C vibration of the benzene ring

at 1700cm-1 remained same in free SZN and its SLNs. The spectrum of SZN (free

drug) and SME-3 formulation showed sharp peaks at 1427 & 1618cm-1 allocated to

symmetric and asymmetric stretching of the carboxylate group. Based on the

calculated and experimental results; assignment of the fundamental frequencies were


84

examined. The difference between the observed and scaled wavenumber values of

most of the fundamentals is very small. Overall, there is no significant change in FT-

IR spectra of SZN free drug and SME-3 nanoformulation, which revealed no


Figure 28. FT-IR Spectra of Pure Sulfasalazine (A) & Processed SZN-SLNs (B)

4.3.9 In-vitro drug release of optimized Sulfasalazine loaded SLNs

It was observed that initially there was burst effect for SME-1, SME-2 and

SME-3 which were 19.46 %, 17.41% about 21.46% respectively in first hour followed

by prolonged release of SZN [Figure 29]. There may be different reasons for burst

release (1) may be the drug was located in shell of SLNs, (2) large surface area, (3)

high diffusion co-efficient, (4) short diffusion distance and (5) low viscosity of SLNs.

When the quantity of drug payload reduced from 200mg to 40mg, the cumulative

percent drug release decreased during the 12 hrs in-vitro study. Hence, it is concluded

that increased drug payload resulted in improved sustained release profile.


85

Figure 29. Percent Drug Release from SZN loaded SLNs formulations

4.3.10 Drug release Mechanism of optimized Sulfasalazine loaded SLNs

By putting the drug release data in different kinetic models like; First- order,

Zero-order, Higuchi model & Kors-Peppa equations, it was observed that SZN-SLNs

followed mixed order kinetics i.e. initially the release pattern was first order kinetic

then followed by zero order kinetics. Release exponent (n) was greater than 0.89,

regarded as Super case-II diffusion mechanism [281]. R2 values and n values are

given in Table 22.

The values of release exponent (n) of SZN loaded SLNs (SME-3) lies in

n>0.89 have been observed, which are regarded as Super case II diffusion mechanism

i.e. diffusion of drug or release pattern of drug obeys relaxation mechanism.


86

Table 22. Different Kinetic Models for Sulfasalazine

4.3.11 Stability Study of optimized Sulfasalazine loaded SLNs

No significant change was observe in size and PDI of SME-3 nanoformulation


was observed at room temperature followed by stabilization for rest of the period

[Figure 30 & 31]. This is also because of the amorphous nature of the SME-3

nanoformulation and might be degradation of both drug and lipid having low glass-

transition temperature. It is also common that particles in amorphous solid deposit on

the surface of the larger ones [274, 278].

FORMULATIONS ZERO

ORDER

(R2)

FIRST

ORDER

(R2)

HIGUCHI

MODEL

(R2)

KORSMEYER-

PEPPAS MODEL

RELEASE

EXPONENT

(R2) (n)

SME−1 0.958 0.912 0.969 0.80696337 0.978

SME−2 0.974 0.956 0.954 0.90638848 0.963

SME−3 0.981 0.972 0.957 0.91271668 0.962

SME−4 0.994 0.977 0.945 0.97277989 0.943

SME−5 0.996 0.982 0.941 0.97109871 0.935


87

Figure 30. Particle size analysis of optimized formulation (SME-3)

Figure 31. PDI analysis of optimized formulation (SME-3)

4.3.12 In-Vivo Study of optimized Sulfasalazine loaded SLNs

After oral dose, plasma concentration−time curves of SME-3 nanoformulation

and marketed drug i.e. marketed drug in rabbits are shown in Figure 32.

Pharmacokinetic parameters are measured and given [Table 24]. It was observed that


88

at all time points, the SZN plasma concentrations were significantly higher in rabbits

treated with SME-3 formulation than for those treated with Salazodine®.

Peak plasma concentration (Cmax) for marketed drug and SME-3

nanoformulationwas1.94±0.3 μgml-1 and 4.72±0.3μgml-1respectively. AUC0→24 for

marketed drug was 1.69 μg.hr.ml-1 whereas for SME-3 was 18.99 μg.hr.ml-1. SME−3

formulation showed 2.43−fold increase in Cmax and 11.23−fold increase in

AUC0→24 compared to marketed drug. Pharmacokinetic study revealed that SZN

absorption was significantly enhanced by taking up SLNs as drug delivery system.

The small particle size of SLNs may have bio−adhesion with gastrointestinal

wall or go through the inter−villar spaces results in rising duration of residence in GIT

[283, 284]. This long term sticking with GIT will result in boosted bioavailability.

