SYNTHESIS OF GLYCOSIDE BASED BIOCOMPATIBLE NONIONIC …prr.hec.gov.pk/jspui/bitstream/123456789/9075/1/74... · 2018. 7. 23. · Wadood Ali Shah, Dr. Abdul Sadiq, Dr. Muhammad Ayaz,

SYNTHESIS OF GLYCOSIDE BASED BIOCOMPATIBLE

NONIONIC SURFACTANTS AND THEIR APPLICATIONS

FOR NIOSOMAL DRUG DELIVERY

MUHAMMAD IMRAN

A thesis submitted to the University of Malakand in partial fulfillment of the

requirements for the degree of Doctor of Philosophy in Pharmacy.

DEPARTMENT OF PHARMACY UNIVERSITY OF MALAKAND

KHYBER PAKHTUNKHWA PAKISTAN

2013-17

SYNTHESIS OF GLYCOSIDE BASED BIOCOMPATIBLE

NONIONIC SURFACTANTS AND THEIR APPLICATIONS

FOR NIOSOMAL DRUG DELIVERY

Submitted by MUHAMMAD IMRAN Supervisor Dr. FARAHT ULLAH

Assistant Professor Department of Pharmacy University of Malakand

Co-Supervisor Dr. MUHAMMAD RAZA SHAH

Professor ICCBS University of Karachi

DEPARTMENT OF PHARMACY UNIVERSITY OF MALAKAND

KHYBER PAKHTUNKHWA PAKISTAN

2013-17

IN THE NAME OF ALLAH, THE BENEFICENT THE MERCIFUL

Read! And thy Lord is the Most Honorable and Most Benevolent, Who taught (to write) by pen, He taught man that which he knew

not

(Surah Al-Alaq 30: 3-5) Al-Quran

DDeeddiiccaattiioonn

To my parents, brother and sisters who supported me throughout my

life. They are my strength and sources of inspiration.

Table of Contents

ACKNOWLEDGEMENT ........................................................................................................ i

List of abbreviations ............................................................................................................... iii

List of Figures.......................................................................................................................... vi

List of Tables ........................................................................................................................... ix

List of Schemes ........................................................................................................................ xi

Summary ................................................................................................................................ xii

1 Introduction .......................................................................................................................1

1.1 Niosomes ......................................................................................................................4

1.1.1 Nonionic surfactants ..............................................................................................6

1.1.2 Additives used in niosomes ....................................................................................8

1.2 Drug delivery applications of niosomes .........................................................................9

1.3 Barriers to oral drug delivery....................................................................................... 11

1.4 Enhancing oral bioavailability through niosomes ........................................................ 12

1.5 Selected drugs for niosomal formulations .................................................................... 16

1.5.1 Levofloxacin ........................................................................................................ 16

1.5.2 Cefixime .............................................................................................................. 16

1.5.3 Moxifloxacin ....................................................................................................... 17

1.5.4 Ciprofloxacin ....................................................................................................... 17

2 Literature Review ............................................................................................................ 19

2.1 Glycoside based nonionic surfactants for niosomal drug delivery ................................ 22

2.2 Bergenin ..................................................................................................................... 25

3 Materials and methods .................................................................................................... 28

3.1 Materials used ............................................................................................................. 28

3.2 Animals used .............................................................................................................. 29

3.2.1 Approval of studies protocols ............................................................................... 29

3.3 Synthesis of nonionic surfactants ................................................................................ 29

3.3.1 Synthesis of Tetradecyl bromide derivative of Bergenin (BRG-BTD-14) ............. 30

3.3.2 Synthesis of Lauroyl chloride derivative of Bergenin (BRG-LRC-12) .................. 31

3.3.3 Synthesis of Bromoundecane derivative of Bergenin (BRG-BRM-11) ................. 31

3.3.4 Synthesis of Bromodecane derivative of Bergenin (BRG-BRD-10) ..................... 32

3.3.5 Synthesis of Bromononane derivative of Bergenin (BRG-BRN-9) ....................... 33

3.4 Characterization of nonionic surfactants ...................................................................... 34

3.5 Biocompatibility studies .............................................................................................. 34

3.5.1 In-vitro cells cytotoxicity studies .......................................................................... 34

3.5.2 Hemolysis assay................................................................................................... 35

3.5.3 In-vivo acute toxicity study .................................................................................. 36

3.6 Preparation of drug loaded niosomal formulations ....................................................... 36

3.6.1 Preparation of Ciprofloxacin loaded BRG-BTD-14 niosomal formulation ............ 37

3.6.2 Preparation of Cefixime loaded BRG-LRC-12 niosomal formulation ................... 37

3.6.3 Preparation of Cefixime loaded BRG-BRM-11 niosomal formulation .................. 38

3.6.4 Preparation of Levofloxacin loaded BRG-BRD-10 niosomal formulation ............ 38

3.6.5 Preparation of Moxifloxacin loaded BRG-BRN-9 niosomal formulation .............. 38

3.7 Characterization of drug loaded niosomal formulations ............................................... 39

3.7.1 Surface morphology, zeta potential, size and PDI ................................................. 39

3.7.2 Drug entrapment efficiency .................................................................................. 39

3.7.3 In-vitro drug release study .................................................................................... 40

3.8 Stability studies ........................................................................................................... 40

3.8.1 Storage stability ................................................................................................... 40

3.8.2 Stability in simulated gastric fluid ........................................................................ 41

3.9 HPLC methods for quantification of drugs in plasma .................................................. 41

3.9.1 HPLC method for Ciprofloxacin quantification .................................................... 41

3.9.2 HPLC method for Cefixime quantification ........................................................... 42

3.9.3 HPLC method for Levoflixacin quantification ...................................................... 43

3.9.4 HPLC method for Moxifloxacin quantification .................................................... 43

3.10 In-vivo bioavailability studies .................................................................................. 44

3.11 Statistical analysis ................................................................................................... 46

4 Results and discussion ..................................................................................................... 47

4.1 Characterization of the synthesized nonionic surfactants ............................................. 47

4.1.1 BRG-BTD-14 ...................................................................................................... 47

4.1.2 BRG-LRC-12....................................................................................................... 49

4.1.3 BRG-BRM-11 ..................................................................................................... 52

4.1.4 BRG-BRD-10 ...................................................................................................... 54

4.1.5 BRG-BRN-9 ........................................................................................................ 56

4.2 Biocompatibility studies .............................................................................................. 59

4.2.1 In-vitro cells cytotoxicity studies .......................................................................... 59

4.2.2 Blood hemolysis assay ......................................................................................... 66

4.2.3 In-vivo acute toxicity ............................................................................................ 69

4.3 Ciprofloxacin loaded BRG-BTD-14 niosomal formulation .......................................... 70

4.3.1 Characterization ................................................................................................... 70

4.3.2 Stability studies .................................................................................................... 75

4.3.3 In-vivo bioavailability studies ............................................................................... 77

4.4 Cefixime loaded BRG-LRC-12 niosomal formulation ................................................. 81




4.5 Cefixime loaded BRG-BRM-11 niosomal formulation ................................................ 89




4.6 Levofloxacin loaded BRG-BRD-10 niosomal formulation .......................................... 96


4.6.2 Stability studies .................................................................................................. 100

4.6.3 In-vivo bioavailability studies ............................................................................. 102

4.7 Moxifloxacin loaded BRG-BRN-9 niosomal formulation .......................................... 104

4.7.1 Characterization ................................................................................................. 104

4.7.2 Stability studies .................................................................................................. 107

4.7.3 In-vivo bioavailability studies ............................................................................. 109

Conclusion .......................................................................................................................... 112

References.............................................................................................................................. 113

i

ACKNOWLEDGEMENT

All praise for almighty Allah, who blessed me to accomplish this enormous task. My special

praises are for the Holy Prophet Hazrat Muhammad (P.B.U.H), the greatest educator, the

everlasting source of guidance and knowledge for humanity. He taught the principles of morality

and eternal values.

I pay special gratitude and sincere thanks to my research supervisor Dr. Farhat Ullah, Assistant

Professor Department of Pharmacy, University of Malakand Chakdara Dir, for his intellectual

inputs and continuous support in completing this research work. His professional insight towards

research and life taught me how to set high standards in life and how to meet them positively.

Indeed without his keen interest and support I would never have been able to complete this

project otherwise.

I wish to acknowledge my co-supervisor Professor Dr. Muhammad Raza Shah, International

Center for Chemical and Biological sciences (ICCBS), HEJ Research Institute of Chemistry,

University of Karachi, who initiated me into the philosophy of synthetic chemistry and drug

delivery research and provided me conducive environment in carrying out the experimental work

in his laboratory at ICCBS. His insightful comments and constructive criticisms at different

stages of my research was quite enough in accomplishing this huge task.

I will always remember the useful guidance of Dr. Waqar Ahmed, Dr. Mir Azam Khan, Dr. Syed

Wadood Ali Shah, Dr. Abdul Sadiq, Dr. Muhammad Ayaz, Dr. Shahzeb Khan, Dr. Muhammad

Shoaib, Dr. Muhammad Junaid, Dr. Munasib Khan and Mr. Jehangir Khan, Department of

Pharmacy, University of Malakand.

ii

I owe special dept of thanks to Mr. Muhammad Amir and Mr. Muhammad Ali, lab assistants in

Center for Bioequivalence Studies and Clinical Research (CBSCR), ICCBS, for their help that

they extended towards me during my two and half year stay in ICCBS. I am also thankful to Mr.

Naveed, Mrs. Namra, Mrs. Sehrosh and Mrs. Rozina, HPLC analysts at CBSCR, ICCBS, for

their help in analytical studies of this research.

I am highly thankful to my friends Mr. Muhammad Rahim, Mr. Shafiullah (PhD scholar, UoM)

Mr. Imdad Ali (PhD scholar, ICCBS), Dr. Mujib Ur Rehman, Assistant professor, ICCBS, Dr.

Muhammad Irfan, Assistant professor, ICCBS, Dr. Zafar Ali Shah, ICCBS, Dr. Farid Ahmed,

ICCBS, for their continuous support during my studies. They always came forward whenever I

was in trouble.

I would like to thank Higher Education Commission (HEC), Pakistan for providing me

scholarship for my PhD studies under indigenous scholarships program. The financial support it

provided to me was enough to continue my research studies.

On the whole, I pay my gratitude to all those in helping me accomplishing my work.

Muhammad Imran

iii

List of abbreviations

ACN Acetonitrile

AFM Atomic Force Microscope

AUC Area under the concentration time curve

AUMC Area under the first moment curve

Cl Clearance

Cmax Maximum plasma drug concentration

d Doublet

DLS Dynamic Light Scattering

DMEM Dulbecco’s Modified Eagle’s medium

DMF Dimethyl Formamide

DMSO Dimethyl sulfoxide

FBS Fetal Bovine Serum

h Hour

HPLC High-performance liquid chromatography

H3PO4 Phosphoric Acid

i.p Intraperitoneally

iv

K2CO3 Potassium Carbonate

LD50 Lethal dose in fifty percent of objects

LUV Large Unilamellar Vesicles

min Minute

MLV Multilamellar Vesicles

mM milimole

MRT Mean residence time

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide

Na2SO4 Sodium Sulfate

PBS Phosphate-buffered saline

PDI Polydispersity Index

PLL Poly-L-lysine

P-gp P-Glycoprotein

rpm Revolution per minute

s Singlet

sec Second

SUV Small Unilamellar Vesicles

v

t Triplet

Tmax Time to reach maximum plasma drug concentration

TLC Thin layer chromatography

vi

List of Figures

Figure 1. 1: Representation of nonionic surfactant based bilayered niosomal vesicle ...................5

Figure 1. 2: Chemical structures of different nonionic surfactants used in niosomal formulations.

