Nanonization and Characterization of Three Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Dexibuprofen and Indomethacin)

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NANONIZATION AND CHARACTERIZATION OF THREE NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (KETOPROFEN, DEXIBUPROFEN AND INDOMETHACIN)

A DISSERTATION SUBMITTED

THE UNIVERSITY OF SARGODHA, SARGODHA

IN PARTIAL FULFILLMENT OF THE REQUIREMENT

FOR

THE DEGREE OF DOCTOR OF PHILOSOPHY IN

PHARMACEUTICS

BY JAHANGIR KHAN COLLEGE OF PHARMACY FACULTY OF PHARMACY UNIVERSITY OF SARGODHA, SARGODHA Session 2011-2014

DEDICATE

THIS THESIS

MY BROTHERS, SISTERS AND WIFE

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APPROVAL CERTIFICATE

It is solemnly described that the dissertation titled “Nanonization and characterization of three non-steroidal anti-inflammatory drugs (ketoprofen, dexibuprofen and indomethacin)” submitted by Jahangir Khan in the partial fulfillment of the requirement for the award of degree of

DOCTOR OF PHILOSOPHY in Pharmaceutics is hereby approved.

Supervisor: ______Co-Supervisor: ______Prof. Dr. Sajid Bashir Dr. Shahzeb Khan Dean Assistant Professor College of Pharmacy Department of Pharmacy Faculty of Pharmacy University of Malakand, Chakdara University of Sargodha, Sargodha

External Examiner: ______

Principal: ______Dean: ______Associate Prof. Dr. Saira Azhar Prof. Dr. Sajid Bashir College of Pharmacy College of Pharmacy University of Sargodha, Sargodha. Faculty of Pharmacy University of Sargodha, Sargodha.

DECLARATION

I declare that the work described in this thesis was carried out by me under the supervision of Prof.

Dr. Sajid Bashir, Dean/Chairman Faculty of Pharmacy University of Sargodha, Sargodha, Pakistan and Dr. Shahzeb Khan Assistant Professor, Department of Pharmacy, University of Malakand,

Chakdara Dir(L), Pakistan, in partial fulfillment of the requirement for the degree of “DOCTOR

OF PHILOSOPHY in PHARMACEUTICS”. I certify that the main content of this thesis accounts for my own research and has not previously been submitted for a degree at any educational institution. Further, it is submitted that the material taken from other sources has been acknowledged.

JAHANGIR KHAN

ACKNOWLEDGEMENTS

I offer my humble praise and gratitude to ALLAH Almighty, The Most Beneficent, The Most

Merciful for sprinkling His uncountable blessings throughout my life and bestowing upon me the intellectual ability and wisdom to explore for its mysteries. Immeasurable salutations upon “The

with Whose Existence and by having the Charity of His ”(ﷺ ) Teacher of the Universe

Knowledge, the cosmos got illuminated with the light of insight and wisdom, and journey of human enlightenment became possible.

I must express my special thanks to my supervisors Prof. Dr. Sajid Bashir, Dean/Chairman

Faculty of Pharmacy, University of Sargodha, Sargodha and Dr. Shahzeb Khan, Assistant

Professor Department of Pharmacy, University of Malakand Chakdara Dir(L) for their continuous support, constant inspiration and precious advice and supervision throughout the course of my research work.

I am immeasurably delighted to place on record my extensive appreciativeness and enthusiastic recognitions to Dr. Muhammad Isreb School of Pharmacy, University of Bradford, UK for his continuous inspiration, friendly supervision, support and priceless efforts throughout my research work to make my project fruitful.

At University of Bradford, I must express my special thanks to Dr. Mohammad Amin Muhammad,

Abdul Rahman Mkia and Yuosef Al Ayoub for their motivations, enthusiasm, worthy piece of advices and guidance. I’m grateful to the Prof. Dr. Anant Pardarkar & his Research team included

Dr. Khaled hafez Assi for providing research facilities and their appreciated assistance. I must acknowledge the technical support of Stuart Fox, David Benson, Toheed raza, Damian Yeadon and other staff members.

I am thankful to the HEC Pakistan for the finical support provide to me under the IRSIP program.

I am also thankful to University of Malakand Chakdara Khyber Pakhtunkhwa for the award of

NOC, study leave and support to pursue my PhD studies.

At University of Malakand I am also thankful to Prof. Dr. Mirazam khan, Prof. Dr. Waqar Ahamd,

Prof. Dr. Rahmat Ali khan, Prof. Dr. Rashid Ahmad and other friends/ colleagues for their help, inspiration and productive suggestions.

I sincerely extend my gratitude to my fellow doctoral colleagues and other friends at UK and

Pakistan especially to Shah Hassan, Muthana Obeed, Dr. Farhat ali khan, Naveed Khan and PhD scholar Sajjad Khan.

I would like to express my warm thanks to my brothers, sisters and my wife whose indispensable support certainly led me towards completion of my PhD studies.

JAHANGIR KHAN

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ABBREVIATIONS

AVG Average

ANOVA Analysis of variance

BCS Biopharmaceutical Classification System cm Centimeter

°C Degrees celsius

COX Cyclooxygenase

Conc Concentration

DLS Dynamic light scattering

DSC Differential scanning calorimetry

Da Dalton

Diclo Diclofenac

DMSO Dimethyl sulfoxide

DXI Dexibuprofen

EPAS Evaporative precipitation into aqueous solutions

ΔG Gibbs free energy

GIT Gastrointestinal tract hr Hour

HPMC Hydroxypropyl methyl cellulose

ΔH Enthalpy change

HPH High pressure homogenization

HP--CD hydroxyl propyl dextrin

H2O Water

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IND Indomethacin

Keto Ketoprofen

LSD Least significant difference test mg Milligram min Minute ml Milliliter mp Melting point mol f Molecular Formula mol wt Molecular weight

NSAIDs Non-steroidal anti-inflammatory drugs

NPXN Naproxen

NLC Nanostructured lipid carriers

P Particle

PXRD Powder X-ray diffraction

PCS Photon correlation spectroscopy

PDI Polydispersity index

PVP Polyvinylpyrrolidone

RESS Rapid expansion from supercritical fluids s Second

SAS Supercritical anti-solvent precipitation

SDS Sodium dodecyl sulfate

SEM Scanning electron microscopy

ΔS Entropy change

SD Standard deviation

TEM Transmission electron microscope

USP United States Pharmacopoeia

UV Ultraviolet

LIST OF FIGURES

Figure I.1: Biopharmaceutical Classification System

Figure 1.2: solubility- diagram (Garside and Davey 2000)

Figure 1.3: Alternation in nuclei magnitude effect free energy (Gibbs 1928)

Figure 1.4: crystal growth Mechanism (Elwell and Scheel 1975; Dirksen and Ring 1991) Figure 1.5: Imaginary crystal faces (i) flat-F (ii) step-S (iii) kink-K (Dirksen and Ring 1991) Figure 1.6: crystal nuclei creation on emerging crystal surface (Garside and Davey 2000) Figure 1.7: creation of spirals initiating from screw dislocation (Mullin 2001) Figure 1.8: Distinguishing features of nanoparticle (Müller, Shegokar et al. 2011). Figure 1.9: Methods of preparation of Nanoparticles.

Figure 1.10: Media Milling (Merisko-Liversidge, Liversidge et al. 2003)

Figure 1.11: High Pressure Homogenization (HPH) (Junghanns and Müller 2008) Figure 1.12: Spray freezing into liquid. (Hu, Johnston et al. 2003)

Figure 1.13: EPAS process (Sinswat, Gao et al. 2005).

Figure 1.14: micro-chip.(Schulte, Bardell et al. 2002).

Figure 1.15: Microfluidic reactor water and Dye (Ali, Blagden et al. 2009)

Figure 1.16: diffusion process liquid streams

Figure 1.17: (Classical DLVO theory). Change in particle distance affects the Potential energy of system. Figure 1.18: absorption comparative study of micro and nanoparticle (Mauludin, Müller et al. 2008). Figure 1.19: Chemical structures of drugs (A) Ketoprofen (B) Dexibuprofen (C) Indomethacin Figure 2.1: Microchannel fluidic reactor

Figure 3.1: Polymers effect on ketoprofen nanocrystals

Figure 3.2: ketoprofen nanoparticle particle size distribution (Poloxamer 407 (1%)

Figure 3.3: polymers effect on Dexibuprofen nanocrystals

Figure 3.4: Dexibuprofen nanoparticle particle size distribution (Poloxamer 407 (0.5%) Figure 3.5: Polymers effect on Indomethacin nanocrystals

Figure 3.6: Indomethacin nanoparticle particle size distribution (PVP k-30 (1%)- HPMC 15cps (0.5%)-SLS (0.5%) Figure 3.7: MR inlet angle Effect on Ketoprofen nanoparticle

Figure 3.8: MR inlet angle Effect on Dexibuprofen nanoparticle

Figure 3.9: MR inlet angle Effect on Indomethacin nanoparticle

Figure 3.10: Effect of flow rate (Antisolvent Variable and solvent constant) on Particle size of ketoprofen nanocrystal Figure 3.11: Effect of Antisolvent and solvent flow rate on size of Dexibuprofen Nanoparticle Figure 3.12: Effect of Antisolvent and solvent flow rate (Antisolvent Variable and solvent constant) on nanocrystal size of Indomethacin Figure 3.13: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of ketoprofen nanoparticle. Figure 3.14: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of Dexibuprofen nanoparticle Figure 3.15: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of Indomethacin nanoparticle. Figure 3.16: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on ketoprofen nanoparticle Figure 3.17: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on Dexibuprofen nanoparticle Figure 3.18: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on Indomethacin nanoparticle Figure 3.19: Drug concentration effect on particle size of Ketoprofen

Figure 3.20: Drug concentration effect on particle size of Dexibuprofen

Figure 3.21: Drug concentration effect on particle size of Indomethacin

Figure 3.22: Effect of Mixing Time on ketoprofen nanoparticle

Figure 3.23: Effect of Mixing Time on Dexibuprofen nanoparticle

Figure 3.24: Effect of Mixing Time on Dexibuprofen nanoparticle

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Figure 3.25: stability of ketoprofen nanoparticle at 2-8ºC

Figure 3.26: stability of Dexibuprofen nanoparticle at 2-8ºC

Figure 3.27: stability of Indomethacin nanoparticle at 2-8ºC

Figure 3.28: stability of Ketoprofen nanoparticle at 25ºC

Figure 3.29: stability of Dexibuprofen nanoparticle at 25ºC

Figure 3.30: stability of Indomethacin nanoparticle at 25ºC

Figure 3.31: Graphical presentation of Zeta potential value of the produced Nanosuspensions Figure 3.32: DSC analysis of processed and un processed Ketoprofen

Figure 3.33: DSC analysis of processed and un processed Dexibuprofen

Figure 3.34: DSC analysis of processed and un processed Indomethacin

Figure 3.35: X Ray Diffractogram of raw and nanocrystals of ketoprofen

Figure 3.36: X Ray Diffractogram of raw and nanocrystals of Dexibuprofen

Figure 3.37: X Ray Diffractogram of raw and nanocrystals of Indomethacin

Figure 3.38: SEM and TEM images (i) Raw Ketoprofen (ii) Ketoprofen nanoparticle Figure 3.39: SEM and TEM images (i) Raw Dexibuprofen (ii) Dexibuprofen nanoparticles Figure 3.40: SEM and TEM images (i) Raw Indomethacin (ii) indomethacin nanoparticle Figure 3.41: Dissolution studies of raw, nanoparticle and marketed product of Ketoprofen Figure 3.42: Dissolution studies of raw, nanoparticle and marketed product of Dexibuprofen Figure 3.43: Dissolution studies of raw, nanoparticle and marketed product of Indomethacin Figure 5.1: effect of milling time on ketoprofen nanoparticles

Figure 5.2: ketoprofen nanoparticle particle size distribution

Figure 5.3: effect of milling time on Dexibuprofen nanoparticles

Figure 5.4: Particle size Distribution of Dexibuprofen nanoparticles

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Figure 5.5: effect of milling time on Indomethacin nanoparticles

Figure 5.6: Particle size Distribution of Indomethacin nanoparticles

Figure 5.7: value of the zeta potential of the selected drugs

Figure 5.8: ketoprofen nanoparticle stability at 2 - 8 ºC

Figure 5.9: Dexibuprofen nanoparticle stability at 2 - 8 ºC

Figure 5.10: Indomethacin nanoparticle stability at 2 - 8 ºC

Figure 5.11: ketoprofen nanoparticle stability at 25 ºC

Figure 5.12: Dexibuprofen nanoparticle stability at 25 ºC

Figure 5.13: Indomethacin nanoparticle stability at 25 ºC

Figure 5.14: DSC analysis of nanocrystal and un processed Ketoprofen

Figure 5.15: DSC analysis of nanocrystal and un processed Dexibuprofen

Figure 5.16: DSC analysis of nanocrystal and un processed Indomethacin

Figure 5.17: X Ray Diffractogram of raw and nanocrystals of ketoprofen

Figure 5.18: X Ray Diffractogram of raw and nanocrystals of Dexibuprofen

Figure 5.19: X Ray Diffractogram of raw and nanocrystals of Indomethacin

Figure 5.20: SEM and TEM images (i) Raw Ketoprofen (ii) Ketoprofen nanocrystal Figure 5.21: SEM and TEM images (i) Raw Dexibuprofen (ii) Dexibuprofen nanoparticles Figure 5.22: SEM and TEM images (i) Raw Indomethacin (ii) indomethacin Nanoparticle Figure 5.23: Dissolution studies of raw, nanoparticle and marketed product of Ketoprofen Figure 5.24: Dissolution studies of raw, nanoparticle and marketed product of Dexibuprofen Figure 5.25: Dissolution studies of raw, nanoparticle and marketed product of indomethacin

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LIST OF TABLES

Table 1.1: Some of the marketed products having Nano-drug (Nijhara and Balakrishnan 2006; Cheong and Boon 2010) (Van Eerdenbrugh, Van den Mooter et al. 2008). Table 3.1: Different polymers used and its effect on ketoprofen Nanocrystals Table 3.2: Different polymers used and its effect on Dexibuprofen nanocrystals Table 3.3: Different polymers used and its effect on Indomethacin nanocrystals Table 3.4: Effect of inlet angle of MR on particle size of Ketoprofen nanoparticle Table 3.5: Effect of inlet angle of MR on particle size of Dexibuprofen nanoparticle Table3.6: Effect of inlet angle of MR on particle size of indomethacin nanoparticle Table 3.7: Effect of Antisolvent and solvent flow rate (Antisolvent Variable and solvent constant) on nanocrystal size of ketoprofen Table 3.8: Effect of Antisolvent and solvent flow rate (Antisolvent Variable and solvent constant) on Dexibuprofen nanoparticle size Table 3.9: Effect of flow rate (Antisolvent Variable and solvent constant) on nanocrystal size Indomethacin Table 3.10: Effect of Antisolvent and solvent flow rate (Antisolvent Constant and solvent variable) on nanocrystal size ketoprofen Table 3.11: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of Dexibuprofen nanoparticle. Table 3.12: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of Indomethacin nanoparticle Table 3.13: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on ketoprofen nanoparticle Table 3.14: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on Dexibuprofen nanoparticle Table 3.15: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on Indomethacin nanoparticle Table 3.16: Drug concentration effect on ketoprofen nanocrystals

Table 3.17: Drug concentration effect on Dexibuprofen nanocrystals

Table 3.18: Drug concentration effect on Indomethacin nanocrystals

Table 3.19: Mixing Time effect on ketoprofen nanoparticle

Table 3.20: Mixing Time effect on Dexibuprofen nanoparticle

Table 3.21: Mixing Time effect on Indomethacin nanoparticle

Table 3.22: Chemical stability of the produced nanosuspensions.

Table 3.23: stability of ketoprofen nanoparticle at 2-8ºC

Table 3.24: stability of Dexibuprofen nanoparticle at 2-8ºC

Table 3.25: stability of Indomethacin nanoparticle at 2-8ºC

Table 3.26: stability of Ketoprofen nanoparticle at 25ºC

Table 3.27: stability of Dexibuprofen nanoparticle at 25ºC

Table 3.28: stability of Indomethacin nanoparticle at 25ºC

Table 3.29: Zeta potential value of the produced nanosuspensions

Table 5.1: Effect of Milling time on ketoprofen nanoparticle

Table 5.2: Effect of Milling time on Dexibuprofen nanoparticle

Table 5.3: Effect of Milling time on Indomethacin particle

Table 5.4: zeta Potential value of the produced nanosuspensions

Table 5.4: Chemical stability of the produced nanosuspensions.

Table 5.5: stability of ketoprofen nanoparticle at 2 - 8 ºC

Table 5.6: stability of Dexibuprofen nanoparticle at 2 - 8 ºC

Table 5.7: stability of Indomethacin nanoparticle at 2 - 8 ºC

Table 5.8: stability of Ketoprofen nanoparticle at 25 ºC

Table 5.9: stability of Dexibuprofen nanoparticle at 25 ºC

Table 5.10: stability of Indomethacin nanoparticle at 25 ºC

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Contents Title i Acknowledgements vi List of Figures x List of Tables xiv Abstract 01 Chapter 01 05 1 Introduction and Literature survey 06 (a) Challenging Condition 06 1.1 Particle Engineering with special reference to crystal 07 1.2 Phase Transition and Polymorphism 16 1.3 Nanoparticles and Its Method of Preparation 18 1.4 Microchannel reactors (MCR): 25 1.5 Features of Nanocrystals 31 1.6 Bioavailability 34 1.7 Marketed Products of nanoparticles 36 1.8 Non-steroidal anti-inflammatory drugs (NSAIDs) 38 1.8.1 Ketoprofen 41 1.8.2 Dexibuprofen 46 1.8.3 Indomethacin 50 1.9 Aims & Objective 58 Chapter 02 60 A. Preparation of Ketoprofen, Dexibuprofen and Indomethacin nanosuspensions through Microchannel Fluidic Reactor 61 2 Materials and Methods 61 2.1 Materials 61 2.2 Method 62 2.2.1 Preparation of Ketoprofen, Dexibuprofen and Indomethacin Nanosuspension 62 2.3 Characterization of the Produced Nanosuspension 66 2.3.1 Drug content Determination 66 2.3.2 Particle size Measurement through DLS 66 2.3.3 Measurement of Zeta Potential 68

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2.3.4 Morphological evaluation through Scanning electron microscopy 69 2.3.5 Transmission electron microscopy 70 2.3.6 Thermal analysis 72 2.3.7 X-ray powder diffraction (XRPD) Analysis 74 2.3.8 Stability studies 76 2.3.9 In-vitro dissolution evaluation 77 2.3.10 Statistical Examination 77 Chapter 03 78 A. Preparation of Ketoprofen, Dexibuprofen and Indomethacin nanosuspension through Microchannel Fluidic Reactor 79 3 Results and Discussion 79 3.1 Evaluation of Polymers 79 3.2 Inlet angle of Microfluidic Reactor 87 3.3 Effect of Flow rate of Antisolvent and Solvent 91 3.3.1 Anti-solvent volume variable and Solvent volume constant 91 3.3.2 Anti-solvent volume constant and Solvent volume variable 95 3.3.3 Equal volume of Anti-solvent and Solvent 99 3.3.4 Batch sizes Scalability 103 3.4 Drug Concentration Effect 104 3.5 Effect of Mixing Time 108 3.6 Stability Studies 112 3.7 Zeta Potential 120 3.8 Thermal and X-ray analysis 122 3.9 Morphology Studies 126 3.10 Invitro dissolution 130 Chapter 04 134 B. Preparation of Ketoprofen, Dexibuprofen and Indomethacin nanosuspensions through Media Milling method 135 4 Materials and Methods 135 4.1 Materials 135 4.2 Method 136 4.2.1 Preparation of Ketoprofen, Dexibuprofen and Indomethacin Nanosuspension 136

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4.3 Characterization of the Produced Nanosuspension 137 4.3.1 Drug content Determination 137 4.3.2 Particle size Measurement 137 4.3.3 Measurement of Zeta Potential 137 4.3.4 Morphological evaluation through Scanning electron microscopy 137 4.3.5 Transmission electron microscopy 138 4.3.6 Thermal Analysis 138 4.3.7 X-ray powder diffraction (XRPD) Analysis 139 4.3.8 Stability studies 139 4.3.9 In-vitro dissolution evaluation 140 4.3.10 Statistical Examination 140 Chapter 05 141 B. Preparation of Ketoprofen, Dexibuprofen and Indomethacin nanosuspensions through Media Milling 142 5 Results and Discussion 142 5.1 Effect of Milling time 142 5.2 Zeta potential 149 5.3 Stability studies 151 5.4 Thermal and X-ray analysis 159 5.5 Morphological Studies 162 5.6 Invitro dissolution 166 Chapter 06 170 6 Conclusion 171 6.1 Future perspective 173 Chapter 07 174 7 Reference 175

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Abstract

The foremost challenge for pharmaceutical scientist is the poor bioavailability of drugs which is the derivative of poor water solubility. The nanocrystal has got prominent consideration in solving the bioavailability problem by increasing the aqueous solubility of the drugs. The nanocrystals can be prepared by bottom up and top down methods. In the current study nanonization has been accomplished by microchannel fluidic reactor (bottom up method) and media milling (top down method).

The current research project has been conducted on three poor water soluble

NSAIDs drugs (ketoprofen, dexibuprofen and indomethacin). In the bottom, up method, drug solutions and polymers solutions were mixed in microchannel fluidic reactor and subsequent emergent nanosuspensions were poured into vials having polymer solutions.

Stable nanocrystals of the ketoprofen, dexibuprofen and indomethacin with particle sizes of 61 nm ± 3.0 with PDI of 0.25 ± 0.07, 45 nm ± 3.0 with PDI of 0.190 ± 0.06, 380 nm ±

5.0 with PDI of 0.290 ± 0.05 were produced. It was observed that antisolvent and solvent flow rate, inlet angle, mixing time and appropriate polymer with specific concentration were the key parameters which greatly affected yielded nanocrystals. The results obtained demonstrates that high antisolvent solvent volume to solvent volume ratio (2.0/0.5 ml/min) produced the relative smaller nanoparticles size. Moreover, it was originated that nanoparticle size increases while increasing the solvent volume and taking antisolvent volume constant. Similarly, at equal ratio of both antisolvent and solvent volumes, it was revealed that low ratio of both liquids produces comparatively smaller nanoparticle size.

The mixing of the nanosuspensions for 60 minutes with subsequent ultra-sonication results in reduction of PDI and nanoparticle sizes. Moreover, small inlet angle 10° produced

smaller nanocrystals in relation to inlet angle 50°. Poloxamer 407 was effective stabilizer for ketoprofen and dexibuprofen nanosuspensions while indomethacin nanosuspension was stabilized with HPMC-PVP-SDS polymer combination. Moreover, the imperative physicochemical characterization of the nanosuspensions were carried out and it was observed that the produced nanosuspensions were stable for two months. The crystallinity of the nanoparticles was confirmed by DSC and PXRD. Furthermore, Morphology examination was carried out through SEM and TEM and it has been substantiated that the produced nanocrystals were homogenously distributed with distinctive crystalline morphology. The produced nanocrystals proved significant dissolution rate correlated to marketed formulations and raw drugs.

In the media milling method, coarse suspension of drug was prepared in the polymer and then was recycled in the milling machine (Dena®). Th effect of milling time on particle sizes of the model drugs was evaluated. The resulted particle sizes were 169 nm

± 1.98 with PDI of 0.194 ± 0.04, 298 nm ± 2.00 with PDI of 0.234 ± 0.05 and 161 nm ±

1.90 with PDI of 0.229 ± 0.06 for ketoprofen, dexibuprofen and indomethacin respectively.

Moreover, the stability studies were carried out and the produced nanosuspensions were stable for two months. The crystallinity of the nanoparticles was established by DSC and

PXRD. The Morphological examination was performed on SEM and TEM and it has been observed that the produced nanocrystals were having distinctive crystalline morphology and homogenously distribution. The dissolution studies revealed that produced nanocrystals have significant dissolution rate in relation to marketed formulations and raw drugs.