Tween−80 may have paid to increase in affinity between stearic acid and intestine

which leads to enhance permeability [285, 286]. From these results, it can be

concluded that SZN absorption was significantly improved by using SLNs

formulation compared to conventional dosage form (marketed drug).

Figure 32. Comparative in−vivo release from SZN-SLNs (SME−3) & Marketed Drug


89

Table 23. Pharmacokinetic parameters of SME−3 & Marketed drug

Micro emulsion technique for the fabrication of poor water-soluble drugs,

which helps to overcome several disadvantages, associated with organic solvent. The

particle sizes of SLNs fabricated by micro emulsion method of both BCS-II and IV

drugs were in the desired nano-metric range, PDI, zeta-potential, EE and DLC.

Stability testing showed that the SLNs were more stable at refrigerated temperature.

Both diffraction & thermal analysis confirmed reduction in crystalline nature of

fabricated nanoformulation. In-vitro release profiles showed sustained release of

Niclosamide and Sulfasalazine. The in-vivo study revealed oral bioavailability

enhancement for both NIC and SZN loaded SLNs in comparison to their respective

marketed products. Statistically the data of all parameters of in-vivo pharmacokinetic

was analysed which confirmed that SLNs formulations enhanced the bioavailability of

NIC and SZN significantly in comparison with marketed drugs [151].

PARAMETERS SZN-SLNS (SME-3) MARKETED DRUG

Cmax (μg mL−1) 4.72 ± 0.3 1.94 ± 0.3

Tmax (h) 12 ± 0.1 06 ±.03

AUC (μg h mL−1) 18.99 1.69

Fr 11.23


90

4.4 Solvent Emulsification Diffusion Technique

4.4.1 Particle size and PDI of Blank SLNs

Blank SLNs were optimized via different variables including concentrations of

surfactant, concentrations of co-surfactant and stirring time. Significant changes were

observed by changing these variables as given in Table 24. Maximum particle size

was observed for BSE-2 i.e. 545.6±5.3 nm and lowest for BSE-12 i.e. 211.8±2.9 nm.

Similarly, the highest PDI was shown by the BSE-1, BSE-2, BSE-6 and BSE-7

(1.00±0.00) and lowest by BSE-11(0.395±0.003) formulation. BSE-11) gave average

particle size 212.2±2.2nm and PDI 0.395±0.003

Different SLNs formulations fabricated on basis of lipid and surfactant ratio showed;

as the concentration of surfactant increases, particle size reduces while there is no

significant change in PDI. Various studies reported that increase in lipid concentration

results in larger particle size and broader particle size distribution [270]. The

formulation BME-4 with Particle size 239.3±1.4nm and PDI 0.537±0.004 was

selected for further optimization on basis of surfactant and co-surfactant ratio.

Blank SLNs formulations prepared on basis of surfactant and co-surfactant ratio

showed that increase in concentration of co-surfactant reduce the particle size up to

BSE-8 while further increase in co-surfactant concentration led to increase in particle

size. Therefore, the BSE-8 with particle size 212.4±3.4nm was selected for further

optimization on basis of stirring time.

Literature shows that stirring time effects PDI while having almost no effect on

particle size [271]. During further optimization process, PDI decreased with increase

in stirring time and after 15 min of stirring, the optimum size (212.2±2.2nm) and PDI


91

(0.395±0.003) were observed for BSE-11 nanoformulation, which was selected for

drug loading.

Table 24. Particle size and PDI of unloaded SLNs

FORMULATION SIZE (nm) PDI

BSE-1 513.9 ± 8.2nm 1.00 ± 0.00

BSE-2 545.6 ± 5.3nm 1.00 ± 0.00

BSE-3 371.7 ± 2.9nm 0.553 ± 0.009

BSE-4 300.3 ± 0.8nm 0.685 ± 0.006

BSE-5 292.7 ± 7.4nm 0.705 ± 0.005

BSE-6 248.0 ± 2.9nm 1.00 ± 0.000

BSE-7 224.1 ± 2.4nm 1.00 ± 0.000

BSE-8 212.4 ± 3.4nm 0.565 ± 0.020

BSE-9 214.4 ± 2.1nm 0.739 ± 0.010

BSE-10 212.6 ± 1.8nm 0.552 ± 0.001

BSE-11 212.2 ± 2.2nm 0.395 ± 0.003

BSE-12 211.8 ± 2.9nm 0.447 ± 0.006

4.5 Fabrication of NIC-SLNS by Solvent Emulsification Diffusion

Technique

4.5.1 Particle size, PDI and Zeta Potential of Loaded Niclosamide SLNs

Five different formulations of Niclosamide loaded SLNs (NSE−1 to NSE−5)

based on drug lipid ratio were fabricated. The optimized NSE-2 nanoformulation

showed particle size 208.6±2.2 nm, PDI 0.376±0.04 and zeta potential -34.11±1.2 mV

[Figure 33 & 34]. The most important factor for evaluation of stability of colloidal

dispersion is Zeta potential (surface charge determination). Recent studies showed

that zeta potential above ±30mV were necessary for electrostatic stability [171]. In the

prepared NIC-SLNs, these values were within the range, which exhibit electrostatic


92

stabilization. Consequently there would be no aggregation, which can potentially led

to prevent Ostwald ripening and particles growth [171].