...................................................................................................................................................7

Figure 1. 3: Diagram representing barriers to oral drug delivery ................................................ 12

Figure 4. 1: Mass spectra of “BRG-BTD-14” ............................................................................ 48

Figure 4. 2: 1H NMR spectra of “BRG-BTD-14” ...................................................................... 49

Figure 4. 3: Mass spectra of “BRG-LRC-12” ............................................................................ 50

Figure 4. 4: 1HNMR spectra of “BRG-LRC-12” ........................................................................ 51

Figure 4. 5: Mass spectra of “BRG-BRM-11” ........................................................................... 53

Figure 4. 6: 1HNMR spectra of “BRG-BRM-11” ....................................................................... 54

Figure 4. 7: Mass spectra of “BRG-BRD-10” ............................................................................ 55

Figure 4. 8: 1HNMR spectra of “BRG-BRD-10” ....................................................................... 56

Figure 4. 9: Mass spectra of “BRG-BRN-9” .............................................................................. 57

Figure 4. 10: 1HNMR spectra of “BRG-BRN-9” ....................................................................... 58

Figure 4. 11: In-vitro cytotoxicity of BRG-BTD-14 against 3T3 cells after (A) 24 and (B) 48 h

where (C) and (D) show its cytotoxicty against HeLa cells after 24 and 48 h respectively ......... 60

vii

Figure 4. 12: In-vitro cytotoxicity of BRG-LRC-12 against 3T3 cells after (A) 24 and (B) 48 h


Figure 4. 13: In-vitro cytotoxicity of BRG-BRM-11 against 3T3 cells after (A) 24 and (B) 48 h


Figure 4. 14: In-vitro cytotoxicity of BRG-BRD-10 against 3T3 cells after (A) 24 and (B) 48 h


Figure 4. 15: In-vitro cytotoxicity of BRG-BRN-9 against 3T3 cells after (A) 24 and (B) 48 h


Figure 4. 16: Hemolysis activity of (A) BRG-BTD-14 and (B) BRG-LRC-12 at various

concentrations. .......................................................................................................................... 67

Figure 4. 17: Hemolysis activity of (A) BRG-BRM-11, (B) BRG-BRD-10 and (C) BRG-BRN-9

at various concentrations ........................................................................................................... 68

Figure 4. 18: AFM images of Ciprofloxacin loaded BRG-BTD-14 niosomal vesicles. ............... 72

Figure 4. 19: In-vitro drug release study of Ciprofloxacin loaded BRG-BTD-14 niosomal

formulation. .............................................................................................................................. 75

Figure 4. 20: Plasma drug concentration of Ciprofloxacin loaded in BRG-BTD-14, commercial

suspension and solution at various time intervals. ...................................................................... 80

Figure 4. 21: AFM images of Cefixime loaded BRG-LRC-12 niosomal vesicles. ...................... 83

Figure 4. 22: In-vitro drug release study of Cefixime loaded BRG-LRC-12 niosomal formulation

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

viii

Figure 4. 23: Plasma drug concentration of Cefixime loaded BRG-LRC-12, Cefiget (Capsule)

and Maxpan (Suspension) at various time intervals. .................................................................. 88

Figure 4. 24: AFM images of Cefixime loaded BRG-BRM-11 niosomal vesicles ...................... 90

Figure 4. 25: In-vitro drug release study of Cefixime loaded BRG-BRM-11 niosomal

formulation ............................................................................................................................... 92

Figure 4. 26: Plasma drug concentration of Cefixime loaded BRG-BRM-11, Cefiget (Capsule)

and Maxpan (Suspension) at various time intervals. .................................................................. 95

Figure 4. 27: AFM images of Levofloxacin loaded BRG-BRD-10 niosomal vesicles. ............... 98

Figure 4. 28: In-vitro drug release study of Levofloxacin loaded BRG-BRD-10 niosomal

formulation. ............................................................................................................................ 100

Figure 4. 29: Plasma drug concentration of Levofloxacin loaded in BRG-BRD-10 nioosmal

formulation, Tablets and solution at various time intervals. ..................................................... 103

Figure 4. 30: AFM images of Moxifloxacin loaded BRG-BRN-9 niosomal vesicles ................ 105

Figure 4. 31: In-vitro drug release study of Moxifloxacin loaded BRG-BRN-9 niosomal

formulation. ............................................................................................................................ 107

Figure 4. 32: Plasma drug concentration of Moxifloxacin loaded in BRG-BRN-9 niosomal

formulation, Tablets and solution at various time intervals. ..................................................... 110

ix

List of Tables

Table 4. 1: Characterization of Ciprofloxacin loaded BRG-BTD-14 niosomes for zeta potential,

size and PDI and encapsulation efficiency ................................................................................. 74

Table 4. 2: Pharmacokinetic parameters of Ciprofloxacin loaded in BRG-BTD-14 based

niosomal formulation, commercial suspension and solution. P values<0.05 was considered

significant ................................................................................................................................. 81

Table 4. 3: Characterization of Cefixime loaded BRG-LRC-12 niosomes for zeta potential, size,

PDI and drug encapsulation efficiency ...................................................................................... 84

Table 4. 4: Pharmacokinetic parameters of BRG-LRC-12 based Cefixime niosomal formulation,

Maxpan (suspension) and Cefiget (Capsules). P values<0.05 was considered significant ........... 89

Table 4. 5: Characterization of Cefixime loaded BRG-BRM-11 niosomes for zeta potential, size

and PDI and drug encapsulation efficiency. ............................................................................... 91

Table 4. 6: Pharmacokinetic parameters of BRG-BRM-11 based Cefixime niosomal suspension,

Maxpan (suspension) and Cefiget (Capsules). P values<0.05 was considered significant ........... 96

Table 4. 7: Characterization of drug loaded BRG-BRD-10 niosomes for zeta potential, size and

PDI and drug encapsulation efficiency. ..................................................................................... 99

Table 4. 8: Pharmacokinetic parameters of Levofloxacin loaded in BRG-BRD-10 based niosomal

formulation, Tablets and solution. P values<0.05 was considered significant ........................... 104

Table 4. 9: Characterization of Moxifloxacin loaded BRG-BRN-9 niosomes for zeta potential,

size and PDI and encapsulation efficiency. .............................................................................. 106

x

Table 4. 10: Pharmacokinetic parameters of Moxifloxacin loaded in BRG-BRN-9 based

niosomal formulation, tablets and solution. P values<0.05 was considered significant ............. 111

xi

List of Schemes

Scheme 3. 1: Synthesis of BRG-BTD-14 ................................................................................... 30

Scheme 3. 2: Synthesis scheme of BRG-LRC-12 ...................................................................... 31

Scheme 3. 3: Synthesis scheme of BRG-BRM-11 ..................................................................... 32

Scheme 3. 4: Synthesis scheme of BRG-BRD-10 ...................................................................... 33

Scheme 3. 5: Synthesis scheme of BRG-BRN-9 ........................................................................ 34

xii

Summary

Niosomes are nonionic surfactant based self-assembling drug delivery systems. They have got

increasing scientific interests due to their higher physical and chemical stability,

biocompatibility, safety, cost-effectiveness and efficient drug loading capability. Currently

available commercial nonionic surfactants have complex chemical structures with varying

degrees of ethoxylation. This leads to complicated chemical analysis and product specifications,

which in turn leads to hazardous effects. This has led the synthetic chemists and drug delivery

scientists to search for biologically safe and effective nonionic surfactants from renewable

resources.

The current research was undertaken for the synthesis of five double tail biocompatible

glycosidic nonionic surfactants for niosomal drug delivery applications. A naturally occurring

glycoside “Bergenin” was chemically derivitized with alkyl halides and acyl halide. These

synthetic nonionic surfactants were labeled as “BRG-BTD-14”, “BRG-LRC-12”, “BRG-BRM-

12”, “BRG-BRD-10” and “BRG-BRN-9”, depending upon the type of alkyl or acyl halide and

hydrocarbon chain length. They were characterized through Mass spectroscopy and 1HNMR. All

the nonionic surfactants were investigated for their biocompatibility through blood hemolysis, in-

vitro cytotoxicit and in-vivo acute toxicity. Niosomal drug delivery applications of the

synthesized nonionic surfactants were explored using Ciprofloxacin, Cefixime, Levofloxacin

and Moxifloxacin as model drugs. The drugs loaded niosomal vesicles of these synthesized

nonionic surfactants were characterized for shape, size, size distribution and surface charge using

atomic force microscope and dynamic light scattering respectively. They were also investigated

for in-vitro drug release and drug entrapment efficiency using HPLC and UV-spectrophotometer

respectively. Niosomal formulations of the selected drugs were assessed for their stability upon

xiii

storage and incubation with simulated gastric fluid. The in-vivo oral bioavailability of these

drugs entrapped in novel niosomal formulations was investigated in rabbits and drugs in plasma

were detected through HPLC.

Mass spectroscopy and 1HNMR analysis confirmed the synthesis of nonionic surfactants. They

were found to be to be nontoxic against 3T3 and HeLa cancer lines even after 48 h incubation

and at 1000 µg/mL concentration. The hemolysis assay showed them to be highly hemo-

compatible. They did not cause any lethality in animals up to 2000 mg/kg body weight dose. All

the surfactants were capable of self-assembling in niosomal nano-vesicles, entrapping the

respective selected drugs. Their drug loaded vesicles were almost spherical with nano-range size.

These vesicles exhibited small variations in their size distribution with highly negative surfaces.

They were able to entrap increased concentrations of drugs. The in-vitro release study confirmed

that they released their entrapped drugs in a sustained and programmed manner and did not show

any abrupt release of their entrapped substances. They were highly stable and retained the

maximum amount of entrapped drugs upon their storage and incubation with harsh simulated

gastric fluid. The drugs in-vivo oral bioavailability was increased in a sustained manner when

they were orally administered in the form of novel niosomal formulations as compared to their

commercial formulations and free solutions.

Findings of this research work confirm that these synthesized nonionic surfactants are highly

biocompatible, safe and effective niosomal nano-carriers. They can be efficient biomaterials for

constructing drug delivery systems to achieve enhanced oral bioavailability and therapeutic

efficacy of drugs having lower water solubility and permeability. They can also be efficiently

used in targeted delivery of drugs to specific sites through their surface functionalization with

active ligands.

Chapter 1 Introduction

1

1 Introduction

Nanotechnology deals with the study of engineering the materials at macromolecular, molecular

and atomic, levels having size in nano-range. It offers unique solutions to problems and exploits

different tools and ideas for various applications in pharmaceutical industry. Devices and

materials fabricated through nano-technological principles are able to interact with the tissues

and cells with greater degree of specificity. Thus, it makes the integration of devices with

biological systems that was not achievable in the past. Nanotechnology encompasses the design,

production, characterization and applications devices, structures and systems by controlling their

size in nano-range. Materials having size in nano-range reveal their unique interesting

characteristics. Their physical properties are changed and their biological functions get enhanced

at this size scale [1, 2].