At the end, this research project demonstrates that model drugs are successfully

produced in nanocrystal form by Microchannel fluidic reactor and media milling methods.

Key words: Ketoprofen, Dexibuprofen, Indomethacin, Microchannel fluidic reactor, media milling, Nanoparticle, Experimental conditions, physicochemical characterization.

Chapter 01

1. Introduction and Literature survey

(a) Challenging Condition

The treatment of a specific disease can be achieved by various therapeutic measures

i.e., chemotherapy, surgical therapy, radiotherapy etc (Recht et al., 1996). The use of

chemotherapy follows the administration of various drugs following various routes. The

most prominently employed route of administration i.e. oral routes is facing myriads of

problems. The only problem which can highly affect the efficacy of a specific drug is the

bioavailability (Wenlock et al., 2003). Moreover, reduced oral bioavailability is main

challenge for the drug to enter pharmaceutical business. This bioavailability depends on

aqueous solubility of the drug because drug should be dissolved or portioned in aqueous

body fluid. Current research proposed that large number of drug acknowledged through

screening process are poorly H2O soluble drugs (Lipinski, 2002). Majority of these active

compounds have affinity of high flow rate through lipophilic membrane but ultimate

absorption from GIT is very slow due to low dissolution rate (Amidon et al., 1995)

Different techniques have been developed to solve the issue of low water solubility.

However, technology of nanoparticle has got high attention of scientist because of high

surface area of the nanoparticle and now become an appropriate platform to address low

water solubility problem. The nanoparticle can be produced by two methods i.e. bottom up

& top down technique (Horn and Rieger, 2001, Rabinow, 2004). These two methods

comprise milling. HPH (Liversidge and Cundy, 1995, Muller and Akkar, 2004) & anti-

solvent precipitation techniques (Bodmeier and McGinity, 1998). Moreover, other methods

such as spray freezing into liquid, Rapid expansion from supercritical to aqueous solution

and rapid expansion of supercritical solution has been also established in last few years

(Rabinow, 2004, de Waard et al., 2010). Above mentioned techniques have different challenging problems e.g. the energy requirement is very large; the process time is very long & the most important one is the nanoparticle growth is uncontrolled. Through this study stable nanosuspension will be prepared with even size distribution. The effect of stabilizer/polymer on nanoparticle growth will be determined.

1.1. Particle Engineering with Special Reference to Crystal

In 1971 Schmidt conducted a photodimerisation reaction in crystalline cinnamic acid & perhaps he used the word crystal engineering for 1st time. Later on it has been defined by different scientist but the Desiraju has given the most up-to-date & complete definition (Desiraju, 2001) which is as under

“The understanding of intermolecular interactions in the context of crystal packing and in the utilization of such understanding in the design of new solids with desired physical and chemical properties”.

The ionic & molecular components of particle have different interaction such as electrical, magnetic etc, which affect the characteristics of particle. These non-covalent interactions can be optimized through crystal Engineering Approach & crystal with appropriate structure can be prepared. In 1962 Hippel comprehensively explained the crystal engineering (von Hippel, 1962). It is clear that Supramolecule of appropriate structure with précis dimension can be obtained through efficient employment of intermolecular H-bonds (Subramanian and Zaworotko, 1995). The Lehn described that atoms & molecules within the molecule are attached with each other as covalent bond & this intermolecular bond chemistry is called supramolecular Chemistry (Lehn, 1988). Now

Many Pharmaceutical & health care sector companies are producing material in crystalline form by utilizing the crystalline Engineering approach(Steed et al., 2000).

The Pharma industries are trying to develop high quality product because they are still having many challenging problems like Drug quality & side effect etc which results in recall of Product from market. Solid dosage form covers 85 % of the product & crystal engineering approach can confirm the quality of these products with ultimate attainment in the market. The Low water solubility also stuck the efficacy of large no of entrant therapeutic molecules. As per scientist reports nearly 70 % of the applicant drugs in development phase, and 40 % of marketed APIs available as oral dosage forms are practically insoluble in water(Shegokar and Müller, 2010). These low water soluble drugs are commonly hydrophobic having more affinity to lipophilic membrane & can easily cross the membrane. Regardless of this affinity of drugs to membrane, its absorption is not fruitful. The only quick dissolution rate can make these drugs effective through oral route

(Amidon et al., 1995). As per Biopharmaceutical Classification System (Figure 1.1

(Amidon et al., 1995) drugs can be classified into four categories. This classification is based on solubility & permeability of the drug. Class-1 compounds have high permeability

& solubility but class-II has high permeability & low solubility. Similarly, class-III has low permeability & high solubility while class-IV has low permeability & low solubility. Class-

II compounds has advantage of high permeability but its low solubility is the big problem because it effects the dissolution rate which consequently effect onset of action. Moreover, it can`t be formulated in parenteral dosage form & hence the low solubility is the chief barrier in new drugs development. So solubility can be enhanced through various techniques & can be utilized orally. The continuous struggle of the drug delivery scientists

have developed and designed a range of strategies to boost the solubility and dissolution of medicines in aqueous media including solubilization (Aungst, 2000), solids dispersions

(Serajuddin, 1999), liposomes (Schwarz et al., 1994), emulsions (Floyd, 1999), micronization (Lawrence and Rees, 2000), micronization (Charoenchaitrakool et al., 2000) and inclusion of complexes cyclodextrins (Loftsson and Brewster, 1996).

Figure I.1: Biopharmaceutical Classification System

All these techniques have different problem therefore crystal engineering technique is used to produce nanoparticle with specific characteristics. These methods include metastable polymorphs, modification at crystal habit, co-crystal design and ultrafine particles (Blagden et al., 2007).

Crystallization:

It can be defined as the practise in which under certain circumstances atoms, molecules, ions gather themselves in 3 dimensional assembly. The critical parameter in the crystallization process is the supersaturation which is the substance concentration in solution relative to equilibrium saturation solubility

푪 Supersaturation ratio = (equation A1) 퐂∗

C= solution concentration

C* = equilibrium saturation solubility

Moreover, in some case e.g. chemical engineering it can be written as

Supersaturation (휟푪) = Css — Ce q (equation A2)

Css = supersaturation concentration

Ceq = equilibrium concentration.

It indicates the large difference among the concentration (conc) will yield into effective crystallization.

When concentration of solute become greater than the molecule equilibrium solubility than crystallization started. It is clear from the figure 1.2 that crystallization started only at supersaturation level. In figure 1.2 there are three regions which are under- saturation, metastable & supersaturation. In under-saturation region, the concentration is very low & crystallization did not occur. In 2nd region, the concentration is beyond the saturation but then again spontaneous nucleation did not occur while crystallization started spontaneously in supersaturation region because conc surpasses equilibrium solubility.

Figure 1.2: diagram of solubility (Garside and Davey, 2000)

In crystallization process, primary phase is the nucleation in which the small bunch is produced & supplemental addition of solute on this bunch yields crystal. There are three types of nucleation which are heterogeneous, homogeneous & secondary nucleation. The nucleation which started spontaneously without any other external molecules is called

Homogeneous nucleation. In such type of nucleation, the no of molecules range is from 10

- 1000 depending upon the supersaturation level. The bunch grows until critical size is attained. In nucleation process the surface free energies are changed along with the phase transformation. This has been investigated by Gibbs (Gibbs, 1928) and Volmer (Volmer,

1939) & summations of changes in surface free energy relate to Phase transformation & nucleus correspondingly.

ΔG= ΔGvol + ΔGsurf (equation A3)

Phase transformation (ΔG)

Nucleus surface (ΔGsurf)

The crystallization method produces the solid particle when supersaturation level is achieved & then addition of solute on bunches occur. The solid phase is more stable as compared to liquid phase so the Phase transformation (ΔG) value decreases & converts into negative which support growth of crystal. However, the nucleate surface has interaction with liquid so the surface free energy of scheme will be increase which retard growth of crystal & support the dissolution of nuclei. This indicates the low value of nucleus surface (ΔGsurf) will support crystal growth & high value will retard its growth with subsequent dissolution of nuclei. Moreover, at critical size (r*) the nuclei surface is little so surface free energy is also less with consequent value of ΔG at maximum level so nucleation started. It can be further cleared from figure 1.3.

Figure 1.3: Alternation in nuclei magnitude effect free energy (Gibbs, 1928)

As compared to homogenous nucleation the heterogeneous & secondary nucleation requires low level of supersaturation. It materializes at less free energy. Normally these

nucleations are encouraged by foreign particles/surface (Marashall, 1987). This external atom(s) /particle(s) has prospective similar construction to internal particle so that less energy is required for nucleation.

Crystal Growth:

Growth in crystal is the critical step in crystal production and can be defined as “the addition of new atoms, ions, or polymer strings into the characteristic arrangement of a crystalline Bravais lattice”.

Growing of crystal occurs at supersaturation level. The molecules of solute move forward from bulk solution near to crystal surface & then deposit on the surface becoming the part of crystal. This process takes place like diffusing process. Basically solute molecules move towards crystal while at same time solvent diffuse out of the crystal & solvation shell region (Figure 1.4) (Dirksen and Ring, 1991b). On the basis of connection/interaction which are possible on the crystal surface, the crystal faces have been categorized into three distinct types of Kinked denoted by (K), Stepped denoted by (S) and

Flat denoted by (F) (Hartman, 1973). Fundamentally, the different faces such as F, S and

K can make single, binary & three bonds respectively (Figure 1.5). As the “K” face of the crystal is the favorite site for bond formation because it can make three bonds at same time.

Growth of crystal can be described by various mechanisms. The few important mechanisms are as under

Convection & Diffusion:

The growth in crystal occurs when a solute molecule reaches to the surface of crystal through diffusion or convection process and become the part of crystal. Similarly, the solvent or solvation shell also diffuses out of crystal surface.

Figure 1.4: crystal growth Mechanism (Elwell and Scheel, 1975, Dirksen and Ring, 1991b) (i) Molecules of solute transfer to crystal surface (ii) surface adsorption, (iii) diffusion (Summ and Evers) step site grouping (v) transmission at step site (vi) Diffusion to crystal frame (vii) Molecules of solvent leave surface of crystal.

Figure 1.5: Imaginary crystal faces (i) flat-F (ii) step-S (iii) kink-K (Dirksen and Ring, 1991b).

Surface Nucleation:

The second mechanism for the crystal growth is surface nucleation. In this mechanism roughness of the surface, imperfection in crystal and bands of growth layers

offer positions for solute molecules to attach with crystal. When the positions for solute decrease then molecules of solute come back towards solution. This position/site can be decreased due to decrease in surface roughness & completion of layer. Additional crystal growth will require new sites, which can be produced by creation of new layer with 2-d surface nucleation Figure 1.6. The nuclei with k face will facilitate the addition of solute molecules. The new nuclei will blowout across surface of crystal & will support the crystal growth evenly.

Figure 1.6: crystal nuclei creation on emerging crystal surface (Garside and Davey, 2000)

Spiral Growth:

The third mechanism for the crystal growth is the dislocation & spiral growth. The dislocation of crystal produces new position for growth. The new emergent outlines of displacement also produce growth patterns. This dislocation may be of screw type, which is very fruitful because it always offers continuous step for addition of solute molecule on crystal surface so produces spiral growth (Figure 1.7). This hypothesis was given by

(Frank, 1949) however later on spiral/screw dislocation was proved by electron microscopy. In 1951 Burton, Caberera and Frank envisioned the theory that screw type

dislocation will produce growth and the growing of crystal will be effected by shape of spiral (Burton et al., 1951).

Figure 1.7: creation of spirals initiating from screw dislocation (Mullin, 2001)

1.2. Phase Transition and Polymorphism

Polymorphism is the phenomenon in which substance exit in more than one (1) crystalline structure and the compound is called polymorphs. The Polymorphs compound has different phases & each phase has a specific characteristic. So presence of polymorphs phase is very important in pharmaceutical product development (Byrn et al., 1994). It is not necessary that all polymorphs forms will be stable. The stable form can be obtained by succeeding transformation & these alterations should follow Ostwald’s rule (Ostwald,

1897). Different factors such as solvent, condition variation at crystallization process and impurities affect the polymorphs form. So, particular polymorphs form can be obtained by specific selection of these parameters. The factor such as presence of specific impurities significantly inhibits the alteration of metastable polymorphs form to stable form. Different material can act as impurities in different condition. For example, Trismisic acid act as impurity in preparation of glutamic acid because it has analogues structure to that of glutamic acid and hence metastable form is obtained instead of stable form of glutamic

acid (P.T. Cardew, 1985). The mechanical & physiochemical proprieties of each polymorph are different (Singhal and Curatolo, 2004). As more than 85 % products of pharma industry have crystalline form of materials (Erdemir et al., 2009). So before product formulation the active & excipients are checked for its polymorphs crystalline structure because the excipient & active of the product are exposed to different condition in product development and each polymorphs of the excipients & active pharmaceutical ingredients have substantial effect on the product characteristics such as dissolution & bioavailability (Zhang et al., 2004). The antibiotic Chloramphenicol has different polymorphs form with different dissolution ant bioavailability rate (Aguiar et al., 1967).

The properties of polymorphs such as high dissolution rate and stability make it highly economical. The conversion of polymorphs from one form to another also takes place during the different process such as grinding, granulation and tableting etc in the product development (Zhang et al., 2004). The research has proved that different factor such pressure, temperature and humidity level causes phase transition of polymorphs materials

(Kitamura, 2002, Moggach et al., 2008). This transformation of polymorphs material is categorized into monotropic and enantiotropic (Giron, 2001). These changes may be reversible (monotropic) and may be irreversible (enantiotropic). When transition takes place from metastable form to stable form due to change in pressure and temperature during different mechanical process like grinding & tableting etc then the transition is termed as monotropic & polymorphs are known as monotrophs. In this type of transformation, the polymorphs substances, which have high melting point, are more stable than those that have low melting point. The reversible phase transition is called enantiotropic transition.

This transition takes place at transition temperature (Tt) and temperature below the

transition temperature (Tt) during the heating procedure will yield A type of polymorphs while temperature above transition temperature (Tt) will yield another type of polymorphs.

1.3. Nanoparticles and Its Method of Production

The particle size is very important in pharmaceutical product because the different characteristics of final product depend on particle size. In this regard, nanoparticles have very minor size and high surface area, which enhances solubility of hydrophobic materials with subsequent escalation in bioavailability (Patravale et al., 2004). The nanoparticles have been defined in different manners by different scientists e.g. the crystalline molecules having at least 1 dimension less than or equivalent to 1000 nm (Fahlman, 2007) while some other scientists claim that crystals materials with below one-micron size and containing drug constituent with least quantity of surfactants essentials for the crystal material stabilization (Gao et al., 2008). Still scientist did not agree on “nano” definition

(Van Eerdenbrugh et al., 2008).

Figure 1.8: Distinguishing features of nanoparticle (Müller et al., 2011).

The distinguishing features of nanoparticle are smallest particle size, high surface area, higher saturation solubility, high velocity of dissolution and cell membrane quick adherence (Müller et al., 2011) as mentioned in (Figure 1.8).

The methods of preparation of Nanoparticle can be broadly classified into two types

The first method is Top Down Technique

The second method is Bottom-up Technique

Through apparatus (e.g, milling apparatus) large particle are cracked & fragmented into smallest units and this procedure is so-called Top Down method (Rabinow, 2004) while in Bottom Up method the atoms are fabricated into molecular size through crystallization process (Patravale et al., 2004). The basic procedure of Nanonization is explained below (Figure 1.9).

Figure 1.9: Methods of preparation of Nanoparticles.

1.3.1: Top down Approaches

This method utilizes the comminution phenomena to produce nanoparticle and comprises different approaches e.g. high pressure homogenization in H2O (Disso Cubes,

Skype Pharma), grinding in media (Nanocrystal R) and combination approach of

Crystallization and High Pressure Homogenization method (Nanoedge, Baxter).

Media milling:

The breaking of particles requires energy and if it is provided by media then approach is called media milling method. In this method we can utilize different medias e.g. zirconium stabilized with yttrium, glass or zirconium oxide, alumina, stainless steel, polystyrene resin (Müller et al., 2001, Liversidge, (1992). Normally this method requires smaller feed size (100 μm) and subsequent product size is also very small (400 nm )

(Liversidge and Cundy, 1995, Merisko-Liversidge et al., 2003, Liversidge, (1992).

Figure 1.10: Media Milling (Merisko-Liversidge et al., 2003)

High Pressure Homogenization (HPH):

In this procedure, the materials (in suspension form) are broken into Nano size by entering into homogenization instrument through high pressure. The instrument has gap of

25 micrometers. When the material along with water is passed with high pressure through small gap, it produces a high shear and cause breaking of particle into Nano size. the particle size is effected by the factors such pressure and shear.

Figure 1.11: High Pressure Homogenization (HPH) (Junghanns and Müller, 2008)

Combined approach:

This method utilizes two practices. First suspension of the material is produced and then through crystallization process nanosuspension is produced along with high-speed stirring. This stirring enhances its stability.

The above mentioned approaches have different merits e.g. preparation of nanosuspensions with different concentration level (low and high) (1 – 400 mg/ml) and also with subsequent produced crystals size of less than 400 nm (Patravale and Kulkarni,

2004b).

1.3.2. Bottom-up Methods

In this method nanoparticle are produced by crystallization process. For this purpose, two solutions are prepared. The first one is hydrophobic material solution which is prepared by solubilized it in organic solvent while polymer solution is prepared by dissolving it in water. Then polymer solution acts as anti-solvent and added to organic solution, which result in production of nanoparticle. Different approaches are available to produce nanoparticle through this method and some of them are as under.

Precipitation by compressed anti-solvent (PCA):

In this approach, CO2 is used in compressed form as anti-solvent. The hydrophobic material & polymer are dissolved in organic solvent in one container while another chamber holds compressed CO2. Then hydrophobic material & polymer solution is inoculated in the chamber which results in the production of nanoparticle (Bodmeier and

McGinity, 1998).

Spray freezing into liquid (SFL):

The hydrophobic material & polymer are dissolved in solvent. Then through atomization process it is sprayed in compressed liquid. Then freeze nanoparticle are produced which is further exposed to lyophilisation process for obtaining solid crystals

(Williams et al., 2002).

Figure 1.12: Spray freezing into liquid. (Hu et al., 2003) [(A: solution cell), (B: high pressure pump), (C: atomizing nozzle), (D: cryogenic liquid cell)].

Evaporative precipitation into aqueous solution (Sepassi et al.):

In this approach, the hydrophobic material is dissolved in organic solvent. The chosen organic solvents have low boiling point. In second container, polymer is solubilized in water & heated. Then hydrophobic material solution will be pushed toward heated channel which will increases the temperature of solution above the boiling point level resulting in spraying of this solution in heated polymer solution. This interaction of organic vapor & aqueous polymer solution will yield nanosuspension (Williams et al., 2002).

Figure 1.13: EPAS process (Sinswat et al., 2005).

All approaches of nanoparticle production have some problems. The basic problem in bottom methods is the growth in crystal size. The basic reason for this growth is high surface area of nanoparticle, which make it thermodynamically unbalanced as compared to large particle with smaller surface area. Due to this instability, these smaller particles will be dissolved is solution and supporting dumping on the surface of large crystal resulting in growth of crystal (Ostwald’s ripening). This problem also results in inconsistent size distribution of nanocrystals, which also affect dissolution rate. To control this Ostwald’s ripening a method has been established in 2004 in which polymer e.g.

Miglyol (a medium chain triglyceride) is used to prevent the growth of crystal (Lindfors,

2004).

All these methods produce nanoparticle but modest and economical approach is the controlled crystallization. In this approach, the critical parameter is the stability of nanoparticle. Moreover, some time another issue also created when drug molecule has little dissolution rate in water and organic solvents.

1.4. Micro-channel reactors (MCR):

A substantial development has been progressively proceeding for processing materials in miniaturization field. In various aspects of our lives, the trend is going to be focused towards smarter and smaller devices. As obvious from the name, microfluidics is a special technique involving minor devices and liquids at micrometers level (Van Der

Woerd et al., 2003). The definition of Microfluidics is (MF) “it is the science and technology in which small amounts of fluids are processed or manipulated in channel with a size range of 10-100µ” (Whitesides, 2006). MF is actually a multidimensional arena of investigation which involve knowledge and contribution in fluidics, material science, microtechnology, physical sciences, chemistry and electromagnetic (Ménétrier-Deremble and Tabeling, 2006). MF is enjoying its infancy stage, as it emerged in the 1990s, and is in progress to develop more and more over the coming few years because of the extensively prominent significance in the various facets including pharmaceutical, chemical and biomedical engineering domains (Ménétrier-Deremble and Tabeling, 2006). Some of the important features of microfluidic reactor are

Microfluidics and laminar flow:

Normally, liquids flow may be turbulent or laminar. It specifically depends upon the ratio between viscous forces to intertia. The relation in this context can be explained by following equation (Weibel and Whitesides, 2006)

pvd 푹풆 = ----- (equation A4) 흁

Re means Reynolds number

P means the Medium density.

V means the flow velocity.

D means the channel diameter.

흁 means the viscosity of the medium

Principally it is clear from that equation that diameter of channel is in directly proportional to Reynolds and MF dimension is small so it produces very low Reynolds numbers. Moreover, in such condition when Reynolds number is very low (< 2000) then viscous forces govern the flow with laminar pattern (Van Der Woerd et al., 2003).

Furthermore, in such a condition if two miscible fluids are moving in microchannel then there will be no eddies or turbulence and fluids will move parallel to each other. The particles of the two fluids will be mixed together by diffusion process. The low value of

Reynolds number with low volume of fluids and micro level of channel positively affects the significant characteristics of the fluids which include high thermal transfer efficiency, high ratios of surface to volume and small diffusion distances (Demello, 2006).

Microreactors and Fluids streams:

The most frequently employed propelling systems in MR have been electrokinetic i.e. the first one is electrophoretic & electroosmotic while second one is the hydrodynamic.

Commonly, fluid flow through electrokinetic mechanism which could be prompted in different channels made of different ingredients and can be charged in lab investigational environments. Fluids which is enclosed will be charged due to possession of ions in higher concentration as compared to counter ions present in bulk fluid. When an electric potential is applied to the walls of channel, the electrodes of opposite charge will attract the charged fluid and consequently convective fluid flow will be observed (Watts and Haswell, 2003).

As far as hydrodynamic pumping is concerned, it uses microscale pumps (conventional)

usually the pumps like syringe which will drive the fluids round the channels network (Fan et al., 2002).

Microfluidic Reactor Construction:

In the very early stages, silicon was most commonly employed for the fabrication of microfluidic devices. Gradually, the attention was diverted towards the glass due to its compatibility with solvent, surface stability and excellent optical properties. However, recently different polymers such as polymethylmethacrylate (PMMA) and polydimethylsiloxane (PDMS) have been extensively used for the fabrication of microfluidic due to their optical clarity and economical preparation (Vanin et al., 2010,

Sommer and Hatch, 2009). In the same way, a technique of soft lithography extensively is utilized for the fabrication of microfluidic systems. Previously in 2004 a detailed study have been reported regarding the microfabrication by soft lithography technique (Song et al., 2004, Kobayashi et al., 2004).

Applications of microfluidics:

(µTAS) Micrometer scale total analysis systems and "lab on a chip" are the additional identical terms used for microfluidics. By using Micrometer scale total analysis systems, all the analyses stages are tackled with the help of chip, which comprises of different stages such as sampling, processing, isolation and finally outcome. By application of other technology i.e. “lab-on-chip”, in very small size such as in square centimeters of chip, a comprehensive lab setup can be launched on it (Figure 1.14).

Figure 1.14: micro-chip.(Schulte et al., 2002).

The exclusive and model characteristics of various devices of microfluidics have gained the attention of users of various fields. Similarly, in chemistry field, miniaturizing reactions can fabricate products with higher purity and relative output in sufficient quantity and short time in comparison to the equal reaction having bulk quantity (Stachowiak et al., 2008).