Figure 33. Average Particle size of NSE−2 formulation

Figure 34. Zeta Potential of NSE−2 formulation

4.5.2 Entrapment Efficiency of Niclosamide Loaded SLNs

To observe the entrapment efficiency of SLNs loaded with NIC, it was found

that maximum percent entrapment efficiency was for NME-2 nanoformulation

(84.4±0.02) whereas for NSE-1 nanoformulation percent entrapment efficiency was


93

36.2±0.04 [Table 25]. As the concentration of lipid decreased the entrapment




(1.6gm) and PVP (0.4gm) were found the effective combination to demonstrate


Table 25. EE% and DLC% of Niclosamide Loaded SLNs Formulation


NSE-1 36.2 ± 0.04 4.25 ± 0.02

NSE-2 84.4 ± 0.02 5.27 ± 0.03

NSE-3 81.1 ± 0.03 3.36 ± 0.04

NSE-4 74.4 ± 0.02 2.40 ± 0.04

NSE-5 62.7 ± 0.01 1.62 ± 0.03

4.5.3 Drug Loading Capacity of Niclosamide Loaded SLNs

Drug Loading Capacity (DLC) of NIC loaded SLNs are given in Table 25.


observed for NSE-2 whereas the formulation NSE-5 showed the lowest loading









94

4.5.4 Scanning Electron Microscopy of optimized Niclosamide Loaded SLNs

SEM micrograph of NIC loaded SLNs evidently denoted that the prepared solid




4.5.5 DSC thermogram of Niclosamide SLNs optimized

DSC thermograms of Niclosamide (free drug), and NSE-2 nanoformulation

were recorded separately. Endothermic peak was observe for pure NIC at 229°C

while for NSE-2 nanoformulation at 180oC [Figure 36]. Tiny peak was observed at

180 °C for NSE-2 nanoformulation, which is because of reduced particle size,

enlarged surface area and closed contact between stearic acid and drug. The melting

point of the drug shifted to lower scale with disappearance of components peaks is

the indication of the complete dispersion of the drug in lipid layers. The shifting of the



Figure 35. SEM micrograph of Niclosamide Loaded SLNs (NSE-2)


95

Figure 36. DSC Thermograms of Pure Niclosamide and NIC-SLNs (NSE-2)

4.5.6 PXRD of optimized Niclosamide loaded SLNs

Powered X-ray Diffraction (P-XRD) pattern showed larger peak counts at highest for

NIC (pure drug) compared NSE-2 nanoformulation [Figure 37]. This study helped to

explore the drug nature after encapsulation. It was observed that drug entrapped in the

SLNs was in the disordered-crystalline or amorphous state. The reduction in

intensities and also disappearance of minute peaks of NSE-2 nanoformulation are

indicative for reduction in crystalline nature [274, 278]. This phase further facilitates

sustained drug release from SLNs [279].

4.5.7 FT-IR Study of optimized Niclosamide loaded SLNs

Major peaks of Pure Niclosamide (NIC) appeared at wave numbers 1572, 1515,

1613, 1285, 1650, and 1218 cm-1 that were also found in NSE-2 nanoformulation

[280]. Based on the calculated and experimental results; assignment of the

fundamental frequencies were examined. The difference between the observed and

scaled wavenumber values of most of the fundamentals is very small. Overall, there is

no significant change in FT-IR spectra of NIC free drug and NSE-2 nanoformulation,


96

which revealed no interaction between drug and other formulation components

[Figure 38].

Figure 37. P-XRD Spectra of Pure Niclosamide and NSE-2

4.5.8 In-vitro drug release of optimized Niclosamide loaded SLNs

It was noted that primarily there was burst release for NSE-1 and NSE-2 about

19.58% and 23.33 % respectively of drug had been released in foremost hour

followed by prolonged release. The rest of three formulations i.e. NSE-3, NSE-4 and

NSE-5 have no burst effect as well as no complete release after 12 hrs [Figure 39].

There may be different reasons for burst release (1) may be the drug was located in

shell of SLNs, (2) large surface area, (3) high diffusion co-efficient, (4) short

diffusion distance and (5) low viscosity of SLNs. When the quantity of drug payload

reduced from 200mg to 40mg, the cumulative percent drug release decreased during

the 12 hrs in-vitro study. Hence, it is concluded that increased drug payload resulted

in improved sustained release profile.