Nanotechnology has been a greater subject of recent scientific interests. It plays a vital role in

advanced medical and biological research especially in the designing of drug delivery systems

that carry drug substances specifically to diseased sites, thus enhancing their therapeutic efficacy

with their lower toxicities on other healthy tissues [3]. Nano-technological developments have

led to the concept of nano-medicines. Nano-medicine covers the production, characterization and

applications of biological, chemical and physical systems in nano-size, ranging from individual

molecules and atoms to nanometers in diameter, followed by the integration of these structures

with biological systems for achieving real-time application. Nano-medicine uses nano-sized tools

for treatment and prevention of diseases and diagnosis to understand their complex patho-

physiology. The concept of nano-medicines has been introduced with the aim to increase life

expectancy chances at lower cost of the treatment with lower risk of their drug side effects [4].

Many active pharmaceutical molecules have been formulated into their commercial nano-


2

medicines in the last decades. This has led the pharmaceutical industries to pay more attention to

nano-medicines for introducing innovative drug dosage forms with higher clinical outcomes of

the drugs at lower cost and less chances of side effects [5].

Currently, nano-medicines are dominated by nano-scale drug delivery systems. As described

earlier, materials in their nano-scale range are able to achieve unique properties that can be

efficiently exploited for various medical purposes. This has opened new avenues for the

designing of innovative and efficient drug delivery formulations [6]. Designing of novel nano-

drug delivery systems have been reported for improving the bioavailability and pharmacokinetic

parameters, resulting in enhanced therapeutic efficacy of the drugs. The problems associated

with drugs like poor aqueous solubility, degradation, bioavailability, toxicological effects, lower

selectivity for the target sites and their erratic release from dosage forms are addressed through

designing novel drug delivery systems [7-10]. Cells have specific receptors on their surfaces that

recognize certain ligands. Drug delivery systems surface modified with certain ligands are

capable of delivering and releasing the loaded drug molecules selectively at the required sites.

Moreover, nano-scale devices can readily interact with bio-molecules due to their large surface

area and size in nano-range [1].

Due to smaller size, unique surface characteristics, peculiar biological and physico-chemical

effects, various nano-carriers systems for delivering and targeting drugs have been developed or

currently under development [11]. Such systems are designed in order for increasing the in-vivo

efficiency of various drugs including anticancer drugs. They are designed drugs with an aim to

protect the drugs from degradation upon their administration, minimize their unwanted side

effects, target them selectively to the required diseased sites and enhance their oral

bioavailability [12]. Among various nano-carriers, vesicular systems have emerged novel means


3

of delivering drug molecules for enhancing their bioavailability in a sustained and controlled

manner, resulting in increased therapeutic efficacy of drugs over a longer time. They have

lamellar structure surrounded by aqueous compartment and are formed due to the self-

assembling of amphiphilic molecules in aqueous medium. They can load and deliver both

lipophilic and hydrophilic drug molecules which are encapsulated in outer lipid layer or interior

hydrophilic compartment respectively [13]. Liposomes and niosomes are the representative

classes of such vesicles made of phospholipids and nonionic surfactants respectively [14].

Liposomes are nano-vesicualr drug delivery systems with spherical morphology and are prepared

from naturally occurring phospholipids with cholesterol as additive. They are widely employed

for biomedical and biotechnological purposes [15]. Liposomal formulations are widely used for

loading and delivering drug molecules of various types, especially anticancer, proteins, genes

and vaccines. They have also been used for diagnosis of various pathological conditions [16-18].

Liposome have been reported to be associated with certain drawbacks like physical, chemical

and biological instabilities, rapid clearance from the body, short half-life, lipid hydrolysis and

oxidation, vesicles fusion and leakage of drugs, lower reproducibility rates, difficulties in

industrial scale production, lower sterility and increased cost of production [19, 20]. Physical and

chemical instabilities have been widely reported for decreasing the in-vivo therapeutic efficacy of

drugs loaded and delivered in liposomes. These instabilities result in the fusion of the vesicles

and abrupt and premature release of the loaded contents in the surrounding environment [21].

The biological fluids also greatly affect the integrity and permeability properties of the

liposomes, thus leading to their inferior clinical outcomes [22].

The above mentioned limitations of liposomal formulations have led the formulations scientists

to develop more stable, cost-effective and efficient vesicular drugs delivery systems. Niosomes,


4

nonionic surfactants based vesicular systems, have emerged best vesicular drug delivery vehicles

and as best alternative to liposomes. They are cost-effective and having higher physical,

chemical and biological stabilities as compared to liposomes [23]. Similarly, niosomes have been

reported to entrap increased concentrations of the drug substances as compared to liposomes.

Moreover, they do not require special storage or handling conditions. Niosomal formulations are

also advantageous over liposomes because of their enhanced permeability and penetration

through biological membranes, resulting in the ultimate increased bioavailability of the drugs

[24-26]. The structural composition, characteristics and drugs delivery applications of niosomes,

especially for enhancing the oral bioavailability, are described in the following section.

1.1 Niosomes

Niosomes are nonionic surfactants based vesicles and were discovered by L’Oreal in the 1970s

[27]. Originally, they were developed to substitute liposomes with such alternatives that are

capable of delivering drugs in a controlled way and can address the problems associated with

liposomes [28, 29]. These vesicles were used for loading and delivering anticancer drugs for the

very first time [30, 31]. Technically, niosomes are nano-range closed bilayer vesicles formed by

the self-assembling of nonionic surfactants when they come in contact with aqueous medium

with cholesterol as an additive [32]. The exact mechanism of vesicles formation of nonionic

surfactants based vesicles is not known. The formation of a closed bilayer structure by the

nonionic surfactants upon their hydration with an aqueous medium is the most widely accepted

theory (Figure 1.1). The closed bilayer architecture requires the application of energy in the

forms of physical agitation or gentle heat [33, 34]. The crux of closed bilayer is that “vesicular

structure” formation requires an amphiphilic molecule in contact with aqueous environment. The

self-assembly of the amphiphilic molecules occurs due to increased interfacial tension between


5

hydrocarbon portion of nonionic surfactant and aqueous medium, leading them to be associated

in a special morphological shape. At the same time, the hydrophilic head groups establish their

contact with the aqueous environment through the steric ionic and hydrophilic repulsion between

the head groups. Thus, supramolecular assembly results due to these opposite forces. The

formation of niosomes also requires the addition of some additives like cholesterol [35].

Figure 1. 1: Representation of nonionic surfactant based bilayered niosomal vesicle

Niosomes can load both lipophilic and hydrophilic drug molecules. They encapsulate hydrophilic

drug substances in the aqueous pool of the vesicles, while the hydrophobic drugs get dissolved in

the lipid bilayers [23]. Depending upon their size and bilayers structures, niosomes are classified

in three different groups. Small unilamellar vesicles (SUV) have single bilayer with a size in the

range of 10–100 nm. Large unilamellar vesicles (LUV) have single bilayer structure and are

mostly found in a size range of 100–3000 nm. Multilamellar vesicles (MLV) are having more


6

than one bilayer in their structures having a size in the 1-20 μm range [36, 37]. Owing to surface

morphology and drug entrapment pattern, niosomes have very closed resemblance with

liposomes. Their cost effectiveness, higher stability and ease in storage have make them as best

alternatives to phospholipids based vesicles [38].

1.1.1 Nonionic surfactants

Nonionic surfactants are surfactants that do not get deionized in aqueous medium due to the

absence of charged groups in the hydrophilic portions of their structures. They are more stable

and biocompatible and have lower toxicities as compared to their amphoteric, cationic and

anionic counterparts [39]. Moreover, they have been advantageous as they maintain pH up to

physiological pH along with their characteristic functions of wetting, solubilization and

permeability enhancement. Being good inhibitors of p-glycoprotein, they are used to enhance the

bioavailability of anticancer and other drugs [13]. Thus, they have greater scientific attention for

formation of stable niosomes for both in-vivo and in-vitro drug delivery applications. Nonionic

surfactants like ester-linked surfactants, crown ethers, glucosyl dialkyl ethers, ethers of

polyglycerol alkyl, Brij, polyoxyethylene alkyl ethers, Tweens and Spans are most commonly

used for the preparation of niosomes. Some commonly and most widely used in nonionic

surfactants in niosomes formation are given in Figure 1.2.


7

Figure 1. 2: Chemical structures of different nonionic surfactants used in niosomal formulations.

The researchers are also trying to synthesize biocompatible synthetic nonionic surfactants for

niosomal drug delivery purposes. Recently, the synthesis of nonionic surfactants with anticipated

desired physico-chemical properties has got much scientific attention. Crown ether amphiphiles

have been extensively synthesized and studied for niosomal drug delivery. They are unique and

can be modulated for desired properties by virtue of their controlled synthesis [40, 41]. Recently,

sugar based nonionic surfactants have got greater scientific attention due to their applications in

many fields. Surfactants of this class are highly biocompatible, biodegradable and are economic

as they are derivitized from renewable resources. This has led to the exploration of a wide


8

variety of sugar based nonionic surfactants for niosomal drug delivery [42, 43]. The availability

of sugar moiety in such surfactants has been highly advantageous. It enhances the hydrophilicity

and adds to the chirality of the surfactants. This in turn modulates and improves the physical

characteristics like increased surfactancy, high biodegradability, lower levels of animals and

human toxicities, higher degree of emulsification and surface interactions. It also leads to

increased antimycotic and antimicrobial potentials of the surfactants [44]. Similarly, cancerous

cells have increased rate of metabolism and they require increased amount of glucose, thus they

uptake increasing amount of glucose as compared to normal cells [45]. Thus, nonionic

surfactants containing glucose or glucose derivatives can be efficiently used for targeting

anticancer drugs to cancerous cells with higher degree of selectivity, resulting in effective

chemotherapy with lower side effects of the drugs [46]. It supports the use of vesicles made

from such surfactants for developing target delivery system [47].

1.1.2 Additives used in niosomes

Cholesterol is used as additive in niosomes as it gives structural stability and influences the

physical properties of niosomes due to its interactions with nonionic surfactants. The interactions

of cholesterol with nonionic surfactants in niosomal vesicles are of greater biological interest.

Being an integral part of the biological membrane, it greatly affects the membrane properties like

permeability, aggregation, fusion process, enzymatic activity, shape, size and elasticity.

Cholesterol is added to niosomes in order to enhance their mechanical strength, cohesion and

permeability towards water. The fluidity of niosomes is enhanced in the presence of cholesterol.

Moreover, the presence of cholesterol in niosomes also increases their rigidity which in turn

protects them in harsh environmental conditions [13, 48].


9

Charged inducers are intended for enhancing the stability of niosomal vesicles. They are added

to the vesicles bilayers in specific concentrations. They prevent the niosomal vesicles

aggregation by increasing the surface charge density. Phosphatidic acid and dicetyl phosphate

are widely used substances for producing negative charges on the surfaces of niosomes. Stearyl

pyridiniumchloride and stearylamine are most commonly used positively charged molecules in

niosomal preparation. The charges substances are usually added to niosomes in a concentration

of 2.5–5 mol%. When the concentration of charge inducers is increased, they prevent the

niosomal vesicles formation [49].

1.2 Drug delivery applications of niosomes

Niosomes are capable of loading and delivering various therapeutically active molecules for the

treatment of different diseases. They can also be used as vehicles for designing novel drug

delivery systems for poorly absorbed drugs. They are able to cross gastrointestinal tract barriers

through transcytosis of Peyer's patches M cells in intestinal lymphatic tissues, thus enhance the

drugs bioavailability [25]. Diseases like leishmaniasis where parasites attack liver and spleen

can be treated efficiently through niosomal drug delivery. The niosomal vesicles have been

reported for their uptake by reticulo-endothelial system. This results in localization of drugs

which in turn helps in the treatment of liver and spleen in an improved way. Similarly,

immunoglobulins like non-reticulo-endothelial systems can also recognize niosomal vesicles due

to their lipid surface [14, 50].