On-chip diagnosis is made possible by microfluidics technologies and secondly the immediate observation of different infections can be done by taking a minute quantity of various fluids of the body. The described technologies make possible the integration of different types of assays into an isolated device (Lee et al., 2010).

The pharmaceutical field also employ microfluidics. A sound attention has been given on

R&D section of pharmaceutical companies to allocate high amount of budgets to improve the lead discovery and optimize various conventional processes. Microfluidics has been proven to be one of the superior technologies both for the formation of novel substances and for their evaluation against a wide variety of biological targets

(http://www.micronit.com).

Microfluidics and crystallization

For the exploration of different methods and crystallization conditions employing minimum quantity of materials in short duration automatic crystallization processes and miniaturizing may be required (Lounaci et al., 2006). The comparatively consumption of small amount of sample and to process numerous experiments in approximately few cubic centimeters of equipment distinguish microfluidics as a smart technique for the initial screening and also for subsequent instigation of crystallization process, particularly for macromolecules. Additionally, microfluidics systems have the ability to sustain a sealed environmental condition for different period such as from days to months.

Correspondingly, offer a complete admittance for the observation and harvesting of crystals after their growth (Van Der Woerd et al., 2003).

As formerly stated, the reduction of dimension of the reactor obviously results in low Reynolds numbers. Likewise, the microreactor demonstrate laminar flow and molecular diffusion provide a force to the microreactors for mixing (Liao et al., 2013,

Tanthapanichakoon et al., 2006). Similarly, the results of previous investigational study of

Ali and Blagden regarding dye tracer demonstrated that the fluids which are introduced to the microreactors through inlets make two separate streams through the Y shape microreactor, which form a distinctive diffusion layer in channel center (Ali et al., 2009a)

(Figure 1.15).

Figure 1.15: Microfluidic reactor water and Dye (Ali et al., 2009a)

For the crystallization process, anti-solvent is carried out within microreactors, it has been published that the reduction in mixing time occurs in relation to time of induction of material crystallization, which is inevitable requisite to fabricate nanocrystals of even dimensions (Zhao et al., 2007b). In the microfluidics, anti-solvent crystallization, the supersaturation is instigated by the process of diffusion and carried by getting mixed between the anti-solvent streams along the interface and drug solution to give rise to nucleation and growth of crystal. The calculation of supersaturation can be performed as under

S = C/C* (equation A5)

S means ratio of supersaturation

C means concentration of solute

C* means solubility equilibrium

Normally there are two regions, one region has high concentration of drug particles while other region has low concentration. So as per diffusion Fick's law, the flow of material particles takes place from high drug concentrated region to low drug concentration region which results in production of diffusion layer. So due to nucleation

and crystal growth, diffusion of drug particles occurs to compensate the depleted solutes

(Figure 1.16).

Figure 1.16: diffusion process of liquid streams

The explanation of crystallization in a fluids movements along with concurrent interrelating experimental parameters (e.g., diameter of microreactors and drug concentration) is not accessible easily via computational fluids dynamics or statistical methods (Ali et al., 2009a).

1.5. Features of Nanocrystals

Nanocrystals Stabilization:

Each molecule has surface free energy depending upon the surface area. The larger molecules have less surface area so energy is less while smaller particle has high surface area with subsequent high surface free energy. This energy is responsible for agglomeration of particle.

∆G = γs/l. ∆A (equation A6)

γs/l = interfacial tension

ΔA = change in surface area

These two parameters (interfacial tension and change in surface area) play important role in the stability of pharmaceutical dosage form. The decrease in interfacial tension can be done by adding of appropriate surfactant. However, surface area will be increased due production of nanoparticle which may affect its stability (Rabinow, 2004).

The stability of nanosuspension greatly depends on Ostwald ripening (Peters and Muller,

1996). The high surface area and broader size distribution are the factors for Ostwald ripening (Patravale et al., 2004). As per Ostwald Freundlich’s equation the saturation solubility depends upon the particle size i.e. the smaller the particle has high saturation solubility. Due to high surface free energy and high saturation solubility, these smaller particles move towards larger particle making the zone more concentrated and supersaturated with succeeding incorporation into larger particle and hence growth in crystal occur. This movement also causes less saturation of the solution adjacent to smaller particle with subsequent dissolution of smaller particle leading to eradication of this zone.

The Ostwald ripening is still challenge but appropriate selection of polymer and surfactant can produce stable nanosuspension by inhibiting this phenomenon. Ostwald ripening is prevented by two mechanisms

1. Steric mechanisms

2. Electrostatic mechanisms

Steric Mechanisms:

In steric mechanism, non-ionic surface-active agents and polymer are used. These agents make a thin coat on the surface of particle and prevent movement of particle towards each other. When particles having an adsorbed film of non-ionic surface-active agent come close to each other, then concentration of these surface-active agents among particles

increases that increase total surface free energy which results in particles repulsion (Van

Eerdenbrugh et al., 2008).

Electrostatic Mechanisms:

In electrostatic mechanism, ionic surface-active agents and polymer adsorption takes place on particle surface and generates charge on particle surface. The particle movement towards each other is stopped because of similar charges on surfaces, which repeal each other and hence Ostwald ripening is prevented. The DLVO theory has been explained the repulsion of particle with similar charges with subsequent increase in electrostatic energy (Derjaguin, 1941) (Figure 1.17).

The particles dispersed in liquid have two types of interaction. The first interaction takes place among the particle and liquid, which is either attractive or repulsive. While other interaction exits among the particle, which is attractive or repulsive in nature. The attractive forces in these interactions are forces of van der Waals whereas repulsive powers are generated by electrical double. This electrical double layer is produced either by adsorption of ionic surfactants on particle surface or particles itself have charges on surface. At optimum particle distance, equilibrium exit between energy of Lifshitz-van der

Waals (Vlw) and energy of repulsive electrostatic (Lounaci et al.) and making the system stable. The particles when comes close to each other than either van der Waals energy or repulsive electrostatic energy is increased which results in agglomeration or repulsion of particles. Hence, those particles, which attained the requisite Lifshitz-van der Waals energy

(Vlw), will agglomerate with consequent growth of crystal. While, the electrical double layer increases repulsive electrostatic energy (Lounaci et al.) with subsequent failure of particles to attain van der Waals energy (Vlw) and hence no agglomeration occurs.

Figure 1.17: (Classical DLVO theory). Change in particle distance affects the Potential energy of system. Vlw represents energy of Lifshitz van der Waals and Vel represents energy of electrostatic

1.6. Bioavailability

Oral Bioavailability is the major issue of hydrophobic drugs. Orally administered drugs first dissolve in body fluid and then absorb into general blood circulation, which is termed as oral bioavailability. Faster dissolution rate results in high absorption. The dissolution rate of material depends on different factors such as surface area, dissolution coefficient and saturation solubility etc.

푑푐 퐶 −퐶 = 퐴. 퐷 ( 푠 푥) (equation A7) 푑푡 ℎ dc/dt = dissolution rate

A= surface area

D= diffusion coefficient

Cs= saturation solubility

Cx= drug concentration at bulk

H= diffusion pathway thickness

The Noyes–Whitney equation of dissolution specifies that direct proportionality of the dissolution rate with surface area and saturation solubility. As particle size is inversely proportional to surface area. So, the nanoparticle has smaller size and high superficial area which results in high dissolution rate. Furthermore, the surface area also increases the saturation solubility but it is effective when particle size is less than 100 nm. This saturation solubility also increases dissolution rate (Müller and Peters, 1998) . The Freundlich

Ostwald equation specifies that decrease in particle radius will increase saturation solubility e.g. the drug molecule below 100 nm can increase 10 – 15 % saturation solubility while 50 % saturation solubility can be increased by using particle size in range of 2.5 micrometer to 300 nanometer (Müller and Peters, 1998). Böhm and Müller explained the association among different parameter such as particle size, vapour pressure and surface curvature through Kelvin equation (Böhm and Müller, 1999). Consulting to equation it has been proved that high surface curvature has higher vapour pressure. Moreover, it has been reported that conversion of molecules from liquid to gas is like phase transformation from liquid to gas with consequential equivalency of vapour pressure to dissolution pressure.

Hence smaller particle has high dissolution rate (Junghanns and Müller, 2008). Due to high surface area, nanocrystals has acquired prominent consideration of researchers for solving bioavailability problem (Muller, 1999).

Figure 1.18: absorption comparative study of micro and nanoparticle (Mauludin et al., 2008).

1.7. Marketed Products of nanoparticles

In August 2000, FDA approved the Immunosuppressant drug (Sirolimus) in nanoform with trade name of Rapamune® by Wyeth. It was prepared through media milling method.

some of the marketed products are as under.

Table 1:1: Some of the marketed products having Nano-drug (Nijhara and Balakrishnan, 2006, Cheong and Boon, 2010) (Van Eerdenbrugh et al., 2008).

Ingredient FDA Therapeutic Method of Trade name, Approval use preparation Company Year Fenofibrate 2005 hypercholesterolemia HPH hypertriglyceridemia Triglide®

Skye Pharma

Fenofibrate 2004 hypercholesterolemia Media hypertriglyceridemia milling TriCor®

Abbott

Aprepitant 2003 Anti-emetic Media (chemotherapy patients) milling Emend®

Merck Sirolimus 2000 Immunosuppressant Media milling Rapamune®

Wyeth Megestrol acetate 2005 Anti-anorexic Media milling Megace ES®

Par Pharmaceuticals

Paliperidone 2011 Schizophrenia Media Palmitate milling

Xeplion®

Johnson & Johnson

Morphine Sulfate 2002 Norcotic analgesic Media milling Avinza®

King Pharm Nabilone 2005 Anti-emetic Precipitation

Cesame® In chemotherapy

Lilly Diltiazem 2002 Anti-angina Media milling Herbesser®

Mitsubishi Tanabe Pharma

1.8. Non-steroidal anti-inflammatory drugs:

Non-steroidal anti-inflammatory drugs (NSAIDs) have been amongst extensively prescribed and useable medications all around the globe. Having proved and enhanced therapeutic efficacy and clinical outcomes for the treatment of pain, they are advised for a larger portion of world population to prevent the symptoms of rheumatoid arthritis and osteoarthritis (Conaghan, 2012). NSAIDs have found their use more than 5000 years back when the musculoskeletal pain was cured with willow bark. Willow bark contains salicin and it was isolated in pure form from it in the year of 1828. This led to the industrial level production of salicylic acid 1874. Then, ultimately, acetylsalicylic acid (aspirin) was synthesized to enhance the salicylic acid palatability in 1897. Later, ibuprofen and indomethacin were developed as the first non-aspirin NSAIDs in 1969 and 1964, respectively. Moreover, various novel groups of NSAIDs were introduced in the market because of extensive research in the field of NSAIDs. These contained naproxen (NPXN) in 1976 and diclofenac (Diclo) in 1974 (Jones, 2001, Brune and Hinz, 2004). NSAIDs have

been reported to be the most widely prescribed drugs in the world as a single day more than

30 million use them for various purposes (Singh, 2000). In USA, NSAIDs get more than

111 million prescriptions annually. This accounts for about 60 % of the USA over-the- counter (Wu et al.) analgesic market (Laine, 2001, Conaghan, 2012).

NSAIDs have been are particular drugs and indicated for relieving the symptoms associated with common analgesia, inflammation and pain (Vane, 2000). The mechanism of action of NSAIDs was described for the very first time by Vane and Piper in 1971. He found that the biosynthesis of prostaglandins is prevented by NSAIDs due to inhibition of binding of arachidonic acid (substrate) to the active site of COX enzyme. Later, it was confirmed that COX enzyme had two isomeric forms. The Characterization of COX-1 and

COX-2 isoenzyme were done in 1976 and 1991 respectively. The catalysis of prostaglandins production is expressed by COX-1. The prostaglandins have been found involved in many physiological functions. These functions include maintaining the renal routine renal function of kidneys, protection of mucosa in GIT and pro-aggregator thromboxane A2 in the platelets. On the other hand, COX-2 expression is induced through the cytokines and many other inflammatory mediators that are present in various tissues like endothelial cells. It has been found to play a vital role in the pain mediation fever an inflammation (Vane and Botting, 1998, Rao and Knaus, 2008). The presence of a third isomer of COX “COX-3” is also speculated. This is believed to elucidate the possible mode of action of acetaminophen, having a very narrow poor inhibitory effect on both COX-1 and COX-2. The Join alternates of COX-1 and COX-2 have been established and denoted by COX-3 however, its humans relevancy is considered low (Davies et al., 2004).

Based on their selectivity for COX inhibition, pharmacological properties and chemical nature, NSAIDs are classified in many different groups or classes. Owing to their chemical nature and molecular patterns, NSAIDs have to share some properties like weak acidity and their lipid solubility. They have found to reveal enhanced bioavailability when taken orally. They are reported for their well-established absorption from gastrointestinal tract. They have also been reported for their decreased hepatic clearance. On the other hand, different NSAIDs have different rates of absorption. This can be related to the appropriateness of different NSAIDs for a specific diseased condition (Tarţău et al., 2012).

Another prevailing classification of NSAIDs is based on their half lives in the in-vivo biological system. They may be having 6.0 hours (hr) half-life (short half-life) or having biological half-life long. Half-life based classification has been helpful in deciding the dosing for a specific NSAID. An NSAID having short half-life is usually given at every 6–

8.0 hr, while an NSAID with long half-life is administered once or two times a day. To achieve an instant therapeutic response, NSAIDs are expected for a rapid absorption so to have a rapid relief. But in case of chronic conditions, NSAIDs are preferred to have not a rapid absorption as they are expected for long term therapeutic efficacy (Rainsford, 2009,

Conaghan, 2012).

1.8.1. Ketoprofen

Ketoprofen (keto) is a NSAID that belongs to the substituted 2-phenylpropionic acids group (Ungprasert et al., 2016). The well-known pharmacological uses of this drug include uses in chronic and acute inflammatory diseases, rheumatoid arthritis, spondylitis, osteoarthritis and severe abdominal pain/cramps linked with menstruation

(Kantor, 1986). It is having a molecular weight (mol wt) of 254.29. It is crystalline solid with Molecular formula (mol f) C16H14O3. Its melting point is 93 – 96 °C. it is freely soluble in acetone, in ethanol, in methylene chloride and practically insoluble in water (Yalkowsky and Dannenfelser, 1992).

For the very first time, RhBne-Poulenc Research Laboratories, Paris synthesized ketoprofen in 1967. Its clinical use in various countries such as in France and United

Kingdom was approved in 1973. Now, throughout the world, it is available in many dosage forms like injections, topical gels and suppositories. In United Kingdom, it is marketed with trade name (Oruvail®) which is controlled-release capsules. Moreover, the safety profile of the drug is well established and much better than aspirin in controlled studies. In

1986, it was approved in capsules dosage form for the conditions of rheumatoid arthritis and osteoarthritis (Kantor, 1986).

It is well established for all NSAIDs, that therapeutic activity of the Keto is believed to achieved due to its interference with the metabolism of arachidonic acid. It has been proved that at plasma levels (EC50 2 μg/L) inhibitory effect on cyclo-oxygenase is utmost powerful. When compared to naproxen it was 6 and 12 times more potent inhibitor of prostaglandin synthesis respectively when used in animal model. It is also more potent than aspirin and phenylbutazone. The ranking of NSAIDs continued same regarding its anti-

inflammatory effect and shows its connection with inhibition of prostaglandin synthesis

(Kubota et al., 1979).

Results of various other studies have reported it as potent inhibitor of prostaglandin synthesis and (Kubota et al., 1979, Higgs et al., 1980, Kantor, 1986).

Besides the above mentioned properties, keto has the pharmacological actions which is pertinent to analgesic and anti-inflammatory activity. For example, it inhibits the bradykinin which is significant pain and inflammation mediator. Similarly, the lysosomal membranes are stabilized by it against osmotic damage. Moreover, it has been found to prevent the lysosomal enzymes release that are held responsible for the mediation of tissue destruction in inflammatory reaction (Migne et al., 1976, Smith, 1978).

Ketoprofen is especially prescribed for treatment of inflammatory nerve pains. Its metabolizes in liver and conjugate with glucuronic acid and reduces its function. keto is used f to tor its anti-inflammatory, analgesic and antipyretic properties. It has been reported that within 0.5 – 2.0 hr it binds to plasma proteins in a ratio of 99 %. Ketoprofen elimination from body is mainly made by renal route, the half-life (1.0 – 4.0 hr) depending on the administration method (Tarţău et al., 2012).

Keto as a prototype API has been encapsulated in to solid lipid nanoparticles which were prepared from a mixture of carnauba wax and beeswax and used emulsifiers were

Tween 80 and egg lecithin. Moreover, the investigation of characteristics of the solid lipid nanoparticles with various surfactant and lipid composition were performed. Solid lipid nanoparticles with size of 75 ± 4 nm and PDI of 0.2 ± 0.02 were achieved by employing

1 % of surfactant mixture at proportion of 40:60 of egg lecithin to Tween 80. The zeta potential of these solid lipid nanoparticles varied from –15 to –17 (mV) which suggested

the presence of analogous border characteristics. 97 % of Keto has been efficiently entrapped which recognized the encapsulation of poorly water soluble drugs by SLNs. It has also been demonstrated that nanoparticles has quicker drug release in formulation having more beeswax as compared to carnauba wax (Kheradmandnia et al., 2010).

The delivery of Ketoprofen performed in oleic acid modified polymeric bilayered nanoparticles in combination with spantide II which represents combined drug delivery system. The preparation of nanoparticles were performed using chitosan and poly(lactic- co-glycolic acid). The particles’ surfaces were transformed with oleic acid and were administered via skin. The quantity of ketoprofen released from nanoparticles, which remained in the dermal layers exhibited increased permeation from nanoparticles in comparison with the plain drug (Shah et al., 2012).

One of the previously conducted studies outlined the effects of interactions between various acrylic polymers and a model drug. The effects were evaluated on nanoparticle physical characteristics which was fabricated through an aerosol flow reactor procedure.

The quantity of model drug i.e., ketoprofen, in the nanoparticles was variable and the nanoparticles were screened for particles morphology, particle size distribution, IR spectroscopy, thermal properties and the release of drug. The nanoparticles which were obtained were spherical in shape and had amorphous and matrix-type structure. The crystallization of ketoprofen was noted after getting the amount of drug more than 33 %

(w/w) in Eudragit L nanoparticles. The transition of glass of the polymer was lowered by the plasticizer effect of drug worked in Eudragit RS and Eudragit E nanocrystals. The nanoparticles fabrication in the aerosol flow reactor method was effected by the thermal

properties of the resulting drug-polymer matrix and the solubility of the drug in the polymer matrix (Eerikäinen et al., 2004).

In the same way one of the previous studies conducted on the preparation of gels of ketoprofen nanoparticle obtained through evaporative precipitation into aqueous solution. For the preparation of nanoparticle of poorly water soluble drug i.e., ketoprofen, liquid droplets of surfactant stabilized ketoprofen which contained residual solvent were thoroughly dispersed in aqueous medium with temperature 60 to 90 °C, which was lower than the melting point of ketoprofen in pure form (Chen et al., 2006).

It has been reported that Francesco et al established novel polyoxyethylene esters of naproxen, ketoprofen, and diclofenac and also assessed them as possible prodrugs of

NPXN, Keto and Diclo in dermal administration. Such esters were acquired by the combination of these drugs with polyoxyethylene glycols with the help of a succinic acid spacer. In-vitro studies were conducted on excised human skin to evaluate the permeation of above mentioned prodrugs. Moreover, they were also evaluated for the topical anti- inflammatory effect in-vivo. The in-vivo results are very significant and good as compared to in-vitro study and demonstrates effective inhibition of methyl nicotinate(Atemnkeng et al., 2007) and also proved earlier in investigation on healthy human volunteers that it produces skin erythema (Bonina et al., 2001).

Likewise, in another report, for topical drug delivery system, multilamellar vesicle liposomes comprising of complex of keto – cyclodextrin were formulated, to exploit the favorable characteristics of mutually carriers simultaneously. Complexes of Drug with hydroxypropyl-βCyd and β-cyclodextrinb produced by combined evaporation and sealed-

heating methods have been characterized by DSC, SEM, hot stage microscopy. Moverover dissolution properties were also investigated. The encapsulation of drug complexes was performed in liposomes and characterized for particle size, encapsulation efficiency employing transmission electron microscopy, dialysis and light scattering techniques. The obtained results verified the deep permeation of the contents into the dermal layers

(Maestrelli et al., 2005).

Similarly, in another investigational study, a new drug delivery system which was based on drug cyclodextrin complexation and which were loaded into nanostructured lipid carriers (NLC) has been prepared to ameliorate the therapeutic efficacy of ketoprofen.

Cyclodextrin was used for its certain properties i.e., stability, solubility, its prolonged release profile, percutaneous absorption enhancing properties of NLC and high tolerability.

The NLC system loaded with Ketoprofen, formulated into a xanthan hydrogel, showed the better drug permeation properties than the plain drug-loaded NLC or plain drug suspension, in virtue of the simultaneous exploitation of the solubilizing properties of cyclodextrin and the penetration enhancing characters of NLC (Cirri et al., 2012).

In the same way, naproxen and Ketoprofen loaded lipid nanoparticles were formulated, using ultrasonication and hot high pressure homogenization techniques, and characterization were performed via differential scanning calorimetry and photo correlation spectroscopy. The behavior of nanoparticle on dermal layers was evaluated, in- vitro, for the determination of drug percutaneous absorption and, in-vivo, for the development of controlled release and active localization abilities. Likewise, an extended anti-inflammatory effect was noticed in the drug loaded nanoparticles with respect to the drug solution (Puglia et al., 2008).

1.8.2. Dexibuprofen

Being one of the important NSAIDs, Ibuprofen is prescribed in many inflammatory conditions like arthritis, rheumatism and mild to moderate pain, fever, primary dysmenorrhea and other various patho-physiological situations (Hao et al., 2005). It is

Crystalline powder. Its mol f is C13H18O2 and mol wt is 206.28. Its melting point is 50 – 53

℃. It is Soluble in DMSO, ethanol, dichloromethane, ethyl acetate, and methanol and insoluble in water (Leising et al., 1996). The drug therapeutically efficacy is greatly dependent upon its water solubility. It is poor water soluble drug (i.e. practically insoluble) with 4.65 value of Pka. So as per Biopharmaceutical Classification it is recognized as class

II drug. After administering through oral route, Dexibuprofen (DXI) absorption takes place in gastrointestinal tract. Hence, the its bioavailability is decreased in the acidic pH of the gastric environment due to its less aqueous solubility. Its bioavailability can be enhanced by increasing its aqueous solubility (Abdelbary and Makhlouf, 2014, Adams et al., 1976)

It is a racemic mixture of S(+) ibuprofen (Dexibuprofen) and R(-) Ibuprofen

(Bonabello et al., 2003). DXI is in-vitro and in-vivo active form of ibuprofen. There has been a proper mechanism in the body that converts the isomers from R to S very slowly

(Hao et al., 2005). Thus, dexibuprofen has been the rapidly acting and is advised to be used in a decreased quantity as compared to its racemic mixture (Kumaresan, 2010). It is also used for the treatment of mucosal gastric damage that is usually caused by the excessive administration of ibuprofen (Abdelbary and Makhlouf, 2014).

It is chemically a 2S-2[4-(2-methyl propyl) phenyl] propanoic acid. Still now, dexibuprofen is not exactly known. It is believed that it achieves its anti-inflammatory action through the inhibition of both COX-1 and COX-2, ultimately leading to the

prevention of the prostaglandin synthesis. Its antipyretic activities can be due to its action on the hypothalamus, leading to enhanced flow of peripheral blood, vasodilatation and ultimately heat dissipation.

The dexibuprofen has gone through various drug delivery investigational studies.

The hydrotropic solubilization procedure was applied for the enhancement of solubility of dexibuprofen.