97

Figure 38. FT-IR Spectra of Pure Niclosamide (A) and NSE-2 formulation (B)

Figure 39. Percent Drug Release from NIC-SLNs Formulations

4.5.9 Drug release Mechanism of optimized Niclosamide loaded SLNs

By putting the drug release data in different kinetic models like; First- order, Zero-

order, Higuchi model & Kors-Peppa equations, it was observed that NIC-SLNs followed

zero order kinetics. Release exponent (n) was greater than 0.89, regarded as Super case-II

diffusion mechanism [281]. R2 values and n values are given in Table 26.


98

Table 26. Different Kinetic Models for NIC loaded SLNs

4.5.10 Stability Study of optimized Niclosamide loaded SLNs

No significant change was observe in size and PDI of NSE-2 nanoformulation



This is also because of the amorphous nature of the NSE-2 nanoformulation and

might be degradation of both drug and lipid having low glass-transition temperature.

It is also common that particles in amorphous solid deposit on the surface of the

larger ones [274, 278]. Statistically data was analysed by two-tailed t-test, which

showed p-value 0.014 for particle size and 0.033 for PDI [Table 27].

4.5.11 In-Vivo Study of optimized Niclosamide loaded SLNs

After oral dose, plasma concentration−time curve [Figure 40] and

pharmacokinetic parameters of NSE-2 nanoformulation and marketed drug in rabbits

are tabulated [Table 28]. It was observed that at all time points, the NIC plasma

concentrations were significantly higher in rabbits treated with NSE-2

nanoformulation than for those treated with Mesan®.

FORMULATIONS ZERO

ORDER

(R2)

FIRST

ORDER

(R2)

HIGUCHI

MODEL

(R2)

KORSMEYER-PEPPAS

MODEL

RELEASE EXPONENT

(R2) (n)

NSE-1 0.9774 0.9109 0.9354 0.82632337 0.9615

NSE-2 0.9941 0.9954 0.9941 0.90632848 0.983

NSE-3 0.9612 0.9577 0.9473 0.91311668 0.942

NSE-4 0.9843 0.9478 0.9455 0.94456989 0.933

NSE-5 0.9762 0.9686 0.9117 0.986809871 0.945


99

Table 27. Stability study of NIC-SLNs (NSE-2)

Peak plasma concentration (Cmax) for marketed drug and NSE-2

nanoformulation was 1.99±0.124 μgml-1 and 4.07±0.124 μgml-1 respectively.

AUC0→24 for marketed drug was 2.005 μg.hr.ml-1 whereas for NSE-2 was 21.19

μg.hr.ml-1. NSE-2 formulation showed 2.04−fold increase in Cmax and 10.59−fold

increase in AUC0→24 compared to marketed drug [Table 28]. The small particle size

of SLNs may have bio−adhesion with gastrointestinal wall or go through the

inter−villar spaces results in rising duration of residence in GIT [283, 284]. This long

term sticking with GIT will result in boosted bioavailability. Tween−80 may have

paid to increase in affinity between stearic acid and intestine which leads to enhance

permeability [285, 286]. From these results, it could be concluded that NIC absorption

was significantly improved by using SLNs formulation compared to conventional

dosage form (marketed drug).

WEEK SIZE (nm)

(5±3⁰C)

SIZE (nm)

(25±2⁰C)

PDI (5±3⁰C) PDI (25±2⁰C)

Zero Week 208.6 208.6 0.376 0.376

1st Week 208.8 213.4 0.376 0.381

2nd Week 209.1 219.3 0.378 0.400

6th Week 209.4 233.2 0.375 0.503

8th Week 210.3 239.5 0.374 0.506

12th Week 211.5 255.5 0.375 0.557

Mean 209.61 228.25 0.37 0.45

±SD 1.09 17.77 0.001 0.07

p-Value 0.014 0.033


100

Table 28. Pharmacokinetic parameters of NSE-2 & Marketed Drug

(n=6, x¯±SD)

Figure 40. Comparative in-vivo drug release from NSE-2 & Marketed Drug

4.6 Fabrication of SZN-SLNs by Solvent Emulsification Diffusion

Technique

4.6.1 Particle size and PDI of Loaded Sulfasalazine SLNs

Five different formulations of SZN loaded SLNs (SSE−1 to SSE−5) based on

drug lipid ratio were fabricated. The optimized SSE-2 nanoformulation showed

particle size 202.3nm±2.2, PDI 0.376±0.02 and zeta potential -35.82mV±2 [Figure

41 & 42]. Results showed that particle size reduced after drug loading, which is due

to decreased free lipid content [272]. In addition, PDI was controlled and reduced by

increasing stirring time [273]. The PDI <0.5 and zeta potential in the range of ±30