Niosomal vesicles are widely used for encapsulation of anticancer drugs in order to enhance their

therapeutic efficacy and minimize their side effects on other normal tissues [51]. Doxorubicin,

an anticancer drug with broad-spectrum anti-tumor efficacy, has been associated with


10

irreversible cardio-toxicity in dose-dependent manner [52, 53]. When delivered in niosomal

vesicles to S-180 tumor bearing mice, doxorubicin was found to increase the mice life span due

to its elevated potentials of decreasing the sarcoma proliferation. In another similar study,

methotrexate entrapped in niosomal vesicles was intravenously administered to S-180 tumor

bearing mice and total regression of tumor was achieved. Moreover, the niosomal vesicles

caused elevated plasma level for the drug for an extended period of time with its lower clearance

from the body [54]. Niosomes formulations have also been exploited for investigating immune

response produced due to certain antigens. The most important characteristic of niosomal drug

delivery is its potential for accumulating the drugs at site action in increased concentration.

Drugs with lower water solubility and narrow therapeutic index have been successfully delivered

with sustained release through niosomal formulations [55].

Transdermal administration of drugs is often required for quick and localized efficacy of drugs.

Various anti-inflammatory drugs such as piroxicam and flurbiprofen and sex hormones such as

levonorgestrel and estradiol are commonly delivered in niosomes through transdermal route for

improving their clinical efficacy [14]. Niosomal drug delivery through transdermal route results

in the enhanced absorption of drugs via connective and epithelium tissues, achieving localized

action of the drugs. This also avoids the systemic toxicities of the drugs and enhances the drugs

therapeutic efficacy many folds at the required sites of action. When encapsulated in niosomal

vesicles, antimonials show enhanced therapeutic efficacy at the site of action with reduced

toxicity due to the uptake of the niosomes by mononuclear cells [51].


11

1.3 Barriers to oral drug delivery

Oral route has been the most convenient and easy way for drug administration. It is preferred

over other routes of drug administration due to several advantages. It is painless and requires no

assistance, thus offers greater patient compliance. Several drugs show their lower therapeutic

efficacy upon their oral administration due to their lower aqueous solubility and permeability

[56, 57]. Poor aqueous soluble drugs are unable to achieve minimum therapeutic concentration in

blood, thus they exhibit lower oral bioavailability [58, 59]. Various factors are involved in the

drugs poor oral bioavailability. Drugs are absorbed when they get solubilized in the aqueous

medium. Lower soluble drugs are poorly absorbed, thus show lower bioavailability upon their

oral administration. Similarly, improper partition coefficient also influences the permeation of

drugs through lipid membrane, resulting in their lower oral bioavailability. First pass effect is

another factor that causes the metabolism of drugs, leading to their poor absorption and low oral

bioavailability. P-glycoprotein (P-gp) mediated efflux has been widely reported for affecting the

oral bioavailability of drugs. Its presence in kidneys, liver and intestine reduces the drugs

absorption from the gastrointestinal tract and enhances their elimination, thus lowers the drugs

oral bioavailability with ultimate inferior therapeutic efficacy. Some drugs are unable to

withstand the harsh enzymatic or acidic environment of stomach and they get degraded. Thus, a

small fraction of such drugs becomes available in the systemic circulation, resulting in their

lower therapeutic efficacy [56, 60]. All the barriers reducing the therapeutic efficacy of orally

administered drugs are depicted in Figure 1.3.

The above mentioned challenges can be resolved by introducing innovative nano-carriers based

drug delivery systems like niosomes. Such systems are highly effective in increasing the drugs

oral bioavailability through enhancing their aqueous solubility, permeability and protection from


12

degradation. They also offer advantages like reduction in doses and dosing frequency and

targeting of drugs to target sites with higher degree of selectivity [61, 62]. Following section

contains a brief review of the reported literature about niosomes used for enhancing oral

bioavailability of drugs.

Figure 1. 3: Diagram representing barriers to oral drug delivery

1.4 Enhancing oral bioavailability through niosomes

Different mechanisms have been reported for enhanced oral bioavailability of drugs delivered

through niosomal vesicles. To reach systemic circulation, the drugs must be first dissolved in

gastrointestinal fluids. This indicates dissolution has been the rate-limiting factor in oral


13

administration of poor aqueous soluble drugs. It results in irregular absorption of the drugs and

thus they become available in the systemic circulation in a small fraction. Such spherical carriers

are intended to enhance the solubility of poor water soluble drugs, thus leading to their increased

oral bioavailability [63]. Hepatic and intestinal first-pass effect also leads to decreased oral

bioavailability of drugs to a greater extent. Niosomal nano-carriers have been reported for

stimulation of lymphatic transport, thus bypass the first-pass metabolism and improve the oral

bioavailability of drugs [64, 65]. Drugs taken are suspected to suffer from inferior oral

bioavailability due to their early clearance from the body and degradation or inactivation by

biological enzymes. Niosomes can improve the oral bioavailability of the drug molecules by

delaying their clearance from blood stream and protecting them from harsh biological fluids [14].

As earlier discussed, P-gp efflux is one of the factors decreasing the oral bioavailability of drugs.

Its presence in kidneys, liver and intestine results in the reduced absorption of drugs from

gastrointestinal tract and increases their clearance from the body. Niosomal vesicles have been

found effective inhibitors of P-gp and have been reported for enhancing the oral bioavailability

of various antiviral and anticancer drugs [13, 39]. Similarly, the size of drug delivery systems in

nano-range also plays a vital role in the increased absorption of drugs gastrointestinal tract.

Small size of the carrier is considered important for improving the drugs permeation through the

epithelial cells and thus lead to elevated bioavailability following oral administration [66].

Surface modifications of niosomal formulations with bile salts and positive and negative charge

inducers has been another reported mechanism for enhancing the oral bioavailability of drugs.

Such modified niosomal formulations interact better with biological systems and get absorbed

and thus ultimately lead to their enhanced oral bioavailability [63].


14

A niosome formulation of acyclovir has been reported for enhancing the in-vivo bioavailability

of the drug in rabbits. The oral bioavailability and mean residence time of acyclovir were

increased more than 2-fold compared to its tablet dosage form [26, 67]. When griseofulvin was

loaded in niosomal formulation and then investigated for in-vivo bioavailability study, its oral

bioavailability was enhanced in a sustained manner [25]. In a study, the niosomes prepared form

Span 40 and coated with bio-adhesive carbopol polymers resulted in enhanced oral

bioavailability of anticancer drug paclitaxel [68]. Glimpiride, an anti-diabetic drug was loaded in

niosomal vesicles prepared form combination of different nonionic surfactants. The niosomal

formulation of the drug caused its elevated oral bioavailability for longer time as compared to

commercial formulation of the drug [69].

The oral bioavailability of poor water soluble docetaxel, an important anticancer drug, was

enhanced through the designing of a novel niosomal nano-carrier. The niosomal system was

modified with D-alpha-tocopheryl polyethylene glycol succinate, an aqueous soluble derivative

of polyethylene glycol, in order to make it long circulating through inhibition of P-gp efflux

system. The novel niosomal formulation caused enhanced oral bioavailability (7.2 times greater)

of docetaxel as compared to its simple solution [70]. Carvedilol is an important antihypertensive

drug that suffers from lower and variable bioavailability. Its different niosomal formulations

were prepared and modified with bile salts and both positive and negative charge inducers. The

niosomes containing bile salts were found to increase the oral bioavailability of carvedilol about

two times in comparison with simple suspension of the drug. Similarly, the positively and

negatively charged niosomal formulations were also found to increase the oral bioavailability of

the drug about 2.3 and 1.7 times higher than that of simple suspension of the drug. The charges

on the surfaces were found responsible for enhancement in the drug absorption [63].


15

Tenofovir disoproxil fumarate has been very important antiviral agent and has been a drug of

choice in the treatment of HIV-1 infection and hepatitis-B in humans. It suffers from lower oral

bioavailability (25%) due to its poor permeability across the biological membrane of

gastrointestinal tract. A niosomal formulation containing cholesterol, dicetyl phosphate and

surfactant in variable molar rations was used for enhancing its oral bioavailability. The

formulation resulted in more than two times increase in the drug oral bioavailability as compared

to its solution. The enhancement in the drug bioavailability was stated to be due to enhanced

absorption of drug loaded niosomes through gastrointestinal track after oral administration [71].

More recently the niosomal formulation of a poor water soluble drug celecoxib was investigated

for oral bioavailability in human volunteers. Findings of the study indicated that the niosomes

greatly enhanced the drug absorption as compared to its commercial capsules [72].

Metformin, an anti-diabetic drug, has been reported with 50% oral bioavailability and get early

clearance in renal tubule secretion. Its oral bioavailability was enhanced through its niosomal

formulations modified with positive and negative charge inducers. The increase in oral

bioavailability of metformin was attributed to muco-adhesive properties of the charges surfaces

of niosomal formulations that led to increased absorption of the drug loaded vesicles. Moreover,

the oral bioavailability was found to be enhanced in a sustained manner over an extended period

of time. This was found to be due to the charges induction that caused the drug release in a slow

and gradual manner [73]. Ganciclovir is an effective inhibitor of human herpes viruses and has

been found highly active against varicella-zoster, cytomegalo and Epstein-Barr viruses. The drug

has low permeability leading to inferior oral bioavailability and therapeutic efficacy. The in-vivo

oral bioavailability studies showed that ganciclovir bioavailability increased significantly upon

its loading in niosomal formulation. This was attributed to various factors like influence of small


16

vesicle size, lipophilicity of niosomal vesicles, greater partitioning of the lipophilic system

towards mucosa and localization of niosomal vesicles at absorption site for longer period of time

[74].

1.5 Selected drugs for niosomal formulations

1.5.1 Levofloxacin

Levofloxacin is an important synthetic fluoroquinolone antibiotic used for the treatment of

various bacterial infections. It prevents DNA synthesis or its repair through the inhibition of

DNA gyrase enzyme, thus resulting in the bacterial lyses [75]. It is commonly used for the

treatment of acute maxillary sinusitis, community-acquired pneumonia and acute exacerbation of

chronic bronchitis. The drug comes from BCS class I, having pH dependent aqueous solubility

[76]. However, it is quickly absorbed and 87% of the oral dose is excreted unchanged in the

urine [77]. This pharmacokinetic behavior of Levofloxacin leads to its oral bioavailability

achieved within short span of time, but with short half-life [78]. Maintaining minimum

therapeutic level of Levofloxacin for an extended period of time would result in better

therapeutic efficacy of the drug. It can be materialized by delivering the drug in a suitable drug

delivery system in order to make it long circulating in the systemic circulation and delay its

clearance from the body.

1.5.2 Cefixime

Cefixime belongs to third generation cephalosporin and is widely used antibiotic against various

gram-positive and gram-negative bacterial strains. It is effective against cefaclor, cephalexin,

trimethoprim-sulfamethoxazole and ampicillin resistant Neisseria gonorrhoeae, Klebsiella


17

pneumonia, Haemophilus influenza and Escherichia coli [79, 80]. Cefixime is aqueous insoluble

due to its weak acidic nature. About 40–50% of its fraction gets excreted through renal and

biliary routes, thus leading to its poor bioavailability with ultimately lower clinical and

therapeutic efficacy [81]. Owing to its lower water solubility and ultimate decreased oral

bioavailability, Cefixime can be a model drug for delivering in a specific drug delivery system.