Actually, the dexibuprofen is literally and practically among the water insoluble

NSAIDs, which possess better anti-inflammatory effect than ibuprofen. For the improvement of dissolution rate and water solubility a mixed hydrotropic solubilization method was employed. Using various conc of hydrotropic materials including urea and sodium citrate dehydrate and nine formulation were formulated. The odor and color of prepared formulae were screened visually and physically. Micromeretic properties, hygroscopicity, pH and solubility for the 1% aqueous solutions were figured out. For in- vitro dissolution USP method type I was used. The nine formulation was assessed through different techniques including DSC, SEM and, infrared spectroscopy. The results obtained demonstrates that formulations were having white color and slightly hygroscopic property but having no odor. The formulae which contained higher amount hydrotropic agents demonstrated a rise in pH. It also showed enhanced solubility and increased amount and rate for dexibuprofen release from dissolution medium (El-Houssieny et al., 2014).

Dexibuprofen has been designed for topical application in the form of emulsion based gel. The dexibuprofen-loaded and ibuprofen-loaded emulsion gels have been fabricated with different excipients such as isopropyl alcohol, propylene glycol, isopropyl myristate, carbopol and Tween 80. Their mechanical properties i.e., adhesiveness and

hardness were evaluated. Furthermore, their dermal permeation, anti-nociceptive and anti- inflammatory potential were assessed by means of Franz diffusion cell with the hairless mouse skin, paw pressure test in rat's hind paws and the carrageenan-induced paw oedema test respectively and then its comparison was made with marketed hydrogel. The emulsion gels of ibuprofen and DXI provided enormously higher adhesiveness and hardness than those of the commercial hydrogel. The DXI emulsion gel was more effective while having

3.5 folds and 2.0 folds high permeability as compared to marketed and ibuprofen gel respectively. This results demonstrats quicker skin permeation. Furthermore, alleviation in carrageenan-induced inflammation was having a descending order i.e., DXI emulsion gel

> commercial hydrogel > ibuprofen emulsion gel. The emulsion gel of DXI furnished expressively higher nociceptive thresholds than emulsion gels of ibuprofen and commercial hydrogel, which led to the utmost enhanced analgesic efficacy (Jin et al.,

2015).

One of the study also employed sucrose to develop furbiprofen loaded nanoparticles. nanoemulsion of flurbiprofen-loaded system have also been reported to give narrow size distribution to uniform emulsion droplets, when prepared by membrane emulsification method. In the study, the nanoemulsion was solidified for the preparation of nanoparticle and the sucrose was used as a carrier in spray drying procedure. The study was conducted to enhance the bioavailability and obtain narrow size distribution. The characterization was carried out via DSC, PXRD and SEM. The bioavailability studies were carried out using rats as experimental animals. The nanoparticles of flurbiprofen prepared showed excellent solidification with no stickiness. The drug solubility was improved approximately 70000 fold for the nanoparticles having AVG P Size of 300 nm.

The DXI crystallinity was lost and converted into amorphous state. Similarly, a significant with higher AUC, Cmax and smaller Tmax was exhibited by the nanoparticles obtained

(Oh et al., 2014).

Similarly, other investigators have reported the preparation of liposomal hydrogel for topical applications of dexibuprofen. The prepared liposomes were examined for diffusion study, particle size measurement, skin permeation study, percent drug entrapment and in vivo study. The study revealed satisfactory fruitful attempt to prepare and evaluate liposome of liposomal gel and dexibuprofen for the provision of sustained delivery of drug.

From the dermal permeability investigations and in vivo study it has been deduced that the prepared liposome of dexibuprofen may be proved as potential candidate for effective and safe sustained drug delivery for a prolonged time, which may be able to decrease dosing frequency (Wasankar et al., 2012).

By using the wet granulation method, mini-matrix tablets containing dexibuprofen have been formulated. The different excipient such as xanthan gum or karaya gum hydroxypropylmethylcellulose (HPMC), Avicel® PH101, talc and Lubritab® lactose,

Encompress® were used to prepare hydrophilic matrix. The mini-matrix tablets were incorporated into hard gelatin capsule shells to obtain Multiple unit dosage forms

(MUDFs). The in-vitro release profile, preparation and release kinetics have also been studied (Cox et al., 1999)

1.8.3. Indomethacin

Being an important member of NSAIDs, indomethacin is also used for effective therapeutic efficacy in various conditions like pyresia, analgesia and inflammation.

Chemically, it is an NSAID of indole-acetic acid derivative. Its chemical name is 1-(p- chlorobenzoyl)25-methoxy-2-methylindole-3-acetic acid (Lucas, 2016). It is commonly used for the management of rheumatic diseases and dermatitis in patients.

It is pale-yellow to yellow-tan, crystalline powder with odorless, or has slight odor

(Osol and Remington, 1980). Its mol f is C19H16ClNO4 and mol wt is 357.787. It is soluble in ethanol, ether, acetone and practically insoluble in water (Budavari, 1989) (Budavari,

1989). Its melting point is 159 – 162 °C Indomethacin is classified as class-II drug owing to its deprived aqueous solubility and increased permeability (Löbenberg and Amidon,

2000). Such drugs always demonstrate decreased absorption and bioavailability, so their dissolution rates and aqueous solubility should be enhanced (Hirasawa et al., 2003). The development of such drugs into efficient dosage forms has been a challenging task for formulation scientists. The case with indomethacin is not different from other such drugs.

It also shows a nonlinear pattern of its bioavailability owing to its poor aqueous solubility.

The deceased solubility of indomethacin is also expected for various side effects like irritation of gastrointestinal tract by virtue of its contact with the mucosa for an extended period of time (Alsaidan et al., 1998).

The indications of NSAIDs, and in particular indomethacin, include arthritis, fever, various headache syndromes, and dysmenorrhea. Indomethacin is also used for closure of patent ductus arteriosus (Heyneman et al., 2000).

Like many other drugs of COX inhibitors, indomethacin has an increased bioavailability of about 98 %. It is mostly metabolized in liver having plasma albumin binding of about 90 %. It achieves its maximum blood plasma concentration after two hours of its administration, which is two short. Its half-life ranges between 3.0 – 10.0 hr (Summ and Evers, 2013, Alván et al., 1975). Its administration through oral route has been advantageous, but also have some complications which include ulceration in the mucosal layers of gastrointestinal tract and the irritations. Moreover, it requires rapid dosing due to its decreased elimination half-life (Helleberg, 1981).

The indomethacin has also undergone various drug delivery studies in which a combination of supercritical CO2 system was used for the preparation of its nanoparticles.

In this study, an improved system was used to overcome the problems associated with decreased solubility of medicinal agents in CO2. The spraying of ethanol solution into a vessel of CO2 was performed, there was no precipitation of IND in the case of CO2 pressure above 15 MPa at 40 oC. This indicated that small droplets of ethanol solution can get dispersed in the high pressure CO2. Secondly, using RESAS device for expansion into distilled water, the same solution gave rise to submicron particles of IND. The SEM images recorded the size of submicron particles to be 300 to 500 nm. The apparatus used in this study, showed an excellent way for the production of IND fine crystals having a high throughput. The IND was undergone various other dissolutions and bioavailability studies using different experimental animals which has been demonstrated to have significant results for nanoparticles as compared to the other techniques (Tozuka et al., 2010).

The literature survey of indomethacin also reveals that this drug has also been undergone through various steps for the enhancement of dissolution and gastric irritancy.

The nanoparticles of Indomethacin has been incorporated into 3D ordered macroporous silica in order to reduce its gastric irritancy and enhance its dissolution. The 3D order silica was employed as a matrix for the nanoparticles of drug for getting control over the size of particles the range of submicrometer, to increase its dissolution rate and minimize the chances of damage to the gastric mucosa. In the matrix of silica the pores were successfully created having size of 200 nm and similarly loaded with indomethacin nanoparticles with various ratios. The profile observed for nanoparticles were rapid in comparison with the micro-sized indomethacin and other commercially available capsules. These characters results in particle surface area increase and the decreased chances of crystallization as well.

Similarly, the decreased chances of damage to the gastric layer was also demonstrated which were attributed to the decreased particle size and encapsulation of drug within 3D matrix of silica gel (Hu et al., 2011).

Through solvent evaporation in micro-fluidization technique Polymeric nanosuspensions containing indomethacin were produced. Different characteristics of the produced nanosuspensions were assessed which include particle size, drug concentration, polymer drug crystallization in the aqueous phase, stability and in-vitro drug release.

Within the polymer phase, more than 98 % of the drug has been found. Within

, the rapid release of indomethacin from ethyl cellulose nanoparticle was noticed.

The nanoparticles which were prepared in intention to provide a prolonged drug release were obtained by employing mixtures of poly methyl methacrylate and ethyl cellulose. The nanosuspension was stabilized by nonionic, anionic, and macromolecular stabilizers at pH of 7.4 while in 0.1 N HCl, the nonionic surfactants were observed as good stabilizers

(Bodmeier and Huagang, 1990).

In another investigational study, the preparation nanosuspensions containing indomethacin were carried out by combined approach i.e. the precipitation-ultrasonication method. The stabilizer used were proteins of food (whey protein isolate, β-lactoglobulin, soybean protein isolate). Dried nanosuspensions were formulated via employing covering of nanosuspensions on top of pellets. The investigation carried out were drug redispersibility, dissolution, morphology and solid-state forms. The stabilization of mean particle size of nanosuspensions were attained by exploiting soybean protein isolate (588 nm), whey protein isolate and (320 nm). The layering of nanosuspensions onto pellets could be successfully performed with high covering efficiency. Similarly, the original nanosuspension and its conversion into dried form retain its particle size and were not affected by fluid-bed coating drying process. Both the original nanosuspension and dried nanosuspensions demonstrated similar dissolution profiles which have been observed to be more quicker than that of the unprocessed particles (He et al., 2013).

Moreover, in another study, the examination was performed for the usage of granules of chitosan as a means to achieve sustained release of indomethacin. Dissolution of powdered and chitosan granules were compared and it was found that sustained release was observed in chitosan granules. A distinguishing characteristic of the chitosan granules was that these granules demonstrates gradual swelling and floating on medium having acidic pH of 1.2. As the chitosan granules has floating characteristic in acidic environment so it can be utilized for different drug formation in sustained-release pattern. The assessment of chitosan granules was done for cross-linking process effects on the drug release pattern. The alteration of the cross-linking process can adjust drug release pattern

(Hou et al., 1985).

The oral indomethacin-HP-Bcyclodextrin loaded chitosan nanoparticles has also been previously prepared and evaluated both in-vivo and in-vitro. As the decreased solubility and the irritation on the gastric line of oral indomethacin greatly affect the therapeutic potential of the indomethacin. The study was designed to enhance the indomethacin solubility via complex formation with hydroxyl propyl dextrin (HP--

CD). The drug was incorporated on the chitosan nanoparticles which has been formed via ionic gelation for the control of release of the drug and enhancement of permeability. The chitosan nanoparticles were coated with alginate for avoidance of release of drug in the stomach, which will lead to decreased contact with the lining of the stomach. By increasing the concentration of HP--CD the solubility of indomethacin was increased with direct proportion. This also provided excellent entrapment efficiency and narrow particle size distribution to the indomethacin. The cumulative release of indomethacin in pH of 1.2 was practically 0 % with better oral drug delivery profile by excellent release of the drug at pH of 6.8. A higher anti-inflammatory effect was demonstrated by the said load nanoparticles and better analgesic potential as well, in comparison with the drug in the market (El-Feky et al., 2013).

The solubility and rate of dissolution of indomethacin have also been enhanced via solid dispersion in PEG 4000 and Gelucire 50/13. The preparation of dispersion system was performed by hot melting method at 1:4, 1:2 and 1:1 of drug to the polymer ratio. For the examination of physical state of the indomethacin, DSC, SEM and XRD were employed. The XRD data exhibited that the drug in solid state was still easily detectable.

The prominent alteration in the melting peak of indomethacin was clearly sorted out with the help of DSC. The whole study demonstrated that the release of indomethacin was

prominently faster from solid dispersion in relation to pure drug in crystalline form. This also revealed that by increasing the amount of polymer in the formulation, the dissolution rate was increased in direct proportion (El-Badry et al., 2009).

Similarly, indomethacin in an ophthalmic delivery system constructed on the perception of ion activated in situ gelation have also been reported. Gelritgellan gum which is a novel vehicle for ophthalmic preparation has been used as gelling agent. This agent has the property that in existence of mono or divalent cations it gels out. These cations exist in the fluid of the eye i.e. lacrimal fluid. The produced preparations were having in drug vitro release in sustained manner over a period of 8.0 hr. These preparations were therapeutically effective and it was based on activity on uveitis induced rabbit eye (Balasubramaniam et al., 2003).

One of the previous studies was carried out to increase the dissolution rate of indomethacin employing liquisolid compacts. The study was conducted for the indomethacin for the improvement of dissolution rate, which is practically insoluble.

Different non-volatile co-solvents were used in the formulation studies of indomethacin.

In all the formulations i.e., 20 formulations the proportion of carrier (microcrystalline cellulose) to coating powder material (silica) was calculated. For the interaction of indomethacin and other components in liquisolid formulations DSC was employed. In comparison to the direct compression method the study demonstrated excellent drug dissolution rate in liquisolid formulation system (Saeedi et al., 2010).

Indomethacin preparation in micelles was prepared by dialysis method through

Amphiphilic diblock copolymer. The copolymer composed of ε-caprolactone (ε-CL) and

methoxypoly (ethylene glycol). The micelle size achieved was less than 200 nm and shows narrow and monodisperse unimodal pattern of size distribution. Dialysis method produced micelles of spherical shapes. The polymer/indomethacin weight ratio was 1/1 and copolymer molecular weight was about 12 000 Dalton (Da). The Nano spheres ware loaded with approximately 42.2 % indomethacin content. The release pattern from Nano sphere was sustained for the period of 15 days and release rate is dependent on drug content entrapment and copolymer mol wt. These reported results indicates that Nano spheres loaded with drug can be used in parenteral dosage form as a novel drug carrier for lipophilic medicine (Kim et al., 1998).

Chemical structures of the selected drugs are as under

A. Ketoprofen.

B. Dexibuprofen

C. Indomethacin

Figure 1.19: Chemical structures of drugs (A) Ketoprofen (B) Dexibuprofen (C) Indomethacin

1.9. Aims & Objective

The issue of poor bioavailability erected from poor water solubility for a list of drug candidates has become a hard challenge for the drug delivery scientists both from academia and research and development sectors. Regarding the resolution of this issue of poor water solubility, the drug delivery scientist has made continuous struggle and developed and designed a range of strategies and various techniques however, among all these strategies and techniques, nanoparticles have been acknowledged distinguishably. In the current study, all the three selected compounds (ketoprofen, dexibuprofen and indomethacin) are insoluble in water which resulted in erratic bioavailability and low absorption. This issue could be addressed by increasing solubility of these drugs by formulating into nanocrystals.

Nanocrystals can be prepared by bottom up method and top down method. The basic goal of this research project is to produce nanosuspensions of the selected drugs by both methods. This research project was aimed to evaluate the use of microfluidic channel reactor to produce nanosuspension of the selected drugs. Yet, this technology is new and needs to be employed for a number of active pharmaceutical ingredients (Hu et al.) to get benefit from this method. The objective included to reveal the imperative parameters such as microfluidic channel reactor inlet angles, solvent and antisolvent flow rates at different level, scalability, mixing time, sonication, polymers and drug concentration effect. This study also aimed to produce nanosuspension of the chosen drugs through media milling method and to investigate the impact of milling time on production of nanoparticles. As nanoparticles during antisolvent crystallization, are produced by controlling the nucleation process. However, in milling process the attrition and impaction forces are involved to reduce the particle sizes with increase of high surface free energies. Both methods has

advantages and disadvantages such Bottom method needs low energy while Top down method requires high energy however, industries normally utilizes top down method (Van

Eerdenbrugh et al., 2009). The characterization of the nanoparticles produced through microfluidic reactor method and media milling method includes particle size, zetapotential, powder X-ray diffraction (PXRD), Differential scanning calorimetery (Byrappa et al.),

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Then the Comparative in-vitro bioavailability study of the produced nanoparticles with the available marketed drug formulations and with the unprocessed drug.

Chapter 02

A. Preparation of Ketoprofen, Dexibuprofen and Indomethacin nanosuspension through Microchannel Fluidic Reactor

2. Materials and Method

2.1. Materials

 Ketoprofen (Batch no: ILKT 140005 infinity lab India),

 Dexibuprofen (Batch no: C102-150100M, Hubei biocause Helen Pharmaceutical

Co Ltd. China),

 Indomethacin (Batch no: BCBP0623V Sigma-Aldrich, UK)

 Poloxamer 407 (Batch no: BCBK50056 Sigma-Aldrich, UK)

 Polyvinylpyrrolidon K30 (PVP k30) (Batch no: 08297052G0, BASF,

Ludwigshafen, Germany)

 Hydroxypropylmethylcellulose viscosity; 15cps (HPMC 15cps) (Batch No:

8028213, Shin-etsu-Japan Chemical Ltd)

 Sodium dodecyl sulfate (SDS) (Batch no: MKBR3557V, Sigma-Aldrich, UK)

 Laboratory Distilled water was acquired at University of Bradford Research

laboratory.

 Other regents used were of analytical grade.

2.2. Method

2.2.1. Preparation of Ketoprofen, Dexibuprofen and Indomethacin Nanosuspension

The nanosuspensions of ketoprofen, dexibuprofen and indomethacin were produced through modified microchannel fluidic reactor (Bonabello et al., 2003) method.

The hydrocortisone nanoparticles reported by (Ali et al., 2009b) were produced by MCFR in which the crystallized nanoparticles were mixed with the buffer solution composed of polymers using stirring and sonication. However, in this new method of MCFR, we used polymeric solution during mixing of the drug solution in microchannel fluidic reactor as well as after processing the nanosuspension. Crystallization of the drug in plain water and polymeric medium is substantially different from each other. In polymeric medium the nucleation and particle size is controlled by polymer molecules which results in low particle size and homogenous size distribution (Khan et al., 2013b).

In this method, Drugs were dissolved in ethanol and polymers dissolved in aqueous phase were introduced into microfluidic channel reactor. Then emergent nanosuspension was poured into vial having aqueous polymer solution and stirred for adequate timing.

Finally, the nanosuspension was sonicated for few minutes.

The batches of for ketoprofen and dexibuprofen were 3.5 ml in which 2.0 ml polymer solution and 0.5 ml drug solution were pumped into MCFR and 1.0 ml polymer solution was kept in vial. Similarly, batches of indomethacin were produced in 4.0 ml in which 2.0 ml polymer solution and 0.5 ml drug solution were pumped into MCFR and 1.5 ml polymer solution was kept in vial.

Figure 2.1: Microchannel Fluidic Reactor

The scalability for ketoprofen and dexibuprofen was checked for 10.5 ml, 21 ml,

49 ml and 98 ml while for indomethacin the scalable perimeter used was 12 ml, 24 ml, 56 ml and 96 ml.

The microfluidic channel reactor with different inlet angle such as 10°, 25° and 50° were used. However, internal diameter used was 0.5 mm.

The mixing times for ketoprofen and dexibuprofen were 30 minutes, 30 minutes and then 5 minutes sonication, 60 minutes and 60 minutes and then 5 minutes sonication.

The mixing times for Indomethacin were 30 minutes, 30 minutes and then 15 minutes sonication, 60 minutes and 60 minutes and then 15 minutes sonication. The stirring speed for mixing was sustained at 1200 rpm.

A range of different polymers were investigated for ketoprofen and dexibuprofen which include Poloxamer 407 with concentration of (0.5 %) and (1 %), PVP k30with concentration of (0.5 %) and (1 %), HPMC 15cps with concentration of (0.5 %) and (1 %),

combination of polymers includes Poloxamer 407 – PVP k30 with concentration of (0.5

%-0.5 %), (1 % - 0.5 %) and Poloxamer 407 - HPMC 15cps with concentration of (0.5 %

- 0.5 %), (1 % - 0.5 %). Another combination of PVP k30 - HPMC 15cps with concentration of (0.5 % - 0.5 %), (1 % - 1 %) and additionally (1.0 % - 0.5 %, 0.5 % - 1.0

% for dexibuprofen only) was also investigated. For Indomethacin, different polymers were investigated which include Poloxamer 407 with concentration of (0.5 %) and (1 %), PVP k30 with concentration of (0.5 %) and (1 %), HPMC 15cps with concentration of (0.5 %) and (1 %) and combination of polymers includes PVP k30 - HPMC 15cps with concentration of (0.5 % - 0.5 %), (1 % - 1 %), Poloxamer 407 - PVP with concentration of

(0.5 % - 0.5 %), (1 % - 0.5 %) and Poloxamer 407 - HPMC 15cps with concentration of

(0.5 % - 0.5 %), (1 % - 0.5 %). The SDS combination with PVP k30 and HPMC was also investigated for Indomethacin and include PVP k-30-HPMC 15cps - SDS with concentration of (1 % - 0.5 % - 0.5 %), (1 % - 0.5 % - 0.1 %), (1 % - 1 % - 0.5 %), (0.5 %

- 0.5 % - 0.5 %).

The effect of drug concentration was also examined. The drug concentration used were (5 mg/ml, 10 mg/ml and 15 mg/ml).

Moreover, effect of flow rate of antisolvent and solvent was checked in three different level. In first level the antisolvent was variable and solvent was constant and ratio include 0.5:0.5 ml/min, 1.0:0.5 ml/min 1.5:0.5 ml/min and 2.0:0.5 ml/min. In second level the antisolvent was constant and solvent was variable and ratio include 2.0:0.5 ml/min,

2.0:1.0 ml/min 2.0:1.5 ml/min and 2.0:2.0 ml/min. In third level the antisolvent and solvent was taken at equal volume and ratio include 0.5:0.5 ml/min, 1.0:1.0 ml/min 1.5:1.5 ml/min and 2.0:2.0 ml/min.

Furthermore, the imperative physicochemical characterization of the produced nanosuspension which includes particle size, zetapotential, powder X-ray diffraction, scanning electron microscopy, Differential scanning calorimetery, Transmission electron microscopy, Physical and chemical stability studies and in-vitro comparative bioavailability with marketed drug and raw drug were performed to explore the special characteristics of nanoparticles in nanosuspensions.

2.3. Characterization of the Produced Nanosuspension

2.3.1. Drug content Determination

The nanosuspensions of selected drugs (ketoprofen, dexibuprofen and indomethacin) were assessed for drugs contents through U.V spectrophotometer (V-630

(JAS.C0) at 260 nm, 222 nm and 320 nm respectively.

2.3.2. Particle size Measurement through DLS

Principle of the DLS

Dynamic Light Scattering Technique (DLS) which is also acknowledged as Quasi elastic scattering (QELS) or Photon-co relation spectroscopy (PCS). This technique is amongst the most popular methods employed to verify size and distribution of the particles of emulsion and molecules dispersed or dissolved in liquids. For example, proteins,

Nanoparticles, polymers, carbohydrates, Micelles and colloidal dispersions.

Fundamentally colloidal dispersions, particles emulsion and Nanosuspension undergo

Brownian motion. This motion is persuaded by the molecules of solvent which are in continuous motion due to their thermal energy. If the particles are illuminated with a Laser, the scattered light intensity fluctuates which is based on the particles size. Smaller particles result in more fluctuation of the scattered light, while big particles produce less fluctuation.

This corresponds to the Brownian motion of the particles. Smaller particles in dispersions are moving fast as compared to the big particles and consequently resulting in different fluctuation. Diameter measured in DLS is called Hydrodynamic diameter. This refers how diffusion of particles within a fluid occurs. In fact, this technique measures the diameter of a fluid having same translational diffusion co-efficient as that of the measured particles. It is the point of attention that the translational diffusion co-efficient not only depends on the

size of the particles core but also on the surface structure as well as concentration and nature of ions present in medium. It means that the size measured by DLS could be larger than the size of the SEM or TEM. The size of the particles could be measured by the

Einstein equation. d [ H ] = kT/3ληD (equation B1)

Where d [ H ] means Hydrodynamic diameter

D means Translational diffusion co-efficient

K means Boltzman’s constant.