PARAMETERS SZN-SLNS (NSE-2) MARKETED DRUG

Cmax (μg mL−1) 4.07 ± 0.124 1.99 ± 0.124

Tmax (h) 12 ± 0.2 06 ± 0.3

AUC (μg h mL−1) 21.19 2.005

Fr 10.595


101

demonstrated that the produced nanoformulation would be stable in nature [274]. In

the prepared SZN-SLNs, these values were within the range, which exhibit

electrostatic stabilization. Consequently there would be no aggregation, which can

potentially led to prevent Ostwald ripening and particles growth [171].

Figure 41. Particle size of SSE-2 Formulation

Figure 42. Zeta Potential of SSE-2 Formulation


102

4.6.2 Entrapment Efficiency of Sulfasalazine Loaded SLNs

To check the entrapment efficiency of SZN-SLNs, it was observed that

maximum percent entrapment efficiency was for SSE-2 nanoformulation whereas for

SSE-1 nanoformulation percent entrapment efficiency was 48.2±0.03 [Figure 43]. As

the concentration of lipid decreased the entrapment efficiency reduced. Drug lipid

ratio (10:1) showed maximum entrapment efficiency whereas further increase in lipid

ratio led to reduce entrapment efficiency. Moreover, concentration of the chosen

excipients including stearic acid (1.0gm), Tween80 (1.6gm) and PVP (0.4gm) were

found the effective combination to demonstrate maximum encapsulation of the drug

with higher loading efficiency [275].

4.6.3 Drug Loading Capacity of Sulfasalazine Loaded SLNs

Drug Loading Capacity (DLC) of SZN loaded SLNs are shown in Figure 43.


observed for SSE-2 whereas the formulation SSE-5 showed the lowest loading







of the SZN with stearic acid that results in maximum drug loading.


103

Figure 43. Entrapment Efficiency & Drug Loading Capacity of SZN−SLNs

4.6.4 Scanning Electron Microscopy of optimized Sulfasalazine Loaded SLNs

SEM micrograph of SZN loaded SLNs evidently denoted that the prepared solid




Figure 44. SEM micrograph of Sulfasalazine Loaded SLNs (SSE-2)


104

4.6.5 DSC thermogram of Sulfasalazine SLNs optimized SLNs

DSC thermograms of SZN (free drug), and SSE-2 nanoformulation were

recorded separately. Endothermic peak was observe for pure SZN at 245°C while for

SSE-2 nanoformulation at 230oC [Figure 45]. Tiny peak was observed at 230 °C for

SSE-2 nanoformulation, which is because of reduced particle size, enlarged surface

area and closed contact between stearic acid and drug. The melting point of the drug

shifted to lower scale with disappearance of components peaks is the indication of the

complete dispersion of the drug in lipid layers. The shifting of the melting point peak

of active pharmaceutical ingredients (API) in SLNs using stearic acid has previously

been reported [277].

Figure 45. DSC thermogram of Pure Sulfasalazine and SSE-2 formulation

4.6.6 PXRD of optimized Sulfasalazine loaded SLNs


highest for SZN (pure drug) compared SSE-2 nanoformulation [Figure 46]. This

study helped to explore the drug nature after encapsulation. It was observed that drug

entrapped in the SLNs was in the disordered-crystalline or amorphous state. The

reduction in intensities and also disappearance of minute peaks of SSE-2


105

nanoformulation are indicative for reduction in crystalline nature [274, 278]. This

phase further facilitates sustained drug release from SLNs [279].

Figure 46. P-XRD Spectra of Pure Sulfasalazine and SZZ−SLNs (SSE-2)


FT−IR spectra of the pure SZN and lSSE−2 nanoformulation were obtained

between 400–2000 cm-1 [Figure 47]. C−O in phenol at 1281 cm-1 of the SZN &

SSE−2 nanoformulation remained the same. Similarly, OH−group of free SZN at

1394 cm-1remained same in SSE−2 nanoformulation [287]. C=C vibration of the

benzene ring at 1700cm-1 remained same in free SZN and its SLNs. The spectrum of

SZN (free drug) and SSE−2 formulation showed sharp peaks at 1427 & 1618cm-1

allocated to symmetric and asymmetric stretching of the carboxylate group. Based on

the calculated and experimental results; assignment of the fundamental frequencies

were examined. The difference between the observed and scaled wavenumber values

of most of the fundamentals is very small. Overall, there is no significant change in

FT-IR spectra of SZN free drug and SSE-2 nanoformulation, which revealed no



106

4.6.8 In-vitro release from optimized SZN loaded SLNs

Cumulative percent drug release during in-vitro study from SSE-1 to SSE-5

formulations were 100%, 100% , 92.31%, 56.35% and 50.15% respectively [Figure

48]. When the quantity of drug payload reduced from 200mg to 40mg, the cumulative

percent drug release decreased from 100% to 50.15% only during the 12 hrs in-vitro

study. Hence, it is concluded that increased drug payload resulted in improved

sustained release profile.