Designing of a niosomal formulation for Cefixime would be a versatile idea for increasing its

water solubility and oral bioavailability.

1.5.3 Moxifloxacin

Moxifloxacin is an important broad spectrum fluoroquinolone and is used against infections

caused by haemophilus influenza, Streptococcus pneumonia and Moraxella catarrhalis. It is also

effective against Mycoplasma pneumonia and Chlamydia pneumoniae [82, 83]. Being an intense

hydrophilic and a member of biopharmaceutical classification system (BCS) class I drugs,

Moxifloxacin oral administration faces several issues such as increased aqueous solubility, early

clearance from the body and pH dependent dissolution. Thus, its oral intake results in

hepatotoxicity and gastrointestinal disturbances. This also results in development of

Moxifloxacin resistance in tuberculosis [84-86]. Thus, Moxifloxacin is a best candidate for

niosomal drug delivery with an aim to enhance its oral bioavailability in a sustain manner along

with elimination of its dose associated toxicities.

1.5.4 Ciprofloxacin

Ciprofloxacin is an effective and broad-spectrum a fluoroquinolone antibiotic and is widely used

against the infections caused by gram-positive cocci and various other gram-negative bacterial

strains. Ciprofloxacin in unique in terms of its superior penetrating potentials into the tissues and


18

gets accumulated in neutophilis and macrophages, thus showing its bactericidal activity in low

environments [87, 88]. These properties make Ciprofloxacin as a drug of choice for treating

infections caused by intracellular pathogens. When it is administered intravenously or orally, it

reaches organs such as spleen, liver, lymph nodes and lungs where intercellular bacterial strains

are found abundantly [89, 90]. Ciprofloxacin is a weak basic BCS class IV drug and is

practically insoluble in water at neutral pH [62]. When given orally, it is absorbed quickly and its

peak plasma concentration (2.5 µg/mL) is achieved within a short period of time (1-2 h) at a 500

mg dose. Thus, the effective therapy with this drug requires the use of multiple doses for

maintaining the minimum therapeutic level of the drug. Moreover, its absorption becomes further

highly decreased when the drug goes down through gastrointestinal tract. This means that

Ciprofloxacin oral bioavailability has been lower due to its poor absorption in the stomach and

small intestine [91]. The drug oral bioavailability can be enhanced by enhancing its aqueous

solubility and absorption from stomach and upper small intestine [92].

Chapter 2 Literature review

19

2 Literature Review

Being versatile nano-vesicular drug carriers, niosomes offer greater opportunities for changes in

their composition through using different suitable surfactants, resulting in preferred

physicochemical and biological features necessary for achieving the desired therapeutic response

[93]. Presently used commercial surfactants have complex chemical structures with varying

degrees of ethoxylation. This leads to complicated chemical analysis and product specifications,

which in turn leads to hazardous effects. Being manufactured from petrochemical products,

presently accepted surfactants are also facing sever environmental concerns [94]. Owing to

limitations of presently used surfactants, various novel surfactants have been synthesized for

achieving innovative niosomes based vesiclular systems aiming at improving the therapeutic

efficacy of delivered drugs [93]. Synthesis of specialized surfactants with structural blocks from

cheap and renewable resources has got scientific interest during last few years [95]. Therefore,

discovering novel biocompatible surfactants with renewable structural blocks and studying their

potentials for drug delivery applications is highly needed.

Literature reports various studies carried out for synthesis of novel nonionic surfactants and their

investigation for niosomal drug delivery. Palmitoyl muramic acid has been reported as a novel

synthetic surfactant. It was used as niosomal drug delivery vehicle for loading and delivering

doxorubicin. The drug loaded vesicles were found with a mean size of 250 nm and exhibited

slight changes in size when incubated at 37 °C for a period of 24 h. The study was highly

interesting as the drug loaded vesicles of the novel nonionic surfactants were found to be

preferentially taken by spleen as compared to liver [96]. N. Ménard et al synthesized lipoamino

acid based surfactants and successfully employed them in the niosomal delivery of anticancer

drug. These surfactants were found less cytotoxic and hemolytic than Tween 80. These novel


20

surfactants solubilized increased amount of the drug and were concluded to be promising carriers

for hydrophobic drug candidates [97].

In a recent study, a series of double-tailed polyglyceryl dialkyl ethers based nonionic surfactants

were synthesized. All the surfactants were screened for their physico-chemical properties

including aqueous phase behavior, effect of carbon chain based lipophilicity and molecules

packing in lamellar phase. All the synthesized surfactants were investigated for their niosomes

forming abilities and they were capable of forming micro-size niosomes by vertex mixing. When

subjected to ultrasonication, they formed niosomal vesicles in nm range. The effects of

cholesterol addition were also studied and it was found that addition of cholesterol caused phase

transition to liquid ordered phase, similar to a phospholipid−cholesterol mixture. The resultant

niosomes with or without cholesterol were subjected to their stability. Stability was determined

in terms of changes in vesicles size and they were found highly stable for 100 days [95].

A novel crown ether nonionic surfactant based on crown ether has been synthesized and reported

for developing a long time stable controlled release niosomal drug delivery system. The system

was used for encapsulating 5-fluorouracil, an anticancer drug [41]. In another study, a similar

new crown ether based new nonionic surfactant was used for the encapsulation and topical

delivery of ammonium glycyrrhizinate, a molecule with anti-inflammatory potentials and

obtained from plants. The new surfactant exhibited a higher degree of tolerability both in-vitro

and in-vivo. It caused increased amount of the drug to be entrapped into its niosomal vesicles

having nano-range size. The niosomal vesicles of the new surfactant were found to enhance the

drug permeation across the skin and its cellular uptake as compared to free drug solution [98].

Similarly, well-defined polyoxyethylene sorbitol oleate was derivitized and exploited as drug

delivery cargo for improving efficacy and safety of anticancer drug docetaxel. The surfactant


21

was found highly biocompatible, causing less hemolysis and histamine release as compared to

the commercially available polysorbate 80. The novel nonionic surfactant delivery systems

improved the safety and efficacy of docetaxel by increasing its targeting towards tumors when

screened for in-vivo imaging in S180 tumor bearing mice [99].

Supramolecular amphiphiles are similar to nonionic surfactants and can form nano-vesicles upon

their self-assembly. They are currently widely used as drug delivery vehicles and are preferred

due to their versatile structural characteristics and drugs molecules encapsulation through host-

guest binary vesicles [100, 101]. Recently, nonionic surfactant like p-phosphonated calix[4]arene

derivative amphiphile was synthesized and explored for delivery of anticancer drug paclitaxel in

the form of nano-vesicles. The paclitaxel loaded amphiphilic calexarene amphiphile nano-

vesicles were further conjugated with folic acid as a tumor targeting ligand. The novel nano-

vesicles were quite capable of delivering the drug specifically to tumor cells due to the over

expression of folate receptors. Moreover, the novel amphiphilic calexarene based vesicles were

also able to unload all the encapsulated paclitaxel contents in the micro-environment of the

tumor cells due to their pH sensitivity, thus leading to enhanced paclitaxel anti-tumor efficacy

with minimum toxic effects on other healthy tissues and cells [102]. Similar supramolecular

nonionic amphiphiles were synthesized by modifying cyclodextrin modified with hydrophobic

chains of intermediate length. The novel nonionic supramolecular amphiphiles were used for the

encapsulation of isoflavone and genistein [103].

Amphiphilic peptides are synthesized from amino acids and are able to self-assemble in nano-

structures of various morphologies. Nonionic surfactants like self-assembling peptides consist of

consecutive lipophilic amino acids as their tail and one or two hydrophilic amino acids as their

head [104]. Recently, an amphiphilic peptide having tetra-tail structure was synthesized for


22

delivering drug selectively to target site. It contained one hydrophilic peptide head group and

four hydrophobic aliphatic tails. It was investigated for its self-assembling potential and was

found cable of forming vesicles in nano-range size in aqueous environment. Doxorubicin and

ibuprofen were used as model hydrophobic drugs for delivering in the nano-vehicles based on

the novel peptide amphiphile. The drugs were found to be released in a sustained release pattern.

The novel drug delivery system was observed to result in a targeted drug delivery due to the

presence of arginine-glycine-aspartic acid sequences, resulting in the specific recognition of

cancerous cells and efficient transport through cell membrane of the constituting peptide.

Moreover, when loaded with porphyrin, the novel peptide based drug delivery system showed

decreased dark toxicity and increased phototoxicity against tumor cells, indicating higher

potentials for efficient photodynamic therapy [105]. Similarly, synthesis of amphiphilic

dipeptides containing conformation-constraining dehydrophenylalanine has been reported for

their formation of self-assembly into nano-vesicles. The vesicles were able to encapsulate small

drug molecules such as riboflavin and vitamin B12. The vesicles were found highly

biocompatible when tested for their toxicity in cancerous cells. The novel vesicles were also

investigated for their cellular uptake study, showing their increased uptake through selected cell

lines [106].

2.1 Glycoside based nonionic surfactants for niosomal drug delivery

Sugar surfactants are distinguished class of surfactants. They carry hydrophobic moieties and

hydrophilic carbohydrate head groups like glucose, sucrose or glycoside. Sugar surfactants with

two or more hydrophobic chains are usually referred to as glycolipids ([107, 108]. Naturally

occurring sugar-based surfactants have obtained increased attention for drug solubilization

because of their interesting characteristics like high surface activity, biodegradability and lower


23

toxicity. Glycosidic moieties in the surfactant represent a strategy for drug targeting since

glycosidic receptors are over-expressed in many types of tumor cells, whereby glycoside-based

drug carriers easily act as tumor targeting ligands. It is also well established that blood–brain

barrier cells express high levels of the glucose transporter GLUT1 [109], therefore, sugar-based

niosomal drug delivery systems could be a valuable approach for achieving an efficient targeted

drug delivery to the brain [110]. Therefore, sugar surfactant systems are gifted candidates for

replacing the traditional surfactants used for pharmaceutical and cosmetic preparations [111-

113].

Novel glycoside based nonionic surfactants from renewable resources are attracting greater

scientific attention due to their cost-effectiveness and their liquid crystalline phase forming

ability. Moreover, they are highly biodegradable, less toxic and having unique chiral properties

[114]. Such nonionic surfactants always contain a hydrophilic sugar ring moiety attached to a

lipophilic tail in the shape of hydrocarbon chain. The physicochemical properties of the

glycoside based nonionic surfactants are determined by the nature of hydrophobic tails, type of

sugar in the head group and its stereochemistry [115]. They are widely used in food,

pharmaceutical and cosmetic industries due to their known safety profiles. Glycoside based

nonionic surfactants have emerged best alternatives to the commercially available

polyoxyethylene surfactants [116]. Currently used commercial nonionic surfactants are from

petro-chemical origin and having complex chemical structures, thus they are associated with

drawbacks such as product specification issues, variations in production batches, complix

chemical analysis and side effects like inflammatory responses due to their histamine releasing

potentials [117].