T means Température

η means liquid Viscosity

Experimental Procedure

The selected drugs (ketoprofen, dexibuprofen, indomethacin) nanosuspensions were assessed for particle size through (PCS) photon correlation spectroscopy (NanoS

Zetasizer®, Malvern Instruments, UK). The zetaszier was calibrated with duke standard

TM with mean diameter of 500 nm ± 0.02 and Nanosphere TM with mean diameter of 59.0 nm ± 2.5. The produced nanosuspensions were analyzed without further dilution. The polydispersity index (PDI) and average particle size diameter (Z-Ave) were determined from three measurements.

2.3.3. Measurement of Zeta Potential

The zeta potential investigation is significant in reference to stability prediction.

The produced nanosuspensions of the selected drugs (ketoprofen, dexibuprofen, indomethacin) were assessed for zeta potential at room temperature (25 ℃) by using the

Nano-zetaszier (Malvern Instruments, UK). Basically, electric field is applied and movement of nanoparticle is measured. The clear disposable zeta cells were used for zetapotential measurements. The sample were dispersed in water and then measurements were taken. Evaluation of produced nanosuspensions of the selected drugs were done in triplicate. Zetaszier was calibrated with zeta potential transfer standard® -68 mV ± 6.8

(Malvern instruments Ltd, UK).

2.3.4. Morphological evaluation through Scanning electron microscopy

Principle of the SEM

The SEM (Scanning Electron Microscopy) method involves the generation of an electron beam which is used to bombard a solid sample and results in high resolution images of the sample surface texture, topography shape and size. An emission of high energy electron beam occurs thermionically in SEM (Scanning electron microscopy) from tungsten cathode and is accelerated towards an anode. The focusing of an electron beam is followed by passing through pair of scanning coils in the objective lens that deflects the beam in horizontal and vertical position to enable sample surface scanning over a rectangular area. Upon impact, electrons from the surface and from the bulk of solid sample are liberated. The liberated electrons travelling in a direction opposite to that of the incident beam of electrons are detected and used to form an image of the solid sample

Experimental Procedure

Morphological evaluation of unprocessed drugs (ketoprofen, dexibuprofen, indomethacin) was done through SEM (Quanta-400, FEI Company, U.K). Different magnifications levels were used for selected drugs images. Gold coating of the unprocessed drugs (ketoprofen, dexibuprofen, indomethacin) particles, using sputter coater was carried out to obtain the clear images. Calibration of the SEM was done through gold grid provided with equipment.

2.3.5. Transmission electron microscopy

Principal of the TEM

Transmission Electron microscopy (TEM) is a worthwhile microscopy procedure with high resolution power where a beam of electron is passed through an ultra-thin specimen and gets interaction with specimen when it passes through. The electrons and specimen interaction is being encountered and is very important, because this leads to formation of images of the particles contained by the specimen. TEMs are capable of producing pretty good images of the tiny particles at a significant resolution range than other microscopic techniques. Emission of electron principally occurs from a “light source” present on the top of the microscope that travel through the vacuum in the column of microscope. In ordinary microscope glass lenses are used to focus the light, in contrast of this TEMs are utilizing electromagnetic lenses to get focus the electrons into a very thin beam and is followed by passing through the desired specimen. The density of the materials is encountered and is very worthwhile because of its electron scattering role. Scattering and disappearance of some of electrons from the beam occurs. The fluorescent screen is finally targeted by the un-scattered electrons at the microscope bottom giving rise to particle images that could be studied directly from the screen or a snap shot is taken by the fitted camera.

Procedure of the Experiment

For external morphology, the produced nanocrystals of ketoprofen, dexibuprofen, indomethacin were characterized at 100 kV through transmission electron microscopy

(JEM - 1200EX, JEOL, Japan). Samples of the selected drugs nanosuspensions was prepared by pouring a drop from nanosuspensions on the copper grid surface having 400

mesh and then dried out at ambient temperature. The negative staining was also applied through an aqueous solution of 2 % magnesium uranyl acetate. Then finally prepared samples were introduced into Microscope (JEM - 1200EX, JEOL, Japan) and assessment was carried out at 100 kV.

2.3.6. Thermal analysis

Principle of the DSC

Differential scanning calorimetery (Byrappa et al., 2008) is a thermos-analytical technique for measuring the variance in the quantity of heat essential for increasing the sample and reference temp as a function of time. Actually, it utilizes the “Null- balance” principle to get ensure that the temperature of the sample is the same as that of a reference pan. Both the sample and reference are heated at predetermined rate. Fundamentally the temp programme for the DSC investigation is planned in a such manner that increase in the temperature of sample holder remains linear as a function of time. The heat capacity of the reference sample must be pretty stable and well defined over the different range of temperatures being analyzed. When a sample is passing through phase transitions process, flow of heat started from or to the sample for maintaining it same as that of the reference temperature. Heat flow to or from the sample actually, relies on whether the process is endothermic or exothermic. For instance, if a sample is going to transfer from solid to liquid, greater amount of heat flow is needed for increasing sample temperature at a rate same to the reference. It is because sample absorb heat as it experiences the phase changeover from solid to liquid endothermically. In contrast of that, if crystallization occur which is an exothermic process, then less amount of heat will be required for raising sample temperature. A signal proportional to the differential power input is fed into a recorder and is translated into a DSC file. Principally, this is graphical presentation of changes in heat flow rate on Y-axis against temperature on X-axis. While the area under the peak is proportional to energy gained, or lost by the sample.

Procedure of the Experiment

Thermal Characteristics of the processed and unprocessed selected drugs

(ketoprofen, dexibuprofen, indomethacin) was assessed by using a differential scanning calorimeter (DSC Discovery, TA Instrument, UK). The DSC instrument was calibrated through indium 99 % (melting Point: 156.6 °C) and Zinc (melting Point: 419.5 °C). The nanoparticles of the selected drugs (ketoprofen, dexibuprofen and indomethacin) were collected from nanosuspensions through optima TL ultracentrifuge (Beckmann, USA). The working temp was 4° C, centrifuge speed was 93000 rpm and operation timing was 60 minutes. The topmost layer was removed and the drying of the remaining residues were performed in desiccator (Ali, 2011). The unprocessed and nanoparticle of ketoprofen, dexibuprofen and indomethacin were checked for thermal examination at rate of 10 °C/min in nitrogen atmospheric condition. The examination was performed at temperature ranged of 0 to 120 °C for ketoprofen, 0 to 100 °C for dexibuprofen and 60 to 190 °C for indomethacin. Evaluation of all samples were done in triplicate.

2.3.7. X-ray powder diffraction (XRPD) Analysis

Principle of the XPRD

Importance of XPRD in term of characterizing solid state phase could not be oversighted. Actually, this method depends on the interaction of crystal plans and atoms with the monochromatic beam of X-rays and its ability to reflect it. If crystal is placed in a monochromatic X-Rays beam, diffraction will occur if the Bragg law (equation B2) is obeyed.

2d sinθ = nλ (equation B2)

Where d = distance between plans,

λ = Wave length of the incident of beam of

n = Order of diffraction pattern.

θ = Angle of beam of reflection

One important condition for the Brag law to be obeyed, the various incident rays must be in phase with each other to avoid destructive interference, however, this is only possible if the path difference of the X-Rays is equal to the wave length or its multiple. If a crystal is rotated, the sooner or later a position would be reached when a particular family of planes will meet the Bragg condition and a diffraction signal would be detected at a diffraction angle. The X-ray diffraction spectrum pattern which results is characterised by peaks of various intensities (y- axis) at different diffraction angles(x-axis). Fundamentally different crystalline substances are having different X-ray diffraction patterns because line position depends upon size of unit cells and a line intensities relies on the type and arrangement of atoms in the crystals. A series of peaks were detected in the powder pattern at various scattering angles. Correlations are found in the angles and their relative intensities with

computed d-spacing to provide a full crystallographic characterization of the powder sample (Brittain et al., 1991). This enables the X-ray diffraction spectra for the identification of the various powder substances. X-Ray diffraction has also been used for crystallinity studies, since the intensities of the peaks are related with diminished crystallinity. Amorphous substances result a diffuse X-Ray diffraction pattern rather than the sharp peaks. In multicomponent system, the peaks with different intensities indicates the presence of different substances which is proportional to their concentration.

Experimental Procedure

The crystallinity of unprocessed and produced ketoprofen, dexibuprofen and indomethacin nanoparticles was determined by X-ray powder diffraction (XPRD equipment Bruker D - 8 (Bruker Kahlsruhl, Germany) with Cu Ka radiation (X = 1.5418

A). The scanning was done at angle with range of 5-50°, step size was 0.05°, count time was 3 s per step, rotation during analysis was 30 rpm and generator was fixed on 40 kV with 30 mA. The un processed samples of the selected drugs were put in plastic sample holder while produced nanocrystals samples of the selected drugs were put in silicon-well sample holder and then positioned in XPRD equipment for running. The XPRD equipment was calibrated through corundum standard.

2.3.8. Stability studies

ketoprofen, dexibuprofen and indomethacin nanosuspensions were assessed for its chemical stability at regular time interval of one week (day-0, day-1, day-3, day-4, day-5, day-6, day-7) and %age of drugs contents was evaluated through U.V spectrophotometer

(V-630 (JAS.C0) at 260 nm, 222 nm and 320 nm respectively. Moreover, physical stability of the selected drugs nanosuspensions were evaluated at refrigerated temperature 2 - 8 ºC and at 25 °C (room temperature) for period of two months (60 days). The variation in the physical appearance were gauged. Moreover, the growth of the particles was observed by determining the Average particle size and PDI values at specific timing periods (0-day, 5- day, 10-day, 15-day 20-day, 30-day, 40-day, 50-day and 60-day) through (PCS) photon correlation spectroscopy (NanoS Zetasizer®, Malvern Instruments, UK).

2.3.9. In-vitro dissolution evaluation

The ketoprofen, dexibuprofen and indomethacin nanosuspensions ware evaluated for in-vitro dissolution and were compared with marketed formulations and raw drugs. For dissolution study USP rotating paddle method was used. The ketoprofen, dexibuprofen and indomethacin samples were put in dissolution vessels holding 900 ml phosphate buffer with pH of 7.2 and kept at 37 ºC ± 0.5 with stirring speed of 100 rpm which been reported by shah et al for nanosuspensions (Nokhodchi et al., 2005, Shah et al., 2016, Khan et al.,

2013a). The sample were collected after specific timing (5, 10, 15, 20, 25, 30, 40, 50, 60 minutes) and were substituted with equal amount of fresh phosphate buffer solution. The assessment of samples (ketoprofen, dexibuprofen and indomethacin) were done on U.V spectrophotometer (V-630 (JAS.C0) at 260 nm, 222 nm and 320 nm respectively.

2.3.10. Statistical Examination

Statistically examination of the experimentally collected data was done by SPSS 18 (SPSS

Inc., USA) The data for all drugs was collected in triplicate. The data presented here are as mean ± standard Deviation. One-way ANOVA test (p < 0.05) and least significant difference test (LSD) were used.

Chapter 03

3. Results and Discussion

A. Preparation of Ketoprofen, Dexibuprofen and Indomethacin Nanosuspension through Microchannel fluidic reactor

In this study, the microfluidic reactor was chosen to produce nanocrystals of the

ketoprofen, dexibuprofen and indomethacin. This is a low energy method which can

produce stable drug nanocrystals. However, process optimization is very paramount to be

taken under consideration. Interestingly, there was observed a variation in particle size of

the model drugs while changing the parameters. These parameters and their impact on the

particle size are described as follow.

3.1. Evaluation of Polymers

In this study a range of polymers were used in stabilizer solution to produce stable

nanocrystals of ketoprofen, dexibuprofen and indomethacin. The effect of polymer types

and concentration was assessed for particle size of ketoprofen, dexibuprofen and

indomethacin nanocrystals. The effect of all polymers on ketoprofen nanoparticles are

accessible in table 3.1. Poloxamer 407 was found the most suitable polymer to produce

ketoprofen and dexibuprofen nanocrystals. For ketoprofen appropriate concentration was

(1 % w/v) and Crystal size and PDI value attained were (61 nm ± 3.0) and (0.25 ± 0.07)

respectively. Data is also presented in Figure 3.1 and particle size distribution is presented

in 3.2. Moreover, for dexibuprofen suitable concentration of Poloxamer 407 was (0.5 %

w/v) and particle size and PDI value attained were (45 nm ± 3.0) and (0.19 ± 0.06)

respectively (Figure 3.3 and Particle size distribution 3.4). The polymers effect on

dexibuprofen nanoparticle are shown in table 3.2. Poloxamer 407 has also been reported

previously, a suitable triblock co polymer to produce stable nanocrystals of Ibuprofen

(Khan et al., 2013b). The favorable interaction due to hydrogen bonding between the two surfaces, including Poloxamer and drugs (ketoprofen and dexibuprofen) can lead to sufficient adsorption of the triblock copolymer onto the surface of ketoprofen. Also, this might be due to the high binding free energies of the polymers and chosen drug compounds

(Seedat et al., 2016). The indomethacin was more difficult and single and two polymers did not stabilize the nanosuspension. Three polymers in combination were used to produce stable indomethacin nanocrystal. The polymers combination used was PVP k30 (1 % w/v)

- HPMC 15cps (0.5 % w/v) - SDS (0.5 % w/v) and nanocrystal obtained with particle size and PDI were (380 nm ± 5.0) and (0.29 ± 0.05) respectively (Figure 3.5). Particle size distribution graph is presented in figure 3.6. The entire polymers effect (Kesisoglou et al.,

2007) on indomethacin nanocrystal is tabulated in table 3.3. Previously PVP - HPMC -

SDS has also been testified, a suitable polymer combination to produce stable nanocrystals of Hydrocortisone (Ali et al., 2009b). The combined impact of PVP and HPMC has also been used for the stability of carbamazepine colloidal system (Douroumis and Fahr, 2007).

Ionic surfactants enhance soundness against temperature effect for suspensions balanced out by polymers (Rabinow, 2004, Kesisoglou et al., 2007)

A. Ketoprofen

Table 3.1: Different polymers used and its effect on ketoprofen nanocrystals

AVG P SIZE

POLYMER (nm) ± SD PDI ± SD

Poloxamer 407 (0.5 %) 80 ± 3.5 0.36 ± 0.06

Poloxamer 407 (1 %) 61 ± 3.0 0.25 ± 0.07

PVP k30 (0.5%) 390 ± 3.0 0.32 ± 0.06

PVP k30 (1%) 131 ± 2.0 1.00 ± 0.07

HPMC 15cps (0.5%) 796 ± 3.5 0.38 ± 0.08

HPMC 15cps (1%) 917 ± 2.0 0.33 ± 0.13

Poloxamer 407 - PVP k30 (0.5% -0.5%) 117 ± 4.0 0.35 ± 0.07

Poloxamer 407 - PVP k30 (1% -0.5%) 59 ± 3.2 0.42 ± 0.06

Poloxamer 407 - HPMC 15cps (0.5% -0.5%) 183 ± 3.5 0.51 ± 0.11

Poloxamer 407 - HPMC 15cps (1% -0.5%) 74 ± 4.5 0.49 ± 0.09

PVP k30 - HPMC 15cps (0.5% -0.5%) 452 ± 3.0 0.39 ± 0.13

PVP k30 - HPMC 15cps (1% -1%) 290 ± 2.5 0.63 ± 0.20

Data is presented as mean ± SD

1200 1.2 AVG P SIZE PDI 1000 1

800 0.8

600 0.6 PDI 400 0.4

200 0.2 AVG AVG SIZE P (nm)

0 0

POLYMER

Figure 3.1: Polymers effect on ketoprofen nanocrystals

Figure 3.2: ketoprofen nanoparticle particle size distribution (Poloxamer 407 (1 %)

B. Dexibuprofen

Table 3.2: Different polymers used and its effect on dexibuprofen nanocrystals

AVG P SIZE

POLYMER (nm) ± SD PDI ± SD

Poloxamer 407 (0.5 %) 45 ± 3.0 0.19 ± 0.06

Poloxamer 407 (1 %) 36 ± 4.0 0.15 ± 0.04

PVP k30 (0.5 %) 286 ± 5.0 0.44 ± 0.19

PVP k30 (1 %) 224 ± 4.0 0.41 ± 0.16

HPMC 15cps (0.5 %) 433 ± 2.0 0.25 ± 0.07

HPMC 15cps (1 %) 556 ± 4.0 0.27 ± 0.08

PVP k30 (1 %) - HPMC 15cps (1 %) 432 ± 6.0 0.34 ± 0.12

PVP k30 (0.5 %) - HPMC 15cps (0.5 %) 398 ± 4.0 0.32 ± 0.10

PVP k30 (0.5 %) - HPMC 15cps (1 %) 485 ± 4.0 0.35 ± 0.15

PVP k30 (1 %) - HPMC 15cps (0.5 %) 453 ± 5.0 0.37 ± 0.18

Poloxamer 407 - PVP k30 (0.5 % - 0.5 %) 121 ± 4.5 0.42 ± 0.19

Poloxamer 407 - PVP k30 (1 % - 0.5 %) 103 ± 4.0 0.40 ± 0.20

Poloxamer 407 - HPMC 15cps (0.5 % - 0.5 %) 256 ± 3.0 0.26 ± 0.22

Poloxamer 407 - HPMC 15cps (1 % -0.5 %) 205 ± 2.5 0.25 ± 0.29

Data is presented as mean ± SD

AVG P SIZE PDI 700 0.50 0.45 600 0.40

500 0.35 PDI 400 0.30 0.25 300 0.20

AVG P SIZE (nm) SIZE P AVG 200 0.15 0.10 100 0.05 0 0.00

POLYMER

Figure 3.3: polymers effect on dexibuprofen nanocrystals

Figure 3.4: Dexibuprofen nanoparticle particle size distribution (Poloxamer 407 (0.5 %))

C. Indomethacin

Table 3.3: Different polymers used and its effect on Indomethacin nanocrystals

AVG P SIZE

POLYMER (nm) ± SD PDI ± SD

Poloxamer 407 (0.5 %) 2120 ± 8.0 0.46 ± 0.20

Poloxamer 407 (1 %) 1281 ± 6.0 0.51 ± 0.14

PVP k30 (0.5 %) 567 ± 5.0 0.38 ± 0.13

PVP k30 (1 %) 489 ± 4.5 0.32 ± 0.09

HPMC 15cps (0.5 %) 620 ± 4.0 0.33 ± 0.09

HPMC 15cps (1 %) 836 ± 4.0 0.39 ± 0.13

PVP k30 (1 %) - HPMC 15cps (0.5 %) - SDS (0.5 %) 380 ± 5.0 0.29 ± 0.05

PVP k30 (1 %) - HPMC 15cps (0.5 %) - SDS (0.1 %) 529 ± 4.5 0.32 ± 0.10

PVP k30 (1 %) - HPMC 15cps (1 %) - SDS (0.5 %) 499 ± 5.0 0.45 ± 0.12

PVP k30 (0.5 %) - HPMC 15cps (0.5 %) - SDS (0.5 %) 431 ± 3.0 0.33 ± 0.11

Poloxamer 407 - PVP k30 (0.5 % - 0.5 %) 822 ± 5.0 0.42 ± 0.20

Poloxamer 407 - PVP k30 (1 % - 0.5 %) 701 ± 4.5 0.44 ± 0.19

Poloxamer 407 - HPMC 15cps (0.5 % - 0.5 %) 915 ± 3.0 0.36 ± 0.18

Poloxamer 407 - HPMC 15cps (1 % - 0.5 %) 989 ± 3.5 0.37 ± 0.12

PVP k30 - HPMC 15cps (0.5 % - 0.5 %) 543 ± 2.5 0.34 ± 0.11

PVP k30 - HPMC 15cps (1 % - 1 %) 468 ± 2.0 0.33 ± 0.09

Data is presented as mean ± SD

AVG P SIZE PDI 2500 0.6

2000 0.5 0.4

1500 PDI 0.3 1000 AVG P SIZE (nm) SIZE P AVG 0.2

500 0.1

0 0

POLYMER

Figure 3.5: Polymers effect on Indomethacin nanocrystals

Figure 3.6: Indomethacin nanoparticle particle size distribution (PVP k30 (1 %) - HPMC 15cps (0.5 %) - SDS (0.5 %)

3.2. Inlet angle of Microfluidic Reactor.

The effect of three different angles on nanoparticle size of ketoprofen, dexibuprofen, indomethacin were investigated. The particle produced by angle 50° ware little bit larger having slightly broader distribution. The Ketoprofen AVG P Size at angle

50° was 97 nm ± 3.5 and PDI was 0.43 ± 0.05 while at angle 10° it was 61 nm ± 3.0 and

PDI was 0.25 ± 0.07 (Figure 3.7). Moreover, dexibuprofen AVG P Size at angle 50° was

86 nm ± 5.0 and PDI was 0.34 ± 0.10 while at angle 10° it was 45 nm ± 3.0 and PDI was

0.19 ± 0.06 (Figure 3.8). The Indomethacin AVG P Size at angle 50° was 437 nm ± 6.0 and PDI was 0.33 ± 0.10 while at angle 10° it was 380 nm ± 5.0 and PDI was 0.29 ± 0.05

(Figure 3.9). It is thought that Keener delta edges (10°) have sharper channel edges which permit the two liquid streams to meet and then stream down the outlet without creating any interruption to either liquid stream (Brook, 2006) while in angle 50° the both liquid streams encounter on smooth plane creating further disturbance of both liquid streams when it is streamed outside.

A. Ketoprofen

Table 3.4: Effect of inlet angle of MCFR on particle size of Ketoprofen nanoparticle

Angle AVG P Size (nm) ± SD PDI ± SD

angle 10° 61 ± 3.0 0.25 ± 0.07

angle 25° 83 ± 2.8 0.41 ± 0.06

angle 50° 97 ± 3.5 0.43 ± 0.05

Data is presented as mean ± SD

120 0.5 0.45 100 0.4 80 0.35 0.3

60 0.25 PDI 0.2 40 0.15 20 0.1 0.05 AVG AVG SIZE P (nm) 0 0 angle 10° angle 25° angle 50°

ANGLE

AVG P Size PDI

Figure 3.7: MCFR inlet angle Effect on Ketoprofen nanoparticle

B. Dexibuprofen

Table 3.5: Effect of inlet angle of MCFR on particle size of dexibuprofen nanoparticle Angle AVG P Size (nm) ± SD PDI ± SD angle 10° 45 ± 3.0 0.19 ± 0.06 angle 25° 62 ± 3.0 0.22 ± 0.09 angle 50° 86 ± 5.0 0.34 ± 0.10

Data is presented as mean ± SD

100 0.40

90 0.35 80 0.30 70 60 0.25

50 0.20 PDI

40 0.15 AVG AVG SIZE P (nm) 30 0.10 20 10 0.05 0 0.00 angle 10° angle 25° angle 50°

ANGLE

AVG P Size PDI

Figure 3.8: MCFR inlet angle Effect on dexibuprofen nanoparticle

C. Indomethacin

Table 3.6: Effect of inlet angle of MCFR on particle size of indomethacin nanoparticle

Angle AVG P Size (nm) ± SD PDI ± SD angle 10° 380 ± 5.0 0.29 ± 0.05 angle 25° 412 ± 5.5 0.31 ± 0.09 angle 50° 437 ± 6.0 0.39 ± 0.10

Data is presented as mean ± SD

500 0.45 450 0.4 400 0.35 350 0.3 300 0.25 250 0.2 PDI 200 0.15 150 100 0.1

AVG AVG SIZE P (nm) 50 0.05 0 0 angle 10° angle 25° angle 50°

ANGLE

AVG P Size PDI

Figure 3.9: MCFR inlet angle Effect on Indomethacin nanoparticle

3.3. Effect of Flow rate of Antisolvent and Solvent

3.3.1. Anti-solvent volume variable and Solvent volume constant

Antisolvent and solvent flow rate was assessed and it was observed that it has dominant effect on nanocrystal size. The nanocrystal of smaller sizes was achieved while changing the antisolvent volume and keeping the drug solution constant. The ketoprofen, dexibuprofen and indomethacin particle sizes achieved at 2.0/0.5 ml/min were 61 nm ± 3.0 with PDI of 0.25 ± 0.07, 45 nm ± 3.0 with PDI of 0.19 ± 0.06 and 380 nm ± 5.0 with PDI of 0.29 ± 0.05 respectively (Figure 3.10, 3.11, 3.12). The obtained relative smaller particle sizes are attributed due to two reasons, the first one is the perfect mixing of drug solutions with antisolvent solutions because antisolvent solutions volume are high as compared to drug solutions (Su et al., 2007). The second one is the higher beginning supersaturation levels which are achieved due to less drugs solutions and high antisolvent concentrations which decreases the solutes concentration on nuclei surfaces (Zhao et al., 2007a).