Figure 47. FT-IR Spectra of Sulfasalazine (A) and SZN−SLNs (SSE-2) (B)

Figure 48. Percent Release from SZN loaded SLNs formulations


107

4.6.9 Drug release Mechanism of optimized Sulfasalazine loaded SLNs

By putting the drug release data in different kinetic models like; First- order, Zero-

order, Higuchi model & Kors-Peppa equations, it was observed that SZN-SLNs followed

mixed order kinetics i.e. initially the release pattern was first order kinetic then followed by

zero order kinetics. Release exponent (n) was greater than 0.89, regarded as Super case-II

diffusion mechanism [281]. R2 values and n values are given in Table 29.

Table 29. Different Kinetic Models for SZN loaded SLNs

4.6.10 Stability Study of optimized Sulfasalazine loaded SLNs

No significant change was observe in size and PDI of SSE-2 nanoformulation



This is also because of the amorphous nature of the SSE-2 nanoformulation and might

be degradation of both drug and lipid having low glass-transition temperature. It is

also common that particles in amorphous solid deposit on the surface of the larger

FORMULATIONS ZERO

ORDER

(R2)

FIRST

ORDER

(R2)

HIGUCHI

MODEL (R2)

KORSMEYER-

PEPPAS MODEL

RELEASE

EXPONENT

(R2) (n)

SSE−1 0.958 0.912 0.969 0.80696337 0.978

SSE−2 0.974 0.956 0.954 0.90638848 0.963

SSE−3 0.981 0.972 0.957 0.91271668 0.962

SSE−4 0.994 0.977 0.945 0.97277989 0.943

SSE−5 0.996 0.982 0.941 0.97109871 0.935


108

ones [274, 278]. Statistically data was analysed by two-tailed t-test, which showed p-

value 0.003 for particle size and 0.004 for PDI [Figure 49 & 50].

.

Figure 49. Change in size of SSE-2 during stability

Figure 50. Change in PDI of SSE-2 during stability

4.6.11 In-Vivo Study of optimized Sulfasalazine loaded SLNs

The plasma concentration-time curve after single oral dose SSE-2

nanoformulation and marketed drug i.e. Salazodine® in rabbits is shown in Figure 51.


109

The oral pharmacokinetic parameters are listed in [Table 30]. It was observed that at

all time points, the SZN plasma concentrations were higher significantly in rabbits

treated with SSE-2 nanoformulation than for those treated with Salazodine®.

Peak plasma concentration (Cmax) for Salazodine® and SSE-2 nanoformulation

was 1.94±0.3 μgmL-1 and 3.62±0.2μgmL-1 respectively. AUC0→24 for Salazodine®

was 6.368 μg/mLh-1 whereas for SSE-2 was 76.2 μg/mLh-1. SSE-2 formulation

showed 1.86-folds increase in Cmax and 8.35-folds increase in AUC0→24 compared to

Salazodine®. After twenty-four hours, SZN plasma concentration was still 1.5±0.3

μgmL-1 for SSE-2 formulation whereas it was untraceable for Salazodine®.

Pharmacokinetic study revealed that SZN absorption was significantly enhanced by

taking up SLNs as drug delivery system. The small particle size of SLNs may have

bio−adhesion with gastrointestinal wall or go through the inter−villar spaces results in

rising duration of residence in GIT [283, 284]. This long term sticking with GIT will

result in boosted bioavailability. Tween−80 may have paid to increase in affinity

between stearic acid and intestine which leads to enhance permeability [285, 286].

From these results, it can be concluded that SZN absorption was significantly

improved by using SLNs formulation compared to conventional dosage form

(marketed drug)From these results, we can conclude that SZN absorption was

improved significantly by employing the SLNs formulation compared with

conventional dosage form (Salazodine®).