24

R. Muzzalupo et al. reported novel niosomes based on alkyl glucopyranoside surfactants for

niosomal delivery of methotrexate for cancer therapy. The surfactants were screened for

biocompatibility and characterized for drug loading efficiency and vesicles size. The drug loaded

vesicles were found to be in 300-500 nm range with low polydispersity. The surfactant loaded

increased concentration of methotrexate, confirming significant interactions between the

niosomal matrices and drug. The glucoside based surfactants vesicles caused delayed release

pattern of the encapsulated drug. Results also confirmed that the surfactant toxicity reduced upon

its vesicles formation [118]. In another study, alkyl polyglucoside was used an alternative

surfactant. Polymeric nanoparticles of phobic human gel filtration fraction 2 phydreptide were

prepared. These peptide loaded polymeric nanoparticles were stabilized with alkyl polyglucoside

surfactant. The stabilization effects of the used surfactant on particle size, drug loading

efficiency and biological activity were investigated in detail. Results revealed that the use of

alkyl polyglucoside surfactant led to smaller size drug particles with enhanced drug entrapment

efficiency, indicating its effective utilization in drug delivery [119].

N.F.K. Aripin et al synthesized novel mixture of alkyl glycosides nonionic surfactants derived

from palm kernel oil or palm oil in 2012. The mixture contained different glycosides with

variable stereoisomers and lipophilic alkyl chains. These glycosides were used as niosomal drug

carrier using vitamin E as active pharmaceutical ingredient. The synthesized glycosidic

surfactants had the ability of self-assembling with a small fraction of dicetyl phosphate as

additive and loaded increased amount of vitamin E. The vesicular formulations of these

surfactant were also screened for their stability and were found highly stable up to 3 months

storage period [113]. Similarly, in another recent study, nonionic surfactants of alkyl glycosides

based on octyl glycoside and maltoside were synthesized and were studied for niosomal drug


25

delivery applications. Both the novel nonionic surfactants were able to form highly stable nano-

range vesicles through their self-assembly in aqueous environment [120]. In a study, a novel

nonionic surfactant having basic structural unit of glucuronic acid was synthesized and used for

delivering two anticancer drugs doxorubicin and 5-fluorouracil. The surfactant was capable of

forming niosomal vesicles with and without cholesterol. The resultant niosomal vesicles of the

surfactant were highly smaller in size demonstrated to encapsulate both 5-fluorouracil and

doxorubicin and released them back in sustained manner. Hemoglobin release study in red blood

cells revealed that the vesicles caused release of hemoglobin in a size dependent manner [42].

2.2 Bergenin

Bergenin is a C-glycoside of 4-O-methyl gallic acid and is widely found in various species of

different families like Mallotus philippinensis, Mallotus japonicas (Euphorbiaceae) and genus

Bergenia (Saxifragaceae) [121, 122]. Bergenin has been a versatile molecule and has been

highly neuro-protective [123], anti-tussive, anit-hypolipidaemic, anti-inflammatory, anti-HIV

and gastro-protective [124, 125]. Similarly, it has elevated free radicals scavenging potentials

and is equally potent to ascorbic acid. It has also been reported for both in-vitro and in-vivo

hepatoprotective potentials [126]. All these studies show that it is highly safe in living system

and can be derivitized for the synthesis of glycoside based nonionic surfactants intended for

niosomal drug delivery applications.

This study has been designed to synthetically derivitize Bergenin with alkyl and acyl halides

having lipophilic chains of different carbon number. The synthesized new nonionic surfactants

have been investigated for their biocompatibility through in-vitro cytotoxicity in different cell

cultures and blood hemolysis assay. The self-assembly of these nonionic biocompatible


26

surfactants was used for niosomal drug delivery of different selected drugs. The drug loaded

niosomal vesicles were characterized for size, polydisersity index, surface morphology, zeta

potential, drug encapsulation efficiency, in-vitro drug release behavior and stability studies.

Niosomal vesicles loaded with selected drugs were used for enhancing their oral bioavailability

in animals’ model. Drugs delivered in these innovative niosomal formulations were quantified in

plasma through HPLC.

Synthesis of such biocompatible new nonionic surfactants from renewable sources has been of

greater scientific interests. Innovative nano-carriers like these niosomal vesicles not only

improve the therapeutic efficacy of drugs, but also reduce the overall cost of treatment strategy.

The preliminary investigations of these niosomal drug delivery vehicles will provide a scientific

base to both the nano-drug delivery technologists and formulation scientists for searching out the

most possible mechanisms for their drug solubilization capabilities, cellular uptake and drug

absorption across mucosal membrane. These results will also open new avenues for formulation

scientists to find their use in disease site specific and targeted delivery, especially in delivering

chemotherapeutic agents by virtue of their glycosidic nature.


27

Aims and Objectives

i. To synthesize glycoside-based biocompatible nonionic surfactants for niosomal drug

delivery applications.

ii. To achieve cost effective and enhanced therapeutic efficacy of selected BCS class I, II

and IV drugs.

Chapter 3 Materials and methods

28

3 Materials and methods

3.1 Materials used

Deionized water was used for preparation of solutions throughout the research. All the organic

solvents consumed in this research were of HPLC grade and were purchased from Sigma-

Aldrich, USA. Dulbecco’s Modified Eagle’s medium (DMEM), Fetal Bovine Serum (FBS),

Polylysine (PLL) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT)

were used in cells cytotoxicity studies and were obtained from Sigma-Aldrich, Germany.

Commercial Tween 80 was used as reference standard and was purchased Merck, Germany.

Cholesterol was used as additive in preparation of niosomal formulations and was purchased

from BDH, UK. Bergenin, 1-bromoundecane, lauroyl chloride, 1-bromodecane, 1-Bromononane,

1-Bromotetradecane, K2CO3 and Na2SO4 were used in the synthesis of new nonionic surfactants

and were all purchased from Sigma-Aldrich, USA.

Different drugs and their commercial formulations were used in in-vivo oral bioavailability

studies. Cefixime commercial suspension (Maxpan) was obtained from Indus Pharma, Pakistan,

and Cefixime commercial capsules (Cefiget) from Getz Pharma, Pakistan. Levofloxacin tablets

(Leflox) were supplied by Getz Pharma, Pakistan. Moxifloxacin commercial tablets (Moxiget)

were obtained from Getz Pharma, Pakistan. Ciprofloxacin commercial suspension (Novidat) was

obtained from Sami Pharma, Pakistan. Cefixime, Ciprofloxacin and Levofloxacin were obtained

from Sigma-Aldrich, Germany. Pure Moxifloxacin was a kind gift from Getz Pharma, Pakistan.


29

3.2 Animals used

Animals for the studies were purchased from Dow University of Health Sciences, Karachi,

Pakistan. Swiss albino mice were used for acute toxicity studies while oral in-vivo bioavailability

studies were performed in rabbit’s local species “Oryctolagus cuniculus”. All the animals were in

good health and disease free condition according to the certificates issued by Animal Facility

Center, Dow University of Health sciences, Karachi, Pakistan. All the animals were housed in

animal house under standard conditions of 12 h day/night cycle and with free access to food and

water.

3.2.1 Approval of studies protocols

Principles of Institutional Animal Care and Use were strictly followed for studies involved use of

animals. Protocols for the studies were approved by the Institutional Ethical Committee for the

use of animals in research, International Center for Chemical and Biological Sciences (ICCBS),

University of Karachi, Pakistan. Protocol “ICCBS-027-CEF-2014” was approved for animals

used in in-vivo studies involving synthesized nonionic surfactants BRG-LRC-12 and BRG-

BRM-11 and drug Cefixime. Similarly, protocol number “ICCBS-2016-0003” was approved for

animals used in in-vivo studies involving synthesized nonionic surfactants BRG-BTD-14, BRG-

BRD-10 and BRG-BRN-9 and drugs Levofloxacin, Moxifloxacin and Ciprofloxacin.

3.3 Synthesis of nonionic surfactants

A series of five different nonionic surfactants was synthesized by chemical derivitization of

Bergenin. They are labeled as BRG-BTD-14, BRG-LRC-12, BRG-BRM-11, BRG-BRD-10, and

BRG-BRN-9.


30

3.3.1 Synthesis of Tetradecyl bromide derivative of Bergenin (BRG-BTD-14)

In a round bottom flask equipped with magnetic stirrer and reflux condenser, Bergenin (656 mg;

2 mM) and K2CO3 (556 mg; 4 mM) were taken in 15 mL dimethyl formamide (DMF) and stirred

for 30 min. Then 1.2 mL (4 mM) Tetradecyl bromide was added to the mixture and further

refluxed for 8 h. Thin layer chromatography (TLC) was used periodically for monitoring the

progress of reaction. The reaction mixture was cooled at ambient temperature upon reaction

completion and extracted with chloroform (3 × 30 mL). The organic solvents were evaporated

and the product was further dried over Na2SO4. A pure white solid product was obtained with a

90% yield.

Scheme 3. 1: Synthesis of BRG-BTD-14


31

3.3.2 Synthesis of Lauroyl chloride derivative of Bergenin (BRG-LRC-12)

Bergenin (492 mg; 1.5 mM) and K2CO3 (415 mg; 3 mM) were refluxed in 15 mL acetone for 45

min in round bottom flask equipped with magnetic stirrer and condenser. Then lauroyl chloride

(656 mg, 3 mM) was added and reaction was monitored through TLC. After 4 h of refluxing,

complete conversion of starting materials to product was observed. The mixture was added water

(30 mL) and extracted with chloroform (3×30 mL). Organic solvents were evaporated under

vacuum and further dried over Na2SO4. A final pure colorless solid product was obtained with

90% yield.

Scheme 3. 2: Synthesis scheme of BRG-LRC-12

3.3.3 Synthesis of Bromoundecane derivative of Bergenin (BRG-BRM-11)

Bergenin (492 mg; 1.5 mmol) and K2CO3 (415 mg; 3 mmol) were refluxed for 45 min in DMF

(15ml) in round bottom flask equipped with a magnetic stirrer and condenser. Then

bromoundecane (534 mg; 3 mmol) was added to the reaction mixture and the progress was


32

monitored using TLC. Upon refluxing the mixture for further 4 h, starting materials were

converted into product. The reaction was stopped by adding water (30 mL) to the reaction

mixture and then extracted with chloroform (3×30 mL). The organic phases were combined and

concentrated under vacuum. The product was obtained as pure colorless solid upon drying over

Na2SO4 with 85% yield.

Scheme 3. 3: Synthesis scheme of BRG-BRM-11

3.3.4 Synthesis of Bromodecane derivative of Bergenin (BRG-BRD-10)

Bergenin (492 mg; 1.5 mmol) and K2CO3 (415 mg; 3 mmol) were refluxed for 45 min in DMF

(10 ml) in n a round bottom flask equipped with a magnetic stirrer and condenser. Then, 1-

Bromodecane 1.25 mL (6 mmol) was added to the reaction mixture and the progress was

monitored using TLC. Starting materials were completed converted to product upon further

refluxing for 4 h. The mixture was cooled at ambient temperature and reaction was stopped by

adding water (30 mL). The mixture was extracted with chloroform (3×30 mL). Organic phases


33

were combined and concentrated under vacuum. Pure colorless product was obtained upon

drying with Na2SO4.

Scheme 3. 4: Synthesis scheme of BRG-BRD-10

3.3.5 Synthesis of Bromononane derivative of Bergenin (BRG-BRN-9)

In a round bottom flask, 328 mg (1.0 milimol) Bergenine and 280 mg (2.0 milimol) K2CO3 were

taken and stirred in 15 ml DMF for 30 minutes. then, 0.4 mL (2.0 milimol) Bromononane was

added to the mixture and refluxed for further 8 h. Reaction progress was monitored with TLC

using Ethylacetate/ n-hexane (7:3 V/V) as solvent system. When the reaction was complete,

reaction mixture was cooled at ambient temperature and was added water (150 mL) following by

its extraction with Ethylacetate (40 mL × 3 times). The organic phase was combined, rotary

evaporated and dried. The crude mixture was run on silica-gel column chromatography using

solvent system EtOAc/ n-hex (1:1 V/V). Final pure product was obtained as a white solid

powder.