Moreover, Complete results are tabulated in table no 3.7, 3.8, 3.9.

A. Ketoprofen

Table 3.7: Effect of Antisolvent and solvent flow rate (Antisolvent Variable and solvent constant) on nanocrystal size of ketoprofen Antisolvent: solvent (ml/min) AVG P SIZE (nm) ± SD PDI ± SD

0.5:0.5 272 ± 5.0 0.39 ± 0.08

1.0:0.5 212 ± 2.5 0.43 ± 0.06

1.5:0.5 147 ± 3.2 0.41 ± 0.05

2.0:0.5 61 ± 3.0 0.25 ± 0.07

Data is presented as mean ± SD

300 0.5 0.45 250 0.4 200 0.35

0.3 PDI 150 0.25 0.2 100

AVG AVG SIZE P (nm) 0.15 50 0.1 0.05 0 0 0.5:0.5 1.0:0.5 1.5:0.5 2.0:0.5 Antisolvent and solvent ratio

AVG P SIZE PDI

Figure 3.10: Effect of flow rate (Antisolvent Variable and solvent constant) on Particle size of ketoprofen nanocrystal

B. Dexibuprofen

Table 3.8: Effect of Antisolvent and solvent flow rate (Antisolvent Variable and solvent constant) on dexibuprofen nanoparticle size Antisolvent: solvent

(ml/min) AVG P SIZE (nm) ± SD PDI ± SD

0.5:0.5 267 ± 3.0 0.36 ± 0.17

1.0:0.5 192 ± 4.0 0.34 ± 0.09

1.5:0.5 107 ± 4.0 0.28 ± 0.10

2.0:0.5 45 ± 3.0 0.19 ± 0.06

Data is presented as mean ± SD

300 0.4 0.35 250 0.3 200

0.25 PDI 150 0.2 0.15

100 AVG AVG SIZE P (nm) 0.1 50 0.05 0 0 0.5:0.5 1.0:0.5 1.5:0.5 2.0:0.5

Antisolvent and solvent ratio

AVG P SIZE PDI

Figure 3.11: Effect of Antisolvent and solvent flow rate on size of dexibuprofen nanoparticle

C. Indomethacin

Table 3.9: Effect of flow rate (Antisolvent Variable and solvent constant) on nanocrystal size Indomethacin Antisolvent: solvent

(ml/min) AVG P SIZE (nm) ± SD PDI ± SD

0.5:0.5 826 ± 3.0 0.47 ± 0.14

1.0:0.5 677 ± 4.0 0.51 ± 0.16

1.5:0.5 553 ± 3.0 0.44 ± 0.23

2.0:0.5 380 ± 5.0 0.29 ± 0.05

Data is presented as mean ± SD

1000 0.6 900 800 0.5 700 0.4

600 PDI 500 0.3 400

300 0.2 AVG AVG SIZE P (nm) 200 0.1 100 0 0 0.5:0.5 1.0:0.5 1.5:0.5 2.0:0.5 Antisolvent and solvent ratio

AVG P SIZE PDI

Figure 3.12: Effect of Antisolvent and solvent flow rate (Antisolvent Variable and solvent constant) on nanocrystal size of Indomethacin

3.3.2. Anti-solvent volume constant and Solvent volume variable

It was observed that keeping antisolvent solution constant and an increasing the volume of drug solution results in larger particles. The maximum results obtained at 2.0/2.0 ml/min and particle size of ketoprofen, dexibuprofen and indomethacin were 885 nm ± 4.0 with PDI of 0.53 ± 0.29, 803 nm ± 3.0 with PDI of 0.57 ± 0.19 and 2446 nm ± 6.0 with

PDI of 0.39 ± 0.31 (figure 3.13, 3.14, 3.15) respectively. This effect is attributed to increase in drug concentration which inflates the solute fixation on the surface of framed nuclei and thus encourage molecular growth and produce bigger crystals (Zhao et al., 2007a, Dirksen and Ring, 1991a). Moreover, detailed results are presented in 3.10, 3.11, 3.12.

A. Ketoprofen

Table 3.10: Effect of Antisolvent and solvent flow rate (Antisolvent Constant and solvent variable) on nanocrystal size ketoprofen Antisolvent: solvent (ml/min) AVG P SIZE (nm) ± SD PDI ± SD

2.0:0.5 61 ± 3.0 0.25 ± 0.07

2.0:1.0 252 ± 2.7 0.49 ± 0.05

2.0:1.5 413 ± 3.5 0.45 ± 0.10

2.0:2.0 885 ± 4.0 0.53 ± 0.29

Data is presented as mean ± SD

1000 0.6 900 0.5 800 700 0.4

600 PDI 500 0.3 400

AVG AVG SIZE P (nm) 0.2 300 200 0.1 100 0 0 2.0:0.5 2.0:1.0 2.0:1.5 2.0:2.0

Antisolvent and solvent ratio

AVG P SIZE PDI

Figure 3.13: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of ketoprofen nanoparticle.

B. Dexibuprofen

Table 3.11: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of dexibuprofen nanoparticle. Antisolvent: solvent (ml/min) AVG P SIZE (nm) ± SD PDI ± SD

2.0:0.5 45 ± 3.0 0.19 ± 0.06

2.0:1.0 218 ± 4.0 0.34 ± 0.14

2.0:1.5 397 ± 4.0 0.39 ± 0.17

2.0:2.0 803 ± 3.0 0.57 ± 0.19

Data is presented as mean ± SD

900 0.70

800 0.60 700 0.50 600

500 0.40 PDI 400 0.30 300

0.20 AVG AVG SIZE P (nm) 200 0.10 100

0 0.00 2.0:0.5 2.0:1.0 2.0:1.5 2.0:2.0

Antisolvent and solvent ratio

AVG P SIZE PDI

Figure 3.14: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of dexibuprofen nanoparticle

C. Indomethacin

Table 3.12: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of Indomethacin nanoparticle Antisolvent: solvent (ml/min) AVG P SIZE (nm) ± SD PDI ± SD

2.0:0.5 380 ± 5.0 0.29 ± 0.05

2.0:1.0 1037 ± 4.0 0.66 ± 0.16

2.0:1.5 1639 ± 6.0 0.57 ± 0.23

2.0:2.0 2446 ± 6.0 0.93 ± 0.31

Data is presented as mean ± SD

2400 1.2 2100 1 1800 0.8

1500 PDI 1200 0.6 900 0.4 600

AVG AVG SIZE P (nm) 0.2 300 0 0 2.0:0.5 2.0:1.0 2.0:1.5 2.0:2.0 Antisolvent and solvent ratio AVG P SIZE PDI

Figure 3.15: Effect of Antisolvent and solvent flow rate (solvent Variable and antisolvent constant) on particle size of Indomethacin nanoparticle.

3.3.3. Equal volume of Anti-solvent and Solvent

At equal ratios of both solutions i.e. drug solution and antisolvent solution it has been observed that at smaller stream rate of both solutions (0.5/0.5 ml/min) results in smaller particle sizes i.e. (Ketoprofen P size 272 nm ± 5.0 with PDI of 0.39 ± 0.08),

(dexibuprofen P Size 267 nm ± 3.0 with PDI of 0.36 ± 0.17) and (Indomethacin P Size 826 nm ± 3.0 with PDI of 0.47 ± 0.14) respectively while at higher stream rate of both solutions

(2.0/2.0 ml/min) results in larger particle sizes (Ketoprofen P size 885 nm ± 4.0 with PDI of 0.53 ± 0.29), (dexibuprofen P Size 803 nm ± 3.0 with PDI of 0.57 ± 0.19) and

(Indomethacin P Size 2446 nm ± 6.0 with PDI of 0.93 ± 0.31 respectively (Figure 3.16,

3.17, 3.18). These results of higher particle sizes at higher stream rate of both solutions

(antisolvent and solvent solutions) are due to inadequate blending of drug solutions with antisolvent solutions as the dwelling time in microchannel fluidic reactor are very short and inadequate diffusion of the two solutions take place (Zhang et al., 2008) and it may produce a non-uniform region supersaturation, subsequently producing bigger particles with more extensive size dispersion (Ali et al., 2009b). Complete results are accessible in table 3.13, 3.14, 3.15.

A. Ketoprofen

Table 3.13: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on ketoprofen nanoparticle Antisolvent: solvent (ml/min) AVG P SIZE (nm) ± SD PDI ± SD

0.5:0.5 272 ± 5.0 0.39 ± 0.08

1.0:1.0 461 ± 4.2 0.44 ± 0.15

1.50:1.50 604 ± 3.5 0.45 ± 0.18

2.0:2.0 885 ± 4.0 0.53 ± 0.29

Data is presented as mean ± SD

1000 0.6 900 0.5 800 700 0.4 600

500 0.3 PDI 400 0.2 300

AVG AVG SIZE P (nm) 200 0.1 100 0 0 0.5:0.5 1.0:1.0 1.50:1.50 2.0:2.0 Antisolvent and solvent ratio

AVG P SIZE PDI

Figure 3.16: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on ketoprofen nanoparticle

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B. Dexibuprofen

Table 3.14: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on dexibuprofen nanoparticle AVG P SIZE (nm) ±

Antisolvent: solvent (ml/min) SD PDI ± SD

0.5:0.5 267 ± 3.0 0.36 ± 0.17

1.0:1.0 404 ± 2.0 0.39 ± 0.18

1.50:1.50 573 ± 4.0 0.43 ± 0.22

2.0:2.0 803 ± 3.0 0.57 ± 0.19

Data is presented as mean ± SD

900 0.7

800 0.6 700 0.5

600 PDI 500 0.4 400 0.3

AVG AVG SIZE P (nm) 300 0.2 200 100 0.1 0 0 0.5:0.5 1.0:1.0 1.50:1.50 2.0:2.0

Antisolvent and solvent ratio

AVG P SIZE PDI

Figure 3.17: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on dexibuprofen nanoparticle

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C. Indomethacin Table 3.15: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on Indomethacin nanoparticle Antisolvent: solvent (ml/min) AVG P SIZE (nm) ± SD PDI ± SD

0.25:0.25 432 ± 4.0 0.33 ± 0.10

0.5:0.5 826 ± 3.0 0.47 ± 0.14

1.0:1.0 1398 ± 5.0 0.54 ± 0.09

1.50:1.50 1875 ± 7.0 0.77 ± 0.13

2.0:2.0 2446 ± 6.0 0.93 ± 0.31

Data is presented as mean ± SD

2700 1.2 2400 1 2100

1800 0.8 PDI 1500 0.6 1200

900 0.4 AVG AVG SIZE P (nm) 600 0.2 300 0 0 0.25:0.25 0.5:0.5 1.0:1.0 1.50:1.50 2.0:2.0

Antisolvent and solvent ratio

AVG P SIZE PDI

Figure 3.18: Effect of Antisolvent and solvent flow rate (Equal ratio of solvent and antisolvent volume) on Indomethacin nanoparticle

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3.3.4. Batch sizes Scalability

Initially batches were produced at 3.5 ml for ketoprofen and dexibuprofen while

4.0 ml was used for indomethacin. The scalability of the batch sizes was also evaluated and the scalable batches for ketoprofen and dexibuprofen were 10.5 ml, 21 ml, 49 ml and 98 ml while for indomethacin the scalable batches were 12 ml, 24 ml, 56 ml and 96 ml.

However, chemical engineering approach will be essential to proceeds the experiments at large scale. The observed results were similar and no significant changes were detected.

The ketoprofen and dexibuprofen results obtained at batch size of 96 ml were 67 nm ± 3.2 with PDI 0.27 ± 0.09 and 50 nm ± 2.8 with PDI 0.23 ± 0.08 respectively. Similarly, indomethacin particle size obtained at 98 ml was 388 nm ± 2.8 with PDI 0.30 ± 0.07.

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3.4. Drug Concentration Effect

The drug concentration effect was evaluated and it was observed that larger particles sizes are obtained at higher concentrations. Generally high concentration produces two types of effect. The first one is production of smaller particles due to a faster nucleation rate while the second one is production of larger particles due to large no of nuclei which subsequently move to agglomeration. The drugs (ketoprofen, dexibuprofen and indomethacin) produces larger particles at higher concentration and data is presented in table 3.16, 3.17, 3.18. Principally higher concentration crops growth of crystal through condensation and coagulation phenomenon (Matteucci et al., 2006, Dong et al., 2009)

Moreover, at higher concentrations the viscosities of drugs solutions are high which decreases mutual diffusion of antisolvent solutions and solvent solutions with consequent creation of non-uniform supersaturation which ultimately produces larger particles through agglomeration or crystal growth. (Zhang et al., 2010). Furthermore, BaSO4 nanocrystals and prednisolone nanoparticles in MCFR have been produced with same effect (Ali et al., 2009b, Su et al., 2007).

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A. Ketoprofen

Table 3.16: Drug concentration effect on ketoprofen nanocrystals

Drug concentration AVG P SIZE (nm) ± SD PDI ± SD

5 mg/ml 61 ± 3.0 0.25 ± 0.07

10 mg/ml 374 ± 3.3 0.38 ± 0.08

15 mg/ml 1232 ± 2.9 0.53 ± 0.09

Data is presented as Mean ± SD

1400 0.6

1200 0.5

1000 0.4 800 0.3 600 PDI 0.2

400 AVG AVG SIZE P (nm)

200 0.1

0 0 5mg/ml 10mg/ml 15mg/ml

Drug Concentration

AVG P SIZE PDI

Figure 3.19: Drug concentration effect on particle size of Ketoprofen

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B. Dexibuprofen

Table 3.17: Drug concentration effect on dexibuprofen nanocrystals

Drug concentration AVG P SIZE (nm) ± SD PDI ± SD

5 mg/ml 45 ± 3.0 0.19 ± 0.06

10 mg/ml 336 ± 5.0 0.37 ± 0.07

15 mg/ml 920 ± 4.0 0.39 ± 0.08

Data is presented as mean ± SD

1200 0.45

0.40 1000 0.35

800 0.30

0.25 600

0.20 PDI AVG AVG SIZE P (nm) 400 0.15

0.10 200 0.05

0 0.00 5mg/ml 10mg/ml 15mg/ml

Drug Concentration

AVG P SIZE PDI

Figure 3.20: Drug concentration effect on particle size of dexibuprofen

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C. Indomethacin

Table 3.18: Drug concentration effect on Indomethacin nanocrystals Drug concentration AVG P SIZE (nm) ± SD PDI ± SD

5 mg/ml 380 ± 5.0 0.29 ± 0.05

10 mg/ml 1545 ± 4.0 0.59 ± 0.15

15 mg/ml 2706 ± 2.0 0.82 ± 0.12

Data is presented as mean ± SD

3000 1.00

0.90 2500 0.80

0.70 2000 0.60

1500 0.50 PDI 0.40 1000

0.30 AVG AVG SIZE P (nm) 0.20 500 0.10

0 0.00 5mg/ml 10mg/ml 15mg/ml

Drug Concentration

AVG P SIZE PDI

Figure 3.21: Drug concentration effect on particle size of Indomethacin

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3.5. Effect of Mixing Time

The resultant nanosuspension produced by MFCR, poured into vial containing polymer solution and mixed with magnetic stirrer. The stirring speed was maintained at

1200 rpm as reported by Khan et al (Khan et al., 2013b). The mixing of 60 minutes produced ketoprofen and dexibuprofen nanoparticles with smaller particle sizes as compared to the mixing time of 30 minutes (Figure 3.22, 3.23). In succeeding to mixing, the produced nanosuspensions was sonicated for 5 minutes which further reduced the particle size and PDI (ketoprofen P Size 61 nm ± 3.0 and PDI 0.25 ± 0.07 and dexibuprofen

P size 45 nm ± 3.0 with PDI of 0.19 ± 0.06 respectively). The optimum timing for indomethacin was 60 minutes with subsequent sonication for 15 minutes and P size obtained were 380 nm ± 5.0 with PDI of 0.29 ± 0.05 (Figure 3.24). Principally high stirring rate and efficient mixing can potentially produce high level of micromixing, supersaturation, rapid nucleation and consequently nanocrystals with relative smaller particle size are produced (Matteucci et al., 2006).

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A. Ketoprofen

Table 3.19: Mixing Time effect on ketoprofen nanoparticle

Mixing time AVG P SIZE (nm) ± SD PDI ± SD

30 min 72 ± 3.5 0.296 ± 0.10

30 min + 5 min sonication 67 ± 3.3 0.274 ± 0.08

60 min 64 ± 3.1 0.265 ± 0.06

60 min + 5 min sonication 61 ± 3.0 0.250 ± 0.07

Data is presented as mean ± SD

80 0.35 70 0.3 60 0.25 50 0.2 PDI 40 0.15 30 20 0.1 AVG AVG SIZE P (nm) 10 0.05 0 0 30 min 30min+5min 60 min 60min+5min sonication sonication Mixing Time

AVG P SIZE PDI

Figure 3.22: Effect of Mixing Time on ketoprofen nanoparticle

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B. Dexibuprofen

Table 3.20: Mixing Time effect on dexibuprofen nanoparticle mixing time AVG P SIZE (nm) ± SD PDI ± SD

30 min 77 ± 4.0 0.36 ± ± 0.12

30 min + 5 min sonication 68 ± 4.0 0.30 ± 0.09

60 min 57 ± 3.0 0.26 ± 0.08

60 min + 5 min sonication 45 ± 3.0 0.19 ± 0.06

Data is presented as mean ± SD

90 0.4

80 0.35

70 0.3 60 0.25 50 0.2 40 0.15 PDI 30 20 0.1

AVG AVG SIZE P (nm) 10 0.05 0 0 30 min 30min+5min 60 min 60min+5min sonication sonication Mixing Time

AVG P SIZE PDI

Figure 3.23: Effect of Mixing Time on dexibuprofen nanoparticle

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C. Indomethacin

Table 3.21: Mixing Time effect on Indomethacin nanoparticle

mixing time AVG P SIZE (nm) ± SD PDI ± SD

30 min 422 ± 6.0 0.39 ± 0.12

30 min + 15 min sonication 411 ± 4.0 0.33 ± 0.09

60 min 389 ± 5.0 0.32 ± 0.08

60 min + 15 min sonication 380 ± 5.0 0.29 ± 0.05

Data is presented as mean ± SD

500 0.45 450 0.4 400 0.35 0.3

350 PDI 0.25 300 0.2 250

0.15 AVG AVG SIZE P (nm) 200 0.1 150 0.05 100 0 30 min 30min+15min 60 min 60min+15min sonication sonication

Mixing Time

AVG P SIZE PDI

Figure 3.24: Effect of Mixing Time on dexibuprofen nanoparticle

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3.6. Stability Studies

Stability of the nanocrystal is very significant because nanonization enhances surface area with increase of surface free energy and consequently stability of nanoparticle is greatly affected (Van Eerdenbrugh et al., 2008). Moreover, little bit high solubility of the drugs at higher temperature, leads to lower supersaturation level with subsequent growth of nanoparticles through Ostwald ripening. In this process, the dissolved particles deposit onto the surface of existing particles with subsequent growth of the particles

(Müller and Peters, 1998) . The van der Waals forces among the nanocrystals prompt to expanded agglomeration and consequentially weakening the nanosuspensions (Wu et al.,

2011). The chemical stability of the produced nanosuspensions of selected drugs was evaluated for one week (seven days). No sign of degradation was observed. The data is presented in table 3.22. Moreover, the produced nanosuspensions were assessed for its physical stability at fridge temp (2 - 8 ºC) and room temperature (25 ºC) for a period of two months (60 days). There was observed, that all three selected drugs (ketoprofen, dexibuprofen and indomethacin) nanosuspension stored at refrigerated temperature (Figure

3.25, 3.26, 3.27) were stable and no significant growth occur. The ketoprofen, dexibuprofen and indomethacin nanosuspensions stored at 25 ºC were also stable (Figure

3.28, 3.29, 3.30). The ketoprofen, dexibuprofen and indomethacin were stable for two months and the particle size did not increase above 99 nm, 76 nm and 412 nm respectively.

The two months data of particle sizes data and PDI value indicate homogenous distribution of the particle and as per Deng et al homogenous distribution prevents Ostwald ripening

(Deng et al., 2010).

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Moreover, regarding nanosuspension stability, Freitas recommended that it should be to be kept in fridge (2 – 8 °C) to retain its particle size (Freitas and Müller, 1998). The comprehensive data is presented in table 3.23, 3.24, 3.25, 3.26, 3.27 and 3.28.