110

Table 30. Pharmacokinetic parameters of SSE−2 & Marketed drug

(n=6, x¯±SD)

Figure 51. Comparative in−vivo release from SSE−2 & Marketed Drug

SLNs can be prepared by solvent emulsification diffusion method which are

used to encapsulate lipophilic drugs. The drug is dissolved in organic phase of the

system, together with the lipid [288]. The particle sizes of fabricated SLNs for both

BCS-II and IV drugs were in the desired nano-metric range, PDI, zeta-potential, EE

and DLC. Stability testing showed that the SLNs were more stable at refrigerated

temperature. Both diffraction & thermal analysis confirmed reduction in crystalline

nature of fabricated nanoformulation. In-vitro release profiles showed sustained

release of Niclosamide and Sulfasalazine. The in-vivo study revealed oral

bioavailability enhancement for both NIC and SZN loaded SLNs in comparison to

their respective marketed products. Statistically the data of all parameters of in-vivo

PARAMETERS SZN-SLNS (SSE-2) MARKETED DRUG

Cmax (μg mL−1) 3.62 ± 0.3 1.94 ± 0.3

Tmax (h) 12 ± 0.1 0 6 ± .03

AUC (μg h mL−1) 76.2 ± 0.003 6.368 ± 0.021

Fr 8.35


111

pharmacokinetic was analysed which confirmed that SLNs formulations enhanced the

bioavailability of NIC and SZN significantly in comparison with marketed drugs

[151].

4.7 Similarity Factor

For similarity factor (f2) determination, in-vitro dissolution studies were

carried out using the paddle method.

Prepared solid dosage form of Niclosamide and Sulfasalazine loaded SLNs

(Microemulsion Technique) and their respective marketed products were processed

under similar conditions. Dissimilarity (f1) and Similarity factor (f2) of the prepared

solid dosage form of the drug loaded SLNs and its marketed product was determined

using the obtained in vitro dissolution data.

4.8 Calculation of f1 and f2 for Niclosamide Nanoformulation

Factors f1 and f2 were being calculated for NME-3 nanoformulation and

marketed product i.e. Mesan®. During this study, f1 was found to be 56 while f2 was

16. The dissolution analysis clearly showed that NME-3 nanoformulation showed

desired better dissolution compared to marketed product. The improved dissolution of

the NME-3 nanoformulation can be envisioned in [Figure 52].


112

Figure 52. Dissolution Profiles of NME-3 and Marketed Drug

4.9 Calculation of f1 and f2 for Sulfasalazine Nanoformulation

Factors f1 and f2 were being calculated for SME-3 nanoformulation and

marketed product i.e. Salazodine®. During this study, f1 was found to be 59 while for

f2 was 15. The dissolution analysis clearly showed that SME-3 nanoformulation

showed desired better dissolution compared to marketed product. The improved

dissolution of the SME-3 nanoformulation can be envisioned in [Figure 53].

Figure 53. Dissolution Profiles of SME-3 and Marketed Drug


113

The results presented in the thesis provide information on the evaluation of an

investigational protocol to screen nanoformulations like Solid Lipid Nanoparticles of

Niclosamide and Sulfasalazine, excipient such as Tween-80, PEG and PVP and new

pharmaceutical compositions. The following practical conclusions can be drawn from

the experimental data summarized in the thesis:

a. The critical parameters for fabrication of SLNs for NIC and SZN have been

optimized using micro emulsion and solvent emulsification diffusion

techniques.

b. Prepared SLNs were characterized by using different techniques like, DSC,

PXRD, SEM, FT-IR etc.

c. Both NIC-SLNs and SZN-SLNs have increased solubility and faster

dissolution rate in physiological conditions.

d. Animal model were successfully used to confirm the enhancement of oral

bioavailability by comparing with commercially available products.

e. Successfully converted the SLNs of both NIC and SZN to solid dosage form.

f. Similarity factors were determined by comparing the dissolution rate of

prepared SLNs to the commercially available products.

g. Biopharmaceutical studies including bioavailability experiments are needed to

test these formulations for clinical testing on humans and also to test

cytotoxicity and toxicokinetics.

h. The relevance of the presented investigational protocol needs to be confirmed

in clinical tests in the future.

i. Our findings may contribute to the development of new pharmaceutical

compositions to formulate hydrophobic drugs.

CONCLUSION

114

Conclusion

The objective of the current study was“to develop economical, safe and

efficient sustained release SLNs loaded with Niclosamide and Sulfasalazine to

enhance their oral bioavailability. Loaded SLNs were successfully converted to solid

dosage form and compared their bioavailability with commercially available products.

Improved pharmacokinetic profile was attained for outdated drugs of both BCS-II

and BCS-IV”.

The SLNs of Niclosamide and Sulfasalazine were fabricated by micro

emulsion and solvent emulsification diffusion methods. Stearic acid was used as

solid lipid with surfactant (Tween-80) and co-surfactants (PEG & PVP). The

chosen concentrations of the excipients were found effective combination to

demonstrate desired results. The particles obtained possessed unique physicochemical

characteristics in terms of particle size, PDI, zeta potential, entrapment efficiency and

drug loading capacity. Spherical shaped SLNs were in nano-metric range, confirmed

by Scanning Electron Microscopy (SEM). Compatibility between drug and excipients

was confirmed by Fourier Transformed Infrared (FT-IR) spectroscopy. Powder X-ray

difrractometry (P-XRD) and Differential Scanning Calorimetry (DSC) showed change

in physical nature of the drug. Prepared drug loaded SLNs were found to be stable at

refrigerated conditions.