34

Scheme 3. 5: Synthesis scheme of BRG-BRN-9

3.4 Characterization of nonionic surfactants

The synthesized nonionic surfactants were characterized for their structures through Mass and

1HNMR spectroscopic techniques.

3.5 Biocompatibility studies

3.5.1 In-vitro cells cytotoxicity studies

Two different cell lines of HeLa (Human Cervical Cancer) and NIH/3T3 (Mouse embryonic

fibroblast) were used for cells cytotoxicity studies. Nonionic surfactants BRG-BTD-14, BRG-

LRC-12, BRG-BRM-11, BRG-BRD-10, and BRG-BRN-9 were investigated for their

cytotoxicity using MTT assay. NIH/3T3 and HeLa cells were cultured in DMEM containing FBS

(10%) and antibiotics (penicillin and streptomycin, 50 units/mL of each) at 37 °C in 5% CO2

humidified atmosphere. Plate wells containing 200 μL medium were seeded with HeLa and 3T3

cells at 6×104 and 8.0×103 cells per density respectively. After 24 h incubation, medium was

replaced with fresh medium (200 μL) containing test samples in various concentrations (62.5-


35

1000 µg/mL). To achieve negative control, cells were grown in the medium without adding test

samples. Cells were further incubated for growth for 24 and 48 h. Each well was added MTT

solution (5 mg/mL, 20 μL) prepared in PBS. Dye that did not react was removed from the

medium after 4 h incubation. Crystal of purple formzan were obtained and were dissolved in

dimethyl sulphoxide (DMSO, 200 μL per well) and the absorbance was measured at 570 nm

using microplate readers (ELx808, BioTek, USA). For positive control and reference standard,

poly (L) lysine (PLL) and Tween 80 were used respectively. Percent cell viability for the test

samples was calculated after 24 and 48 h using the following formula:

Percent cells viability =

(Test sample mean Absorbance) / (Negative control mean absorbance) × 100

3.5.2 Hemolysis assay

Hemo-compatibility of BRG-BTD-14, BRG-LRC-12, BRG-BRM-11, BRG-BRD-10, and BRG-

BRN-9 was investigated in blood obtained from viens of human volunteers. Fresh blood was

subjected to centrifugation for 10 min at 700 g for erythrocytes separation through

sedimentation. The erythrocytes pellet was washed three times with 7.4 pH phosphate buffer

solution (PBS) and subjected to centrifugation for 10 min at 700 g followed by its suspending in

PBS in 1:10 ratio (Erythrocytes: PBS, w/v). Erythrocytes suspension (0.2 mL) was mixed with 4

mL of test samples having concentrations in the range of 62.5-1000 μg/mL. Tween 80 was tested

as the reference standard. The samples were further incubated for h at 37 °C followed by their

centrifugation (700 g for 10 min) for removal of non-lysed erythrocytes. Supernatants containing

released hemoglobin were carefully collected and then read spectrophotometrically (Shimadzu,

UV-240, Hitachi U-3200, Japan). Hemoglobin released from the cells as a function of


36

synthesized nonionic surfactants destabilization effects on cell membrane was detected at 540

nm. PBS and distilled water was incubated with erythrocytes suspension for achieving 0% and

100% hemolysis respectively. Percent hemolysis was determined using the following formula:

Percent Hemolysis = (Abs - Abs0) / (Abs100 - Abs0) × 100

Abs100, Abs0 and Abs represent absorbance of solution of 100% hemolysis, solution of 0%

hemolysis and test sample respectively [127].

3.5.3 In-vivo acute toxicity study

Test samples were investigated for their in-vivo acute toxicity studies in two phases using Swiss

albino mice (18-25g) of either sex. Prior to conduct the experiments, mice were fasted for a

period of 12 h. During first phase investigation, mice were divided into three groups (n=4) and

they were injected intraperitoneally (i.p.) with surfactants BRG-BTD-14, BRG-LRC-12, BRG-

BRM-11, BRG-BRD-10, and BRG-BRN-9 at doses of 100, 500 and 1000 mg/kg body weight.

All the animals were observed for muscles paralysis, abdominal cramps and mortality. Dose

causing deaths in fifty percent mice population (LD50) was determined. During second phase

investigation, two groups of mice (n=4) were taken and treated i.p. with BRG-BTD-14, BRG-

LRC-12, BRG-BRM-11, BRG-BRD-10, and BRG-BRN-9 at doses of 1500 and 2000 mg/kg

body weight. All the animals were observed for muscles paralysis, abdominal cramps and

mortality. Dose causing deaths in fifty percent mice population (LD50) was determined [128].

3.6 Preparation of drug loaded niosomal formulations

All the five synthesized nonionic surfactants were screened for their niosomal drug loading

potentials. BRG-LRC-12 and BRG-BRM-11 were used for loading Cefixime in their niosomal


37

vesicles. BRG-BTD-14, BRG-BRD-10, and BRG-BRN-9 were used for loading Ciprofloxacin,

Levofloxacin and Moxifloxacin respectively in their niosomal vesicles. Drug loaded niosomal

vesicles of the synthesized nonionic surfactants were prepared through thin film re-hydration

method.

3.6.1 Preparation of Ciprofloxacin loaded BRG-BTD-14 niosomal formulation

BRG-BTD-14 (30mg) and cholesterol (10 mg) were dissolved in 20 mL mixed solvent system of

methanol and chloroform (4:6, v/v). Drug ciprofloxacin (10 mg) was dissolved in 20 mL

methanol, and mixed well with cholesterol and BRG-BTD-14 solution. The organic solvents

were evaporated using a rotary evaporator (BUCHI, 131 Rotavapor, Switzerland). A thin lipid

film was obtained and was further dried under reduced pressure. The thin lipid film was hydrated

with 10 mL of PBS (pH 7) at 45 °C for 30 min. To reduce the size of the drug loaded vesicles,

vesicles suspension was subjected to sonication in ultrasonicator (LABSONIC L, B. Braun

Biotech International, PA, USA) for 4 min at 25 °C with 5 s on/off cycle. The niosomal

suspension was stored at 4 °C for further experiments.

3.6.2 Preparation of Cefixime loaded BRG-LRC-12 niosomal formulation

BRG-LRC-12 (40 mg) and cholesterol (20 mg) were dissolved in 20 mL mixed solvent system

of methanol and chloroform (4:6, v/v). Cefixime (10 mg) was dissolved in 20 mL methanol, and

added to cholesterol and BRG-LRC-12 solution. The organic solvents were evaporated and drug

loaded niosomal vesicles of the nonionic BRG-LRC-12 surfactant were prepared in the same

way as mentioned above.


38

3.6.3 Preparation of Cefixime loaded BRG-BRM-11 niosomal formulation

BRG-BRM-11 (40 mg) and cholesterol (20 mg) were dissolved in 20 mL mixed solvent system

of methanol and chloroform (4:6, v/v). Cefixime (10 mg) was dissolved in 25 mL methanol, and

added to cholesterol and BRG-LRC-12 solution. Organic solvents were evaporated and drug

loaded niosomal vesicles of the nonionic BRG-BRM-11 surfactant were prepared in the same

way as mentioned above.

3.6.4 Preparation of Levofloxacin loaded BRG-BRD-10 niosomal formulation

Nonionic surfactant BRG-BRD-10 (30 mg) and cholesterol (15 mg) were dissolved in 25mL

chloroform and methanol solvent system (4:2 v/v). Levofloxacin (10 mg) was dissolved in 20mL

methanol, and then added to BRG-BRD-10 and cholesterol solution and mixed well. Organic

solvents were evaporated and drug loaded niosomal vesicles of the nonionic BRG-BRD-10

surfactant were prepared in the same way as mentioned above.

3.6.5 Preparation of Moxifloxacin loaded BRG-BRN-9 niosomal formulation

BRG-BRN-9 (25 mg) and cholesterol (12.5 mg) were dissolved in chloroform and methanol

mixed solvent system (40 ml, 2:2, v/v). Drug Moxifloxacin (10 mg) was dissolved in methanol

(15 ml). Both the drug and lipid phase solutions were mixed and organic solvents were

evaporated and drug loaded niosomal vesicles of the nonionic BRG-BRN-9 surfactant were

prepared in the same way as mentioned above.


39

3.7 Characterization of drug loaded niosomal formulations

3.7.1 Surface morphology, zeta potential, size and PDI

Surface morphology of the niosomal vesciles loaded with selected drugs were investigated

through atomic force microscope (AFM, Agilent 5500). Niosomal formulation was properly

diluted with deionized water and its drop was placed on mica slide. The slide was carefully dried

at room temperature in dust fee cabin. The slide was mounted on microscope and was visualized

in a non-contact mode. Vesicles surface morphology was observed and imagined. Size and

polydispersity index (PDI) of the niosomal vesicles in formulations were determined through

DLS (Zetasizer-90, Malvern Instruments, UK). Samples were properly diluted with deionized

water and taken in disposable plastic cuvette and size was measured at 25 °C using water as

dispersant. For Zeta potential measurement, diluted samples were slowly injected to capillary

disposable cells and read with the instrument at 25 °C.

3.7.2 Drug entrapment efficiency

The prepared niosomal formulations were investigated for their drug entrapment efficiencies

using HPLC. Niosomal formulations containing specific amount of Cefixime, Levofloxacin,

Moxifloxacin or Ciprofloxacin were centrifuged (Universal 16, Hettich, Germany) for 25 min at

12,000 rpm. The supernatants containing free drugs were removed and the isolated pellets of

niosomal vesicles were dissolved in known volume of methanol. The resulting solution of each

drug was run on HPLC using respective mobile phase and chromatographic conditions as

mentioned in the coming section. Drug entrapment efficiency was determined for each drug

according to equation indicted below:


40

EE% = (Amount of drug entrapped/ Total amount of drug) ×100

3.7.3 In-vitro drug release study

Drug loaded niosomal vesicles containing specific amount of Cefixime, Levofloxacin,

Moxifloxacin or Ciprofloxacin were taken in PBS of 7.4, 6.8, 4 or 1.2 pH and packed in dialysis

membrane (12,000 KDa). The membrane containing niosomal vesicles was suspended in a

beaker containing PBS (25 ml) of 7.4, 6.8, 4 or 1.2 pH. The beaker was shaken at 1000 rpm

while maintaining the temperature at 37 °C. Samples (2 ml) were regularly withdrawn at pre-

determined time intervals and same volume was added to media for prevention of saturation.

Cefixime, Levofloxacin, Moxifloxacin or Ciprofloxacin were detected at 289 nm, 298 nm, 294

nm and 278 nm respectively and was quantified in the samples using UV spectrophotometer

(Shimadzu, UV-240, Hitachi).

3.8 Stability studies

3.8.1 Storage stability

Drug loaded niosomal formulations based on the synthesized nonionic surfactants were

investigated for their storage stability up to 30 days. Briefly, 10 mL formulation was taken in a

glass vial, sealed and stored at 4 °C. Samples (2 mL) were taken at pre-determined time intervals

and investigated for the percent drug retained. Drug quantification was carried out using UV-

visible spectrophotometric method earlier described for quantification of drug in the in-vitro

release study.