Chemical stability

Table 3.22: Chemical stability of the produced nanosuspensions. day-0 day-1 day-2 day-3 day-4 day-5 day-6 day-7

Drugs %age of the drug content ± SD

97.8 97.8 97.6 97.5 97.4 97.2 97.1 97.0

Ketoprofen ± 2.0 ± 2.3 ± 2.2 ± .97 ± 2.3 ± 2.2 ± 2.0 ± 2.5

97.5 97.4 97.4 97.3 97.1 97.0 96.9 96.9

Dexibuprofen ± 2.2 ± 2.0 ± 1.95 ± 2.1 ± 2.4 ± 1.98 ± 1.8 ± 2.6

97.7 97.6 97.5 97.4 97.3 97.2 97.1 97.0

Indomethacin ± 1.95 ± 2.1 ± 2.4 ± 2.6 ± 2.2 ± 1.90 ± 2.1 ± 2.3

Data is presented as mean ± SD

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A. Ketoprofen

Table 3.23: stability of ketoprofen nanoparticle at 2 - 8 ºC

Days AVG P Size (nm) ± SD PDI ± SD

fresh 61 ± 3.0 0.250 ± 0.07

After 5 days 64 ± 2.9 0.254 ± 0.06

After 10 days 66 ± 3.1 0.256 ± 0.05

After 15 days 68 ± 3.2 0.258 ± 0.06

After 20 days 70 ± 3.0 0.261 ± 0.05

After 30 days 73 ± 3.3 0.263 ± 0.04

After 40 days 75 ± 3.5 0.266 ± 0.05

After 50 days 78 ± 3.5 0.267 ± 0.04

After 60 days 80 ± 3.2 0.268 ± 0.06

Data is presented as mean ± SD

150 0.350

0.300 120 0.250

90 0.200 PDI 60 0.150 0.100 30

0.050 Avg P SIZE P SIZE Avg(nm) 0 0.000 fresh After 5 After After After After After After After days 10 15 20 30 40 50 60 days days days days days days days DAYS AVG P Size PDI

Figure 3.25: stability of ketoprofen nanoparticle at 2 – 8 ºC

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B. Dexibuprofen

Table 3.24: stability of dexibuprofen nanoparticle at 2 - 8 ºC

Days AVG P Size ± SD PDI ± SD fresh 45 ± 3 0.190 ± 0.06

After 5 days 47 ± 3 0.193 ± 0.06

After 10 days 49 ± 4 0.195 ± 0.06

After 15 days 50 ± 3 0.196 ± 0.07

After 20 days 52 ± 3 0.198 ± 0.07

After 30 days 55 ± 2 0.200 ± 0.07

After 40 days 57 ± 3 0.203 ± 0.06

After 50 days 59 ± 3 0.205 ± 0.05

After 60 days 61 ± 3 0.206 ± 0.06

Data is presented as mean ± SD

200 0.400

0.350 160 0.300

120 0.250 PDI 0.200

80 0.150

0.100 40 AVG P SIZE AVG P SIZE (nm) 0.050

0 0.000 fresh After 5 After After After After After After After days 10 15 20 30 40 50 60 days days days days days days days DAYS AVG P Size PDI

Figure 3.26: stability of dexibuprofen nanoparticle at 2 - 8 ºC

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C. Indomethacin

Table 3.25: stability of Indomethacin nanoparticle at 2 - 8 ºC Days AVG P Size (nm) ± SD PDI ± SD

fresh 380 ± 5.0 0.290 ± 0.05

After 5 days 383 ± 4.6 0.293 ± 0.04

After 10 days 385 ± 4.3 0.295 ± 0.05

After 15 days 387 ± 4.2 0.297 ± 0.06

After 20 days 389 ± 4.8 0.299 ± 0.05

After 30 days 392 ± 4.5 0.301 ± 0.04

After 40 days 395 ± 4.4 0.302 ± 0.05

After 50 days 397 ± 4.5 0.304 ± 0.04

After 60 days 399 ± 5.0 0.306 ± 0.04

Data is presented as mean ± SD

0.400 400 0.350 350 0.300 300

0.250 PDI 250 0.200 200 150 0.150

100 0.100 AVG P SIZE AVG PSIZE (nm) 50 0.050 0 0.000 fresh After 5 After After After After After After After days 10 15 20 30 40 50 60 days days days days days days days DAYS AVG P Size PDI

Figure 3.27: stability of Indomethacin nanoparticle at 2 - 8 ºC

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AA. Ketoprofen

Table 3.26: stability of Ketoprofen nanoparticle at 25 ºC

Days AVG P Size (nm) ± SD PDI ± SD

fresh 61 ± 3.0 0.250 ± 0.07

After 5 days 67 ± 3.5 0.254 ± 0.05

After 10 days 73 ± 3.2 0.258 ± 0.06

After 15 days 79 ± 3.2 0.260 ± 0.05

After 20 days 84 ± 3.1 0.261 ± 0.06

After 30 days 88 ± 3.2 0.263 ± 0.04

After 40 days 91 ± 2.8 0.266 ± 0.06

After 50 days 95 ± 3.0 0.270 ± 0.04

After 60 days 99 ± 3.5 0.273 ± 0.05

0.300 250 0.270 0.240 200 0.210

0.180 PDI 150 0.150 0.120 100 0.090 50 0.060 Avg P SIZE AvgP SIZE (nm) 0.030 0 0.000 fresh After After After After After After After After 5 days 10 15 20 30 40 50 60 days days days days days days days DAYS AVG P Size PDI

Figure 3.28: stability of Ketoprofen nanoparticle at 25 ºC

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BB. Dexibuprofen

Table 3.27: stability of dexibuprofen nanoparticle at 25 ºC

Days AVG P Size (nm) ± SD PDI ± SD fresh 45 ± 3.0 0.190 ± 0.06

After 5 days 49 ± 2.8 0.193 ± 0.05

After 10 days 55 ± 2.9 0.195 ± 0.07

After 15 days 60 ± 3.0 0.197 ± 0.05

After 20 days 64 ± 3.2 0.198 ± 0.04

After 30 days 67 ± 2.8 0.201 ± 0.05

After 40 days 70 ± 2.9 0.203 ± 0.06

After 50 days 72 ± 3.1 0.207 ± 0.04

After 60 days 76 ± 3.0 0.209 ± 0.05

Data is presented as mean ± SD

0.400 240 0.350 200 0.300 160 0.250 0.200

120 PDI 0.150 80 0.100 40 0.050

Avg P SIZE AvgP SIZE (nm) 0 0.000 fresh After After After After After After After After 5 days 10 15 20 30 40 50 60 days days days days days days days DAYS AVG P Size PDI

Figure 3.29: stability of dexibuprofen nanoparticle at 25 ºC

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CC. Indomethacin

Table 3.28: stability of Indomethacin nanoparticle at 25 ºC

Days AVG P Size (nm) ± SD PDI ± SD fresh 380 ± 5.0 0.290 ± 0.05

After 5 days 386 ± 5.0 0.293 ± 0.06

After 10 days 391 ± 3.0 0.296 ± 0.04

After 15 days 395 ± 3.3 0.298 ± 0.05

After 20 days 398 ± 3.1 0.299 ± 0.06

After 30 days 401 ± 4.3 0.301 ± 0.05

After 40 days 404 ± 3.0 0.302 ± 0.04

After 50 days 408 ± 3.2 0.307 ± 0.05

After 60 days 412 ± 4.5 0.310 ± 0.06

Data is presented as mean ± SD

450 0.6 400 0.55 0.5 350 0.45 300 0.4 250 0.35

0.3 PDI 200 0.25

150 0.2 Avg P SIZE AvgP SIZE (nm) 100 0.15 0.1 50 0.05 0 0 fresh After 5 After After After After After After After days 10 15 20 30 40 50 60 days days days days days days days DAYS AVG P Size PDI

Figure 3.30: stability of Indomethacin nanoparticle at 25 ºC

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3.7. Zeta Potential Investigation:

The value of zeta potential of the produced nanosuspensions of the selected drugs were determined at 25 ℃ through Malvern zetaszier. The zeta potential of the ketoprofen was -17.2 mV ± 2.0 which is nearly close to commonly accepted value of ± 20mV (Yang et al., 2008, Jacobs and Müller, 2002) while the dexibuprofen and indomethacin zeta potential value were -21.5 mV ± 2.4 and -24.3 mV ± 1.8 respectively which is in the accepted region. Correspondingly, at higher batch sizes zeta potential value for ketoprofen, dexibuprofen and Indomethacin were -17.9 mV ± 1.9, -22.1 mV ± 2.2 and -25.0 mV ± 1.8 respectively. Actually, Zeta potential value are used to foresee the physical stability of the nanosuspensions (Patravale and Kulkarni, 2004a). The polymer used for ketoprofen and dexibuprofen is Poloxamer 407 which is already reported for stability of Ibuprofen nanosuspension (Khan et al., 2013b). Similarly polymers used for indomethacin were PVP

– HPMC - SDS combination which is also previously reported for stability of

Hydrocortisone nanosuspension (Ali et al., 2009b).

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Table 3.29: Zeta potential value of the produced nanosuspensions

Zeta potential (mV Zeta potential (mV) ± SD ± SD Formulation Batch sizes Formulation Batch sizes (3.5 ml for keto & DXI) (98 ml for keto & DXI) Drugs and (4.0 ml for IND) and (96 ml for IND) Ketoprofen Nanosuspension -17.2 ± 2.0 -17.9 ± 1.9 Dexibuprofen nanosuspension -21.5 ± 2.4 -22.1 ± 2.2 Indomethacin nanosuspension -24.3 ± 1.8 -25.0 ± 1.8 Data is presented as mean ± SD

Drugs nanosuspension

Ketoprofen Dexibuprofen Indomethacin nanosuspension nanosuspension nanosuspension 0

-5

-10

-15 Zeta Zeta potential -20

-25

Figure 3.31: Graphical presentation of Zeta potential value of the produced nanosuspensions

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3.8. Thermal and X-ray analysis

The crystallinity of the produced nanocrystals of ketoprofen, dexibuprofen and indomethacin was assessed and compared with the raw counterparts using DSC and PXRD.

These studies confirmed that the produced nanoparticles were crystalline in nature. All the raw drugs and produced nanoparticles have sharp melting endotherm. There was observed a reduction in melting point of ketoprofen, dexibuprofen and indomethacin nanocrystals related to the raw drugs. The melting point of raw ketoprofen was 95.0 ℃ while the melting point of ketoprofen nanocrystal was 90.0 ℃. (Figure 3.32). Similarly melting points of raw dexibuprofen and nanocrystals dexibuprofen were 53.0 ℃ and 50.0 ℃ respectively (Figure

3.33). Correspondingly the melting points of raw Indomethacin was 161.6 ℃ and nanoparticle Indomethacin were 159.0 ℃ (Figure 3.34)This is because of the small particle size and lower packing density of the nanocrystals lattice compared to the raw particles

(Patravale and Kulkarni, 2004a, Valleri et al., 2004). Basically, peak intensity shifted to lower level because of minor angle reflection by the nanocrystal. Furthermore, due to sizing effects the peak for the nanocrystal was also found little broadened compared to the raw drugs. The PXRD studies also confirmed the crystalline characteristics of the ketoprofen, dexibuprofen and indomethacin nanoparticles. The PXRD Diffractograms of the raw ketoprofen, dexibuprofen and indomethacin were found with sharp and high intensity peaks (Figure 3.35, 3.36, 3.37). On the other hand, for nanocrystals of ketoprofen, dexibuprofen and indomethacin, there was observed a disappearance of some peaks and decrease in the intensity of the peaks as well. Owing to small particle size effect, the diffraction patterns are different compared to the raw ketoprofen, dexibuprofen and

122

Indomethacin because peak intensity has been shifted to lower level due to minor angle reflection by the nanocrystal (Bunjes et al., 2000).

123

A. DSC of Ketoprofen, Dexibuprofen and Indomethacin Ketoprofen raw Ketoprofen nano

0 20 40 60 80 100 120 140 Temperature

Figure 3.32: DSC analysis of processed and unprocessed Ketoprofen

Dexibuprofen raw Dexibuprofen nanoparticle

0 20 40 60 80 100 120 Temperature

Figure 3.33: DSC analysis of processed and unprocessed dexibuprofen

Indomethacin Raw Indomethacin nanoparticle

50 70 90 110 130 150 170 190 210 Temperature

Figure 3.34: DSC analysis of processed and unprocessed indomethacin

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B. PXRD of Ketoprofen, Dexibuprofen and Indomethacin

0 10 20 30 40 50 60 2 Theta ketoprofen raw unprocessed Ketoprofen Nanoparticle

Figure 3.35: X Ray Diffractogram of raw and nanocrystals of ketoprofen

0 10 20 30 40 50 60 2 Theta

Dexibuprofen unprocesses Dexibuprofen nanoparticle

Figure 3.36: X Ray Diffractogram of raw and nanocrystals of dexibuprofen

0 10 20 30 40 50 60 2 Theta Indomethacin Raw Indomethacin Nanoparticle

Figure 3.37: X Ray Diffractogram of raw and nanocrystals of Indomethacin

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3.9. Morphological Studies

SEM and TEM were used for the morphological studies of raw and processed ketoprofen, dexibuprofen and indomethacin. SEM images [Figure 3.38-(i), 3.39-(i) and

3.40-(i)] shows that all the selected raw drugs are crystalline in nature. The raw ketoprofen particles are noticed as triangular and irregular in shape (Dixit et al., 2012) while raw dexibuprofen particles were rectangular in shape with smooth surface (Walser et al., 1997).

Moreover, the raw indomethacin particles were also observed of irregular shape (El-Badry et al., 2009). In addition, the images of nanocrystals obtained by TEM [Figure 3.38-(ii),

3.39-(ii) and 3.40-(ii)] clearly shows that the nanoparticles sizes of ketoprofen and dexibuprofen are below 0.100 micron (100 nm) while indomethacin nanoparticles are below 0.400 micron (400 nm) with homogenous size distribution which can potentially have faster dissolution rate. TEM images of ketoprofen, dexibuprofen and indomethacin clearly demonstrates the defined morphology indicative of crystalline materials.

126

 SEM and TEM Images of Ketoprofen (i)

(ii)

Figure 3.38: SEM and TEM images (i) Raw Ketoprofen (ii) Ketoprofen nanoparticle

127

 SEM and TEM Images of Dexibuprofen (i)

(ii)

Figure 3.39: SEM and TEM images (i) Raw Dexibuprofen (ii) Dexibuprofen nanoparticles

128

 SEM and TEM Images of Indomethacin (i)

(ii)

Figure 3.40: SEM and TEM images (i) Raw Indomethacin (ii) indomethacin nanoparticle

129

3.10. In-vitro dissolution

The in-vitro dissolution investigation of the produced nanocrystals demonstrated enhanced dissolution rate compared to the raw ketoprofen, dexibuprofen and indomethacin and marketed tablets of ketoprofen, dexibuprofen and indomethacin i.e. 100 mg, 200 mg and 25 mg respectively). Figures 3.41, 3.42, 3.43 show that approximately 84.7 % of ketoprofen nanocrystals, 85.4 % of dexibuprofen nanocrystals and 78.8 % of indomethacin nanocrystals were dissolved in first 5 minutes. On the other hands, the raw ketoprofen, dexibuprofen, indomethacin showed the dissolution rate in first 5 minutes around 22.9 %,

25.4 %, 18.3 % while marketed tablets of the selected drugs showed the dissolution rate in first 5 minutes around 33.1 %, 35.6 % and 29.2 % respectively. This significant increase in dissolution rate of ketoprofen, dexibuprofen and indomethacin nanocrystals exhibited that controlled nucleation produced nanocrystals with increased surface area and consequently enhanced dissolution rate (Khan et al., 2013a, Patravale and Kulkarni,

2004b). Moreover, it is also already proved that as per Freundlich Ostwald equation the nanoparticle with size below 100 nm have quicker dissolution rate (Müller and Peters,

1998).

130

120

100

40 Average Average of % drug dissolved 20

0 0 10 20 30 40 50 60 70 Time (Minutes)

Average % of unprocessed ketoprofen dissolved Average % of ketoprofen nanosuspension dissolved Average % of ketoprofen tablet dissolved

Figure 3.41: Dissolution studies of raw, nanoparticle and marketed product of Ketoprofen

131

120

100

Average Average of % drug dissolved 40

0 0 10 20 30 40 50 60 70 Time (Minutes)

Average % of unprocessed Dexibuprofen dissolved Average % of Dexibuprofen nanosuspension dissolved Average % of Dexibuprofen tablet dissolved

Figure 3.42: Dissolution studies of raw, nanoparticle and marketed product of dexibuprofen

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120

100

Average Average of % drug dissolved 40

0 0 10 20 30 40 50 60 70 Time (Minutes) Average % of Indomethacin unprocessed drug dissolved Average % of Indomethacin nanosuspension dissolved Average % of Indomethacin tablet dissolved

Figure 3.43: Dissolution studies of raw, nanoparticle and marketed product of Indomethacin

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134

Chapter 04

B. Preparation of Ketoprofen, Dexibuprofen and Indomethacin nanosuspensions through Media Milling method

4. Materials and Methods

4.1. Materials

 Ketoprofen (Batch no: ILKT 140005 infinity lab India),

 Dexibuprofen (Batch no: C102-150100M, Hubei biocause Helen Pharmaceutical

Co Ltd. China),

 Indomethacin (Batch no: BCBP0623V Sigma-Aldrich, UK)

 Polyvinylpyrrolidon K30 (Batch no: 08297052G0, BASF, Ludwigshafen,

Germany)

 Hydroxypropylmethylcellulose viscosity; 6cps (HPMC 6cps) (Batch No:

(8028213, BASF, Ludwigshafen, Germany)

 Sodium dodecyl sulfate (Batch no: MKBR3557V, Sigma-Aldrich, UK)

 Laboratory Distilled water was acquired at University of Bradford Research

laboratory.

 Other regents used were of analytical grade.

135

4.2. Method

4.2.1. Preparation of Ketoprofen, Dexibuprofen and Indomethacin Nanosuspension

Nanosuspensions of selected drugs (ketoprofen, dexibuprofen and indomethacin) were prepared through a size reduction system called Dena® DM100 (Sulaiman, 2007).

The system of Dena® DM100 (BK Ltd, England) consists of soft polymeric fast rotating conical rotor sitting within conical polymeric sleeve. In-between the rotor and outer sleeve, a narrow gap is formed upon filling the rotor indentations with grinding media (0.2 µm yttrium reinforced zirconium beads). Within the narrow gap, the production of high shear and turbulence provides potential for particles rupturing and shearing which results in ultrafine product formation in the size ranges of sub-micron/Nano. During the process, a suspension is produced which is recycled incessantly through a screen of stainless-steel retaining the milling media and preventing contamination of product. Finally, desired nanosuspension are collected and characterized.

In the current study, first of all, 250 ml stabilizer solutions were prepared by mixing of 3 polymers i.e. Sodium dodecyl sulfate (0.1 % w/w), 6 cps grade of HPMC (0.5 %w/w) and PVP K30 (0.5 %w/w) and water and then selected drug(s) was dispersed in polymer solution for 5minutes in stirring and then sonicated for 5min. Upon mixing of stabilizer solution and drug(s) materials gives 350 ml coarse suspension which contain the drug substance as 3.5 %w/w. The obtained coarse suspension was then kept in the feed stock hopper of media milling machine. Inside the size reduction chamber, the suspension was recycled. The effect of the milling time on nanoparticle size was determined by taking samples from the media milling machine at different interval of time and with the help of

Zetasizer Nano instrument (Malvern Instruments Ltd, UK) particle sizes were measured.

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4.3. Characterization of the Produced Nanosuspension

4.3.1. Drug content Determination

The nanosuspensions of selected drugs (ketoprofen, dexibuprofen and indomethacin) were assessed for drugs contents through U.V spectrophotometer (V-630

(JAS.C0) at 260 nm, 222 nm and 320 nm respectively.

4.3.2. Particle size Measurement

The nanosuspensions of chosen drugs (ketoprofen, dexibuprofen, indomethacin) were assessed for particle size through (PCS) photon correlation spectroscopy (Malvern

Instruments, UK). The zetaszier was calibrated as per standard default parameters mentioned in chapter no 2 (2.3.2). The polydispersity index (PDI) and average particle size diameter (AVG P size) were determined from three measurements.

4.3.3. Measurement of Zeta Potential

The produced nanosuspensions of the selected drugs (ketoprofen, dexibuprofen, indomethacin) were assessed for zeta potential at room temperature (25 ℃) by using the

Nano-zetaszier (Malvern Instruments, UK). The sample were dispersed in water and then measurements were taken. Evaluation of produced nanosuspensions of the selected drugs were done in triplicate. Calibration was done as per procedure mentioned in chapter no 2

(2.3.3.)

4.3.4. Morphological evaluation through Scanning electron microscopy

137

out to obtain the clear images. Calibration of the SEM was done through gold grid provided with equipment.

4.3.5. Transmission electron microscopy

The produced nanocrystals of ketoprofen, dexibuprofen, indomethacin were characterized for external morphology at 100 kV through transmission electron microscopy

(JEM - 1200EX, JEOL, Japan). Samples of the selected drugs nanosuspensions was prepared by pouring a drop from nanosuspensions on the copper grid surface having 400 mesh and then dried out at ambient temperature. The negative staining was also applied through an aqueous solution of 2 % magnesium uranyl acetate. Then finally prepared samples were introduced into Microscope (JEM - 1200EX, JEOL, Japan) and assessment was carried out at 100 kV.

4.3.6. Thermal analysis

Thermal Characteristics of the processed and unprocessed selected drugs

Scientific, Germany) with working temp of 4 °C, speed rotation of 20000 rpm for 45 mins.

The topmost layer was removed and the drying of the remaining residues were performed in desiccator. The unprocessed and nanoparticle of ketoprofen, dexibuprofen and indomethacin were checked for thermal examination at rate of 10 °C/min in nitrogen atmospheric condition. The examination was performed at temperature ranged of 0 to 120

138

°C for ketoprofen, 0 to 100 °C for dexibuprofen and 60 to 190°C for indomethacin.

Evaluation of all samples were done in triplicate.

4.3.7. X-ray powder diffraction (XRPD) Analysis

A). The scanning was done at angle with range of 5 - 50°, step size was 0.05°, count time was 3 s per step, rotation during analysis was 30 rpm and generator was fixed on 40 kV with 30 mA. The un processed samples of the selected drugs were put in plastic sample holder while produced nanocrystals samples of the selected drugs were put in silicon-well sample holder and then positioned in XPRD equipment for running. The XPRD equipment was calibrated through corundum standard.

4.3.8. Stability studies

(V-630 (JAS.C0) at 260 nm, 222 nm and 320 nm respectively. Moreover, physical stability of the selected drugs nanosuspensions was evaluated at refrigerated temperature 2 - 8 ºC and at 25 °C (room temp) for period of two months (60 days). The variation in the physical appearance were gauged. Moreover, the growth of the particles was observed by determining the Average particle size and PDI values at specific timing periods (0-day, 5th- day, 10th-day, 15th-day, 20th-day, 30th -day, 40th -day, 50th -day and 60th -day) through (PCS) photon correlation spectroscopy (Nano S Zetasizer®, Malvern Instruments, UK).

139

4.3.9. In-vitro dissolution evaluation

4.3.10. Statistical Examination

Statistically examination of the experimentally collected data was done by SPSS

18 (SPSS Inc., USA). The data for all drugs was collected in triplicate. The data presented here are as mean ± standard Deviation. One-way ANOVA test (p < 0.05) and least significant difference test (LSD) were used.

140

141

Chapter 05

5. Results and Discussion

B. Preparation of Ketoprofen, Dexibuprofen and Indomethacin nanosuspensions through Media Milling method

5.1. Effect of Milling time

In the top down approach, large sizes particles are reduced into smaller particles sizes by different mechanisms such as impaction and attrition. Basically energy is generated by shearing and turbulent of the milling media and particles with consequent in particles reduction (Merisko-Liversidge et al., 2003) The nanosuspension of the selected drugs (ketoprofen, dexibuprofen and indomethacin) were prepared by media milling method. The particle sizes of the coarse suspensions of ketoprofen and indomethacin decreases abruptly in first 10 minutes and then there is gradual decrease in size. Whereas particles of dexibuprofen coarse suspension decreases gradually and did not follow the earlier abrupt and gradual pattern. The particle sizes of the nanosuspensions of Ketoprofen and Indomethacin were accomplished at 60 minutes while the dexibuprofen was difficult in reducing its particle sizes and finally nanosuspension was achieved at 150 minutes. The obtained particle sizes of ketoprofen and Indomethacin were 169 ± 1.98 nm with PDI of

0.194 ± 0.04 and 161 ± 1.90 nm with PDI of 0.229 ± 0.06 respectively. The obtained particle size of dexibuprofen nanosuspension was 298 ± 2.0 with PDI of 0.234 ± 0.05.

Primarily it has been proved that shear stress has been increased with increase in milling time with consequent further decrease of particle sizes (Stenger and Peukert, 2003). The detailed data is accessible in table 5.1, 5.2, 5.3. Moreover, data is also presented in figure

5.1, 5.3, 5.5. Particle size distribution graph is offered in 5.2, 5.4, 5.6 The results obtained demonstrates that selected drugs can be produced at Nano level by media milling process

142

and the size differences are due to the difference mechanisms involved in bottom method and top down method (Verma et al., 2009).