In-vitro study of all SLNs formulation showed that increase in drug payload

resulted in enhanced sustained release behaviour. The SLNs formulations were

successfully converted to oral solid dosage form. Similarity study showed better

results in comparison to commercially available products. In-vivo release study

confirmed that this system is best fitted to augment oral bioavailability of

Niclosamide and Sulfasalazine in term of increased water solubility and permeability.

CONCLUSION

115

These results validated that, SLNs as drug delivery system satisfies improved oral

bioavailability with sustained release, which offers new diagonal to formulate

hydrophobic drugs.

Future Work

After successful conversion of SLNs in to suitable dosage form, further studies

are required to check the release and bioavailability of coated granules in form of

spansules and assessment of these formulations have to be performed in order to

check its safety and toxicological parameters.

The in-vitro and in-vivo evaluation of the formulation is required in order

to study the continuous and long-term exposure of the drug to the animal models

to clarify any doubts regarding the safety and toxicity of lipid and drug in chronic

administration.

All these studies must be carried out in order to bring the work forward

before being considered for clinical studies. Moreover, it is necessary to conduct

the clinical trials in human volunteers to evaluate the pharmacokinetics parameters

of solid lipid nanoparticles.

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116

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Publications

1. PUBLICATION FROM THESIS

Maqsood ur Rehman, Mir Azam Khan, Waheed S. Khan, Muhammad Shafique,

Munasib Khan (2017). Fabrication of Niclosamide loaded solid lipid

nanoparticles: In-vitro Characterization and Comparative in-vivo Evaluation.

Artificial cells, nanomedicine, and biotechnology 1-9.

DOI:10.1080/21691401.2017.1396996 (Impact Factor 5.605)

Mir Azam Khan, Maqsood-ur-Rehman, Waheed S. Khan, Shahzeb Khan,

Waqar Ahmad, Muhammad Shafique (2017) Fabrication of sulfasalazine

loaded solid lipid nanoparticles, in-vitro Characterization and Comparative in-

vivo Evaluation to enhance oral bioavailability Evaluation. Acta

Pharmaceutica (Accepted) (Impact Factor 1.26)

Maqsood ur Rehman, Waheed S. Khan, Mir Azam Khan, Muhammad

Shafique, Ayesha Ihsan. Explore solid lipid nanoparticles to augment oral

bioavailability of Niclosamide: pharmaceutical and stability study. (Under

Review)

Mir Azam Khan, Maqsood ur Rehman , Waheed S. Khan, Muhammad

Shafique, Waqar Ahmad. Solid lipid nanoparticles for sulfasalazine:

fabrication, characterization, in-vitro and in-vivo assessment for enhanced oral

bioavailability. (Submitted).

2. PUBLICATIONS FROM SAME PROJECT

Muhammad Shafique, Mir Azam Khan, Waheed S. Khan, Maqsood-ur-Rehman,

Waqar Ahmad, and Shahzeb Khan (2017) Fabrication, Characterization, and In

https://doi.org/10.1080/21691401.2017.1396996

REFERENCES

155

Vivo Evaluation of Famotidine Loaded Solid Lipid Nanoparticles for Boosting

Oral Bioavailability. Journal of Nanomaterials. (Impact Factor 1.871)

Muhammad Shafique, Mir Azam Khan, Waheed S. Khan, Maqsood-ur-Rehman,

Shahzeb Khan, Waqar Ahmad, (2017) Famotidine loaded solid lipid

nanoparticles: Physico-chemical characterization and in vivo evaluation of boosted

oral bioavailability Acta Pharmaceutica (Accepted) (Impact Factor 1.26)

Muhammad Shafique, Mir Azam Khan, Maqsood-ur-Rehman, Waqar Ahmad,

Shahzeb Khan “Enhancing oral bioavailability of roxithromycin by nano-

emulsifying drug delivery system”. (Submitted)

Muhammad Shafique , Mir Azam Khan, Waqar Ahmad, Maqsood-ur-Rehman,

Shahzeb Khan “Fabrication and characterization of roxithromycin loaded solid

lipid nanoparticles: comparative in-vivo evaluation confirming enhanced oral

bioavailability” (Submitted)

Documents

prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/9272/1/Maqsood ur Rehm… · II CERTIFICATE OF APPROVAL This is to certify that the research work presented in this thesis,