Table 5.1: Effect of Milling time on ketoprofen nanoparticle

Milling Time AVG Particle size (nm) PDI

(minutes) ± SD ± SD

5 642 ± 3.0 0.429 ± 0.11

10 574 ± 2.7 0.401 ± 0.05

15 424 ± 2.5 0.373 ± 0.04

20 339 ± 2.3 0.312 ± 0.04

25 289 ± 2.1 0.287 ± 0.05

30 254 ± 2.2 0.261 ± 0.04

35 221 ± 2.1 0.250 ± 0.03

40 201 ± 2.2 0.244 ± 0.05

45 187 ± 2.3 0.237 ± 0.03

50 179 ± 2.1 0.201 ± 0.04

55 170 ± 1.98 0.195 ± 0.05

60 169 ± 1.98 0.194 ± 0.04

Data is presented as Mean ± SD

143

1000 0.5 900 0.45 800 0.4 700 0.35 600 0.3

500 0.25 PDI 400 0.2

AVG AVG P SIZE(nm) 300 0.15 200 0.1 100 0.05 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (minutes)

p size PDI

Figure 5.1: effect of milling time on ketoprofen nanoparticles

Figure 5.2: ketoprofen nanoparticle particle size distribution

144

Table 5.2: Effect of Milling time on dexibuprofen nanoparticle

Milling Time AVG Particle size (nm) PDI

(Minutes) ± SD ± SD

5 1374 ± 4.0 0.512 ± 0.22

10 1206 ± 3.8 0.456 ± 0.12

20 1006 ± 2.8 0.405 ± 0.06

30 858 ± 1.80 0.387 ± 0.04

45 705 ± 3.6 0.359 ± 0.03

60 681 ± 3.10 0.321 ± 0.04

75 555 ± 2.90 0.285 ± 0.05

90 462 ± 1.90 0.265 ± 0.03

105 382 ± 2.00 0.247 ± 0.05

120 315 ± 1.70 0.239 ± 0.04

135 300 ± 2.10 0.236 ± 0.04

150 298 ± 2.00 0.234 ± 0.05

Data is presented as Mean ± SD

145

0.6 1400 1200 0.5 1000 0.4

800 0.3 PDI 600 0.2

Avg Avg (nm) P Size 400 200 0.1 0 0 5 10 20 30 45 60 75 90 105 120 135 150 Time (minute)

p size PDI

Figure 5.3: effect of milling time on dexibuprofen nanoparticles

Figure 5.4: Particle size distribution of dexibuprofen nanoparticles

146

Table 5.3: Effect of Milling time on Indomethacin particle Milling AVG Particle size

Time (nm) PDI

(minute) ± SD ± SD 5 550 ± 3.0 0.442 ± 0.09

10 481 ± 2.5 0.345 ± 0.05

15 423 ± 2.2 0.289 ± 0.05

20 379 ± 2.0 0.287 ± 0.06

25 308 ± 2.4 0.279 ± 0.05

30 274 ± 2.5 0.274 ± 0.06

35 227 ± 2.3 0.263 ± 0.05

40 203 ± 2.2 0.256 ± 0.4

45 187 ± 2.0 0.229 ± 0.5

50 172 ± 1.95 0.230 ± 0.04

55 162 ± 2.00 0.229 ± 0.05

60 161 ± 1.90 0.229 ± 0.06

Data is presented as Mean ± SD

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0.5 900 0.45 0.4 750 0.35 600 0.3 0.25 450 PDI 0.2

AVG AVG (nm) P Size 300 0.15 0.1 150 0.05 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Time (minutes)

Avg P size PDI

Figure 5.5: effect of milling time on Indomethacin nanoparticles

Figure 5.6: Particle size Distribution of Indomethacin nanoparticles

148

5.2 Zeta potential

The assessment of Zeta potential is the vital parameter in scheming of the predicted stability (Patravale and Kulkarni, 2004a). The media milling process is absolutely change from precipitation process of nanonization and have different mechanisms (disintegration of larger particles into smaller particles or building of particle from molecule or atoms) and also have different problems from each other such as probability of contamination in product, engineering of particles below 100 nm and involvement of energy etc (He et al.,

2010) so consequently the same polymers used in both process produces different type of results. The achieved zeta potential value of the selected drugs was in the testified accepted level i.e. ± 20 mV. This zeta potential value has been recognized as accepted value for stability of the nanosuspension (Ali et al., 2009c).Moreover, the polymers combination

(PVP-HPMC-SDS) used in this method is already reported for stability of Ibuprofen,

Glipalamide and Artemisinin nanosuspensions by Dena® (Khan et al., 2014, Shah et al.,

2016). The detailed data is present in table 5.4.

149

Table 5.4: zeta Potential value of the produced nanosuspensions

S.No Drug Zeta Potential ± SD

1 Ketoprofen Nanosuspension -22.0 ± 2.25

2 Dexibuprofen Nanosuspension -24.3 ± 2.05

3 Indomethacin Nanosuspension -26.0 ± 1.95

Data is presented as Mean ± SD

Drugs nanosuspension

Ketoprofen Dexibuprofen Indomethacin nanosuspension nanosuspension nanosuspension 0

-5

-10

-15

-20 Zeta Zeta potential -25

-30

Figure 5.7: value of the zeta potential of the selected drugs

150

5.3. Stability studies

The produced nanosuspensions were evaluated for chemical and physical stabilities. Chemical stability was assessed for one week while physical stability was measured for two months. There was no sign of degradation. The data of the chemical stability is organized in table 5.4. Principally nanoparticles have high surface area with high surface free energy (Van Eerdenbrugh et al., 2008) and tries to agglomerate to decrease energy i.e. surface free energy. The physical stability of the produced nanosuspensions was assessed at two temperature, the first one at fridge temp (2 - 8 ºC) and second one at room temperature (25 ºC). It was observed, that all three selected drugs

(ketoprofen, dexibuprofen and indomethacin) nanosuspension stored at refrigerated temperature and at room temperature (Figure 5.8, 5.9, 5.10, 5.11, 5.12 and 5.13) were stable. The comprehensive data is presented in table 5.5, 5.6, 5.7, 5.8, 5.9, and 5.10. The

HPMC-PVP-SDS combination has been proved for stability of the drug in top down method (Plakkot et al., 2011). Moreover, it has been observed that HPMC - PVP - SDS combination adsorbs on the surface of the nanocrystal and stabilizes it (Khan et al., 2014).

Furthermore, the PDI value indicates the proper and consistent distribution of particles which avoids Ostwald ripening, so particle size is retained (Deng et al., 2010).

151

Table 5.4: Chemical stability of the produced nanosuspensions. day-0 day-1 day-2 day-3 day-4 day-5 day-6 day-7 Drugs %age of the drug content ± SD

98.2 98 97.9 97.7 97.6 97.4 97.3 97.2 Ketoprofen ± 2.2 ± 2.4 ±1.90 ± 2.4 ± 2.6 ± 2.5 ± 2.1 ± 2.5

97.9 97.7 97.7 97.6 97.5 97.1 96.8 96.6 Dexibuprofen ± 1.90 ±1.98 ±1.92 ±1.90 ± .85 ± 2.4 ± 2.0 ± 2.8

97.1 96.9 96.6 96.5 96.3 96.2 96.1 96.0 Indomethacin ± 2.0 ± 2.1 ± 2.0 ±1.95 ±1.90 ± 2.2 ± 2.5 ± 3.0

Data is presented as mean ± SD

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A. Ketoprofen

Table 5.5: stability of ketoprofen nanoparticle at 2 - 8 ºC

Days AVG P Size (nm) ± SD PDI ± SD fresh 169 ± 1.98 0.194 ± 0.04

After 5 days 170 ± 2.05 0.195 ± 0.05

After 10 days 170 ± 2.20 0.195 ± 0.06

After 15 days 171 ± 2.25 0.197 ± 0.04

After 20 days 173 ± 2.30 0.198 ± 0.05

After 30 days 175 ± 2.25 0.199 ± 0.03

After 40 days 176 ± 2.30 0.200 ± 0.06 after 50 days 177 ± 2.35 0.201 ± 0.05 after 60 days 178 ± 2.30 0.201 ± 0.04

Data is presented as Mean ± SD

200 0.250 180 160 0.200 140

120 0.150

100 PDI 80 0.100

Avg Avg (nm) P Size 60 40 0.050 20 0 0.000 fresh After 5 After 10After 15After 20After 30After 40after 50 after 60 days days days days days days days days Days

AVG P Size PDI

Figure 5.8: ketoprofen nanoparticle stability at 2 - 8 ºC

153

B. Dexibuprofen

Table 5.6: stability of dexibuprofen nanoparticle at 2 - 8 ºC

AVG P Size (nm)

Days ± SD PDI ± SD

fresh 298 ± 2.00 0.234 ± 0.05

After 5 days 299 ± 2.12 0.238 ± 0.04

After 10 days 301 ± 2.08 0.240 ± 0.05

After 15 days 304 ± 1.99 0.242 ± 0.04

After 20 days 305 ± 1.95 0.243 ± 0.03

After 30 days 307 ± 1.92 0.245 ± 0.05

After 40 days 309 ± 1.85 0.246 ± 0.04

after 50 days 310 ± 2.15 0.247 ± 0.05

after 60 days 311 ± 2.18 0.249 ± 0.05

Data is presented as Mean ± SD

350 0.35

300 0.3

250 0.25

200 0.2

150 0.15 PDI

Avg Avg Size P (nm) 100 0.1

50 0.05

0 0 fresh After 5 After 10After 15After 20After 30After 40 after 50 after 60 days days days days days days days days Days

AVG P Size PDI

Figure 5.9: Dexibuprofen nanoparticle stability at 2 - 8 ºC

154

C. Indomethacin

Table 5.7: stability of Indomethacin nanoparticle at 2 - 8 ºC

AVG P Size (nm)

Days ± SD PDI ± SD

fresh 161 ± 1.90 0.229 ± 0.06

After 5 days 162 ± 2.05 0.230 ± 0.06

After 10 days 164 ± 2.00 0.232 ± 0.06

After 15 days 164 ± 2.20 0.233 ± 0.03

After 20 days 166 ± 2.05 0.235 ± 0.04

After 30 days 167 ± 2.10 0.236 ± 0.05

After 40 days 169 ± 2.05 0.237 ± 0.04

after 50 days 171 ± 2.10 0.238 ± 0.05

after 60 days 172 ± 2.15 0.239 ± 0.05

Data is presented as Mean ± SD

200 0.3 180 160 0.25 140 120 0.2 100

0.15 PDI 80

60 0.1 AVG AVG (nm) P Size 40 0.05 20 0 0 fresh After 5 After 10 After 15 After 20 After 30 After 40 after 50 after 60 days days days days days days days days Days

AVG P Size PDI

Figure 5.10: Indomethacin nanoparticle stability at 2 - 8 ºC

155

AA. Ketoprofen

Table 5.8: stability of Ketoprofen nanoparticle at 25 ºC

AVG P Size (nm) ±

Days SD PDI ± SD

fresh 169 ± 1.98 0.194 ± 0.04

After 5 days 175 ± 2.16 0.195 ± 0.05

After 10 days 181 ± 2.37 0.196 ± 0.03

After 15 days 184 ± 2.01 0.197 ± 0.07

After 20 days 188 ± 2.09 0.198 ± 0.04

After 30 days 190 ± 2.11 0.199 ± 0.05

After 40 days 193 ± 2.15 0.201 ± 0.04

after 50 days 197 ± 2.00 0.203 ± 0.03

after 60 days 199 ± 2.03 0.204 ± 0.04

Data is presented as Mean ± SD

250 0.240 200 0.220 0.200 150

0.180 PDI 100 0.160

Avg Avg (nm) P Size 0.140 50 0.120 0 0.100 fresh After 5 After 10 After 15 After 20 After 30 After 40 after 50 after 60 days days days days days days days days Days

AVG P Size PDI

Figure 5.11: ketoprofen nanoparticle stability at 25 ºC

156

BB. Dexibuprofen

Table 5.9: stability of dexibuprofen nanoparticle at 25 ºC

AVG P Size (nm) ±

Days SD PDI ± SD

fresh 298 ± 2.00 0.234 ± 0.05

After 5 days 307 ± 2.20 0.239 ± 0.06

After 10 days 313 ± 2.30 0.241 ± 0.05

After 15 days 315 ± 2.50 0.242 ± 0.04

After 20 days 318 ± 2.35 0.244 ± 0.05

After 30 days 322 ± 2.45 0.246 ± 0.04

After 40 days 325 ± 2.40 0.250 ± 0.03

after 50 days 328 ± 2.30 0.256 ± 0.03

after 60 days 330 ± 2.35 0.260 ± 0.04

Data is presented as Mean ± SD

350 0.350

300 0.300

250 0.250

200 0.200

150 0.150 PDI

Avg Avg (nm) P Size 100 0.100

50 0.050

0 0.000 fresh After 5 After After After After After after 50after 60 days 10 days 15 days 20 days 30 days 40 days days days Days

AVG P Size PDI

Figure 5.12: Dexibuprofen nanoparticle stability at 25 ºC

157

CC. Indomethacin

Table 5.10: stability of Indomethacin nanoparticle at 25 ºC

AVG P Size (nm) ±

Days SD PDI ± SD

fresh 161 ± 1.90 0.229 ± 0.06

After 5 days 167 ± 2.20 0.230 ± 0.05

After 10 days 176 ± 2.40 0.233 ± 0.04

After 15 days 180 ± 2.15 0.234 ± 0.05

After 20 days 181 ± 2.20 0.235 ± 0.4

After 30 days 182 ± 2.11 0.236 ± 0.06

After 40 days 184 ± 1.80 0.238 ± 0.05

after 50 days 185 ± 2.40 0.241 ± 0.04

after 60 days 187 ± 2.45 0.244 ± 0.05

Data is presented as Mean ± SD

250 0.350

200 0.300

150 0.250 PDI

100 0.200 Avg Avg (nm) P Size 50 0.150

0 0.100 fresh After 5 After After After After After after 50after 60 days 10 days 15 days 20 days 30 days 40 days days days Days

AVG P Size PDI

Figure 5.13: Indomethacin nanoparticle stability at 25 ºC

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5.4. Thermal and X-ray analysis

The crystallinity of the nanocrystals of ketoprofen, dexibuprofen and indomethacin produced through milling method was judged and compared with the raw counterparts using DSC and PXRD. The evaluation confirmed the crystallinity of nanocrystals. The raw drugs and nanocrystals have sharp melting endotherm. There was observed a reduction in melting point of ketoprofen, dexibuprofen and indomethacin nanocrystals related to the raw drugs. The melting point of raw ketoprofen was 95.0 ℃ while the melting point of ketoprofen nanocrystal was 93.0 ℃. (Figure 5.14). Similarly melting points of raw and nanocrystals dexibuprofen were 53.0 ℃ and 51.0 ℃ respectively (Figure 5.15).

Correspondingly the melting points of raw Indomethacin was 161.6℃ and nanoparticle

Indomethacin were 158.0 ℃ (Figure 5.16). This is because of the small particle size and lower packing density of the nanocrystals lattice compared to the raw particles (Patravale and Kulkarni, 2004a, Valleri et al., 2004). Basically, peak intensity shifted to lower level because of minor angle reflection by the nanocrystal. Furthermore, due to sizing effects the peak for the nanocrystal was also found little broadened compared to the raw drugs. The

PXRD investigation also established the crystalline characteristics of the ketoprofen, dexibuprofen and indomethacin nanoparticles. The PXRD Diffractograms of the raw ketoprofen, dexibuprofen and indomethacin were found with sharp and high intensity peaks (Figure 5.17, 5.18, 5.19). On the other hand, for nanocrystals of ketoprofen, dexibuprofen and indomethacin, there was observed a decrease in the intensity of the peaks.

Owing to small particle size effect, the diffraction patterns are different compared to the raw ketoprofen, dexibuprofen and indomethacin because peak intensity has been shifted to lower level due to minor angle reflection by the nanocrystal (Bunjes et al., 2000).

159

C. DSC of Ketoprofen, Dexibuprofen and Indomethacin Ketoprofen raw Ketoprofen nanocrystal

0 20 40 60 80 100 120 140 Temperature

Figure 5.14: DSC analysis of nanocrystal and un processed Ketoprofen

Dexibuprofen Raw Dexibuprofen nanocrystal

0 20 40 60 80 100 120 Temperature

Figure 5.15: DSC analysis of nanocrystal and un processed dexibuprofen

Indomethacin Raw Indomethacin nanoparticle

50 70 90 110 130 150 170 190 210 Temperature

Figure 5.16: DSC analysis of nanocrystal and un processed Indomethacin

160

D. PXRD of Ketoprofen, Dexibuprofen and Indomethacin

0 10 20 30 40 50 60 2 Theta

ketoprofen raw ketoprofen nanocrystal

Figure 5.17: X Ray Diffractogram of raw and nanocrystals of ketoprofen

0 10 20 30 40 50 60 2 Theta

Dexibuprofen unprocesses Dexibuprofen nanocrystal

Figure 5.18: X Ray Diffractogram of raw and nanocrystals of dexibuprofen

0 10 20 30 40 50 60 2 Theta

Indomethacin Raw indomethacin nanocrystal

Figure 5.19: X Ray Diffractogram of raw and nanocrystals of Indomethacin

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5.5. Morphological Studies

SEM and TEM were used for the morphological studies of raw and processed ketoprofen, dexibuprofen and indomethacin. SEM images [Figure 5.20-(i), 5.21-(i) and

5.22-(i)] shows that all the selected raw drugs are crystalline in nature. The raw ketoprofen particles are noticed as triangular and irregular in shape (Dixit et al., 2012) while raw dexibuprofen particles were rectangular in shape with smooth surface (Walser et al., 1997).

Moreover, the raw indomethacin particles were also observed of irregular shape (El-Badry et al., 2009). In addition, the images of nanocrystals obtained by TEM [Figure 5.20-(ii),

5.21-(ii) and 5.22-(ii)] clearly shows that the nanoparticles sizes of ketoprofen and indomethacin are below 200 nm while dexibuprofen nanoparticles are below 400 nm with homogenous size distribution which can potentially have faster dissolution rate. TEM images of ketoprofen, dexibuprofen and indomethacin clearly demonstrates the defined morphology indicative of crystalline materials.

162

 SEM and TEM Images of Ketoprofen (i)

(ii)

Figure 5.20: SEM and TEM images (i) Raw Ketoprofen (ii) Ketoprofen nanocrystal

163

 SEM and TEM Images of Dexibuprofen (i)

(ii)

Figure 5.21: SEM and TEM images (i) Raw Dexibuprofen (ii) Dexibuprofen nanoparticles

164

 SEM and TEM Images of Indomethacin (i)

(ii)

Figure 5.22: SEM and TEM images (i) Raw Indomethacin (ii) indomethacin Nanoparticle

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5.6. In-vitro dissolution

The produced nanocrystals of the selected drugs demonstrated enhanced in-vitro dissolution rate compared to the raw ketoprofen, dexibuprofen and indomethacin and marketed tablets of ketoprofen, dexibuprofen and indomethacin i.e. 100 mg, 200 mg and

25 mg respectively). Figures 5.23, 5.24, 5.25 show that approximately 78.6 % of ketoprofen nanocrystals, 76.9 % of dexibuprofen nanocrystals and 85.6 % of indomethacin nanocrystals were dissolved in first 5 minutes. On the other hands, the raw ketoprofen, dexibuprofen, indomethacin showed the dissolution rate in first 5 minutes around 22.9 %,

25.4 %, 18.3 % while marketed tablets of the selected drugs showed the dissolution rate in first 5 minutes around 33.1 %, 35.6 % and 29.2 % respectively. This significant increase

(P < 0.05, one way ANOVA) in dissolution rate of ketoprofen, dexibuprofen and indomethacin nanocrystals displayed that produced nanocrystals has smaller sizes with increased surface area and consequently enhanced dissolution rate (Khan et al., 2013a,

Plakkot et al., 2011, Junghanns and Müller, 2008). Moreover, high dissolution rate also proved that nanoparticle did not agglomerate and retained its surface area.

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120

100

Average Average of % drug dissolved 20

0 0 10 20 30 40 50 60 70 Time (Minutes)

Average % of unprocessed drug dissolved Average % of nanosuspension dissolved Average % of tablet dissolved

Figure 5.23: Dissolution studies of raw, nanoparticle and marketed product of ketoprofen

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120

100

40 Average Average of % drug dissolved

0 0 10 20 30 40 50 60 70 Time (Minutes)

Average % of unprocessed drug dissolved Average % of nanosuspension dissolved Average % of tablet dissolved

Figure 5.24: Dissolution studies of raw, nanoparticle and marketed product of dexibuprofen

168

120

100

20 Average Average of % drug dissolved

0 0 10 20 30 40 50 60 70 Time (Minutes)

Average % of unprocessed drug dissolved Average % of nanosuspension dissolved Average % of tablet dissolved

Figure 5.25: Dissolution studies of raw, nanoparticle and marketed product of indomethacin

169

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Chapter 06

6. Conclusion

Today large no of drugs is facing poor bioavailability issue and the major cause of which is the poor water solubility. The pharmaceutical scientists are struggling to address this issue of poor water solubility by applying diverse approaches. In this respect

Nanonization technique has been recognized as a most appropriate and beneficial approach in solving poor water solubility issue. The pragmatic method for nanonization are top down and bottom up techniques. Both methods have different problems such as particle size distribution and its growth, scaling up of the batch size, product contamination, energy requirement and processing time etc. The current research has been conducted with aimed to produce nanosuspensions of chosen drugs through microfluidic channel reactor and media milling and to optimize the important parameters of the process. The study has been conducted on three NSAIDs drug (ketoprofen, dexibuprofen and indomethacin). Both methods have produced nanoparticles with good size distribution and have adequate stability. The ketoprofen have smaller nanoparticle size (61 nm ) in MCFR method while have larger nanoparticle size (169 nm ) in media milling method. Similarly, nanocrystal of dexibuprofen have smallest size (45 nm ) in MCFR method in relation to media milling method size (298 nm ). Contrarily Indomethacin have attained smaller nanoparticle size

(161 nm ) in media milling method as comparative to size (380 nm ) of MCFR method.

This study demonstrated that MFCR is a low energy and novel bottom up technology which can potentially produce the stable nanocrystals. However, the experimental and process conditions need to be properly controlled. The optimization of the parameters was done. It was determined that antisolvent and solvent flow rate greatly

171

affect the particle sizes. The high antisolvent volume to solvent volume produced the excellent and optimum results with smaller particle sizes. The inlet angle was the other substantial parameter and It was observed that low inlet angle produced nanocrystals with smaller particle size. The high mixing time with high stirring rate was the also the key factor which is required for fruitful results. Additionally, drug concentration and the appropriate selection of the polymers/surfactant are also the key factors to control the particle size during nucleation. The mixing of polymer solution with drug solution in microchannel reactor is important to get smaller particle. The obtained particle size of produced nanosuspensions of the chosen drugs were 61 nm, 45 nm and 380 nm respectively. MFCR is a technology which produces the nanocrystals without changes in the particles attributes especially crystallinity of the particles. Produced nanoparticle were stable for two months at 2 – 8 ℃ and 25 ℃. The higher dissolution rate of the produced nanocrystals compared to raw and marketed drug showed that nanocrystal has retained the sizes and PDI. The MCFR established the nanonization of the selected drugs and proved that it is simple, low energy and straightforward technique and can be utilized for variety of Active Pharmaceutical Ingredient.

Top Down method was also employed for the selected drugs. Normally Top method is normally utilized by industry. The chosen drugs (ketoprofen, dexibuprofen and indomethacin) were successfully nanonized through media milling. The milling time effect was evaluated on the chosen drugs and it was noticed that initially ketoprofen and indomethacin particle sizes decreases abruptly and then there was gradual decrease in particle size while dexibuprofen particle was decreased with gradual pattern instead of abrupt and gradual pattern. The nanocrystals of ketoprofen and dexibuprofen were

172

produced at sizes of 169 nm and 161 nm respectively while dexibuprofen nanocrystal was

298 nm. The crystalline attributes of the chosen drugs have been retained at nano-level.

The raw drugs of ketoprofen and Indomethacin were recycled for 60 min while dexibuprofen was recycled for 150 min. The produced nanosuspensions were stable for two (02) months (60 days) Moreover, the nanocrystal demonstrates higher dissolution rate as compared to marketed and raw drugs. This higher rates of dissolution also indicates that the produced nanoparticles have low particle size with high surface area with subsequent conformation of homogenous distribution of particles instead of agglomeration of particles.

6.1. Future perspective

The following point has been raised from this study

 The MCFR technology is new and needs to be employed for several active

pharmaceutical ingredients (APIs).

 To understanding the atomistic level mechanism of early stages of precipitation in

MCFR and calculation of surface free energy by Coarse grand modelling or other

modelling technique.

 The Scaling up of the batch sizes can be done via using large no of MCFR in

parallel by employing controlled parameters.

 The large-scale preparation in media milling and MCFR while using engineering

and industrial approach.

 Conversion of nanocrystals attained in MCFR and Media Milling into solid dosage

form (capsule and tablet).

 Measurement of in-vivo studies.

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174

Chapter 07

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