41

3.8.2 Stability in simulated gastric fluid

Simulated gastric fluids (SGF) was prepared according to the reported method [129]. Briefly, 7

mL HCl, 2 g NaCl and 3.2 mg/mL pepsin were added to water (1 L final volume) and its pH was

adjusted to 2.0 with 1.0 M HCl solution. Niosomal formulations containing specific amount of

respective drugs were diluted with GSF (1: 10 v/v, niosomes: SGF) and incubated at 37 °C with

100 rpm agitation speed. Then samples were withdrawn at 10, 20, 40 and 60 min time intervals

and were investigated for percent drug retained following the method used for drug loading

efficiency.

3.9 HPLC methods for quantification of drugs in plasma

3.9.1 HPLC method for Ciprofloxacin quantification

Stock solutions (1 mg/mL) of Ciprofloxacin and Levofloxacin (internal standard) were prepared

in deionized water and acetonitrile (ACN) respectively. Different concentrations (0.05-7 µg/mL)

of Ciprofloxacin were prepared and run for construction of calibration curve.

HPLC system (Shimadzu, LC20A, Kyoto, Japan) with Phenomenex C18 column (5 µ; 250×4.6

mm) and UV detector was used for Ciprofloxacin separation and detection. The column oven

temperature was set at 50.0 ±0.2 °C. The mobile phase consisted of 0.025M Phosphoric acid

buffer solution, ACN and tetrahydofuran in 88: 10: 2 V/V/V ratios. The flow rate was fixed at 1

mL/min. An injection volume of 25 µL was selected for injection into HPLC. Levofloxacin

(internal standard) concentration was fixed as 3 µg/mL. The sample run time was optimized as 9

min. Retention times for Ciprofloxacin and Levofloxacin were optimized as 7 ±1 and 6 ±1 min

respectively.


42

For extraction of drug from plasma, plasma (200 µL) containing spiked drug was added to 30 µL

of internal standard followed by addition of 100 µL of 0.04 M acetic acid. The mixture was

vertex mixed for 60 sec. It was heated at 96 °C for 10 min, cooled to room temperature and then

centrifuged at 12000 rpm for 10 min. Supernatant (50 µL) was taken, diluted with 450 µL of

0.025 M phosphoric acid buffer and vortex mixed for 30 sec. An aliquot of 100 µL was taken

and 25 µL was injected into HPLC.

3.9.2 HPLC method for Cefixime quantification

Cefixime stock solution (250 µg/mL) was prepared by dissolving its weighed amount in

methanol. HPLC system (Shimadzu, LC20A, Kyoto, Japan) with Phenomenex C8 RP column (5

µ; 150×4.6 mm) and UV detector was used for separation and detection of Cefixime. Mobile

phase consisted of methanol and 0.4 M H3PO4 in 15:85 in V/V ratio. The pH of the mobile phase

was maintained at 3.0. The optimized flow rate of the mobile phase was 1.0 mL/min. The

column oven temperature was set at 50 °C temperature. The retention time for Cefixime was

13.5 ± 1.5 min. Cefixime was detected at 288 nm. Blank plasma was spiked with Cefixime

standard solution to obtain its different concentrations (2–10 µg/mL) for calibration curve.

Drug was extracted from plasma through protein precipitation method. Plasma (200 µL)

containing specific concentration of drug was added to 600 µL ACN and vortex mixed for 1 min.

The mixture was then centrifuged for 10 min at 10,000 rpm. Supernatant (600 µL) was taken and

dried in concentrator. It was then reconstituted in 100 µL mobile phase and its 50 µL aliquot was

injected into HPLC.


43

3.9.3 HPLC method for Levoflixacin quantification

Stock solutions (1 mg/mL) of Levofloxacin and internal standard Ciprofloxacin (1mg/mL) were

prepared by dissolving 5mg of each drug in 5 mL ACN and deionized water, respectively.

Levofloxacin working solutions were prepared from stock solution in 0.05–7 µg/mL

concentrations for calibration curves.

The mobile phase consisted of 0.025M phosphoric acid buffer solution, ACN and

tetrahydrofuran in 88:10:2 V/V/V ratios. HPLC system (LC20A, Shimadzu, Japan) was equipped

with phoenomenex C8RP column (5 µ; 150 × 4.6 mm). Chromatography was carried out at a

flow rate of 1 mL/min at a column temperature of 50.0 ±0.2 °C. All solutions were filtered

through a 0.45 µm membrane (Sartorious, Germany) before use. The injection volume and

running time were 25 µL and 10 min respectively. Levofloxacin was detected at 298 nm.

Retention times were 6.3–7.3 and 7.5–8.5 min for Levofloxacin and Ciprofloxacin, respectively.

Plasma (200 µL) was added to 0.04M acetic acid (100 µL) and vortex mixed for 30 s, followed

by heating at 96 °C for 10 min. Then the mixture was centrifuged at 14006 g for 10 min.

Supernatant (50 µL) was taken and diluted with 950 µL phosphoric acid buffer solution

(0.025M) and vortex-mixed for 30 s. An aliquot of 200 µL was taken and 25 µL was injected

into HPLC.

3.9.4 HPLC method for Moxifloxacin quantification

Stock solutions (1 mg/mL) of Moxifloxacin and internal standard Levofloxacin were prepared in

deionized water. Working solution (8 μg/mL) of Moxifloxacin was prepared in plasma.

Similarly, working solution (0.5 μg/mL) of Levofloxacin was prepared in acetic acid. Different


44

concentrations (0.05-6.4 μg/mL) of Moxifloxacin were also prepared for construction of its

calibration curve. The mobile phase consisted of 0.025M H3PO4 buffer solution and ACN in

75:25 V/V ratios. The pH of mobile phase was maintained at 3.0. Phenomenex C18 column

(250cm x 4.6mm, 5µm) equipped with HPLC system (LC20A, Shimadzu, Japan) was used.

Chromatographic separation was carried out at 1.2 mL/min flow rate with column oven

temperature of 50 °C. An injection volume of 25 µL was found suitable for the process. The

sample was run for 7.5 min. Retention times were optimized as 5.0 ±1.0 min and 3.0 ±1.0 min

for Moxifloxacin and Levofloxacin respectively. The wave lengths of 295 nm and 500 nm were

selected as excitation and emission respectively.

Plasma (200 μL) spiked with drug was added to 0.2 mL acetic acid containing 0.5 μg/mL

internal standard and vertex mixed for 10 s. Then the mixture was heated at 96 °C for 10 min.

The mixture was cooled at room temperature and centrifuged at 12000 rpm for 10 min.

Supernatant (50 μL) was taken and added to 950 μL mobile phase followed by vertex mixing for

60 s. A volume of 25 μL was injected into HPLC.

3.10 In-vivo bioavailability studies

Rabbit’s local species “Oryctolagus cuniculus” were used for investigating the in-vivo oral

bioavailability studies. Three groups of animals (n=6) were kept under standard laboratory

conditions having access to water and food. Before conducting studies, all the animals were

fasted having access to water only. For Cefixime, its BRG-LRC-12 and BRG-BRM-11 based

niosomal formulations were given to first group at 6 mg/kg body weight oral dose. Its

commercial capsules “Cefiget” (powder suspended in 0.5% Tween 80) and suspension

“Maxpan” were given to second and third groups respectively at the same oral dose. For


45

Moxifloxacin, its BRG-BRN-9 based niosomal formulation was given orally to first group at 8

mg/ml per kg body weight. Similarly, drug solution and its commercial tablets (Moxiget)

(powder suspended in 0.5% Tween 80) were given to animals in second and third groups

respectively at the same oral dose. For Levofloxacin, its BRG-BRD-10 based niosomal

formulation was given orally to first group at 10 mg/ml per kg body weight. Levofloxacin

solution and its tablets (Leflox) (powdered and suspended in 0.5% Tween 80) were given to

animals in second and third group at the same oral dose. For Ciprofloxacin, its BRG-BTD-14

based niosoaml formulation was given orally to first group at 7 mg/ml per kg body weight.

Ciprofloxacin solution and its commercial suspension (Novidat) were given to animals in second

and third groups at the same oral dose.

At pre-determined time intervals (0-24 h), 1 mL blood samples were collected in heparinized

tubes form the marginal ear vein of animals. Plasma was immediately separated from blood by

its centrifugation at 4000 rpm for 5 min and kept at -80 °C. Different pharmacokinetic parameters

were determined from the individual drug plasma concentration-time curves of drugs after their

oral administration in the form of their solutions, commercial dosage forms or niosomal

formulations. Values of maximum drug plasma concentration (Cmax) and time to reach maximum

drug plasma concentration (Tmax) were obtained from plasma drug concentration time curves

directly. The values of drug plasma concentrations at different time intervals were further used

for calculating various pharmacokinetic parameters like clearance (Cl), mean residence time

(MRT), area under concentration–time curve to the last point (AUC0-24) and area under the first

moment Curve (AUM0-24).


46

3.11 Statistical analysis

Data was presented as mean ±SEM of the experiments performed in triplicate. Student’s

independent sample t-test at 95% confidence level was used for comparing pharmacokinetic

parameters of niosomal formulations of the drugs with drugs solutions or their commercially

available formulations. The two-way ANOVA followed by post bonferroni test was used for

finding statistical significance among multiple groups in blood hemolysis and cells cytotoxicity

studies. P values less than 0.05 were considered statistically significant.

Chapter 4 Results and discussion

47

4 Results and discussion

4.1 Characterization of the synthesized nonionic surfactants

4.1.1 BRG-BTD-14

1H NMR (400 MHz, CDCl3): δ 7.41 (s, 1H, ArH); 4.74 (d, J = 10.4 Hz, 1H, CH); 3.98 (m, 8 H);

3.9 (s, 3H, OCH3); 1.82 (m, 11 H); 1.43 (m, 4H, -CH2) 1.23 (m, 44 H, CH2); 0.86 (t, J= 6.8 Hz,

6H, CH3). FAB-MS [M-H], m/z: 719.2.

The proton NMR and FAB negative spectra of the synthesized compound confirm its successful

synthesis. A peak at 719.2 m/z is observed for the synthesized compound BRG-BTD-14 as

shown in Figure 4.1 which is consistent with the theoretical calculated molecular weight of the

compound (M-1). Similarly the proton NMR spectra show singlet for aromatic proton at 7.41

ppm, a triplet is observed at 0.86 ppm for the terminal six aliphatic protons of the attached

carbon chains and a singlet peak for OCH3 is observed at 3.9 ppm as shown in Figure 4.2. A

multiplet peak for the remaining 44 protons of the attached carbons tails is observed at 1.23 ppm.

All other peaks are found on their respective places. This data confirms the successful synthesis

of the compound BRG-BTD-14.


48

Figure 4. 1: Mass spectra of “BRG-BTD-14”


49

Figure 4. 2: 1H NMR spectra of “BRG-BTD-14”

4.1.2 BRG-LRC-12

EI-MS m/z: 692.4

1H NMR (400 MHz, CDCl3): δ 0.85 (t, J=4.8 Hz, 6H, CH3), 1.23 (m, 32H, CH2), 2.32 (t, J=7.6

Hz, 4H, CH2CO), 2.56 (q, 4H, CH2 ), 3.55 (d, 1H, CH), 3.86 (s, 3H, OCH3), 3.94 (d, J=6.8 Hz,

1H, CH), 4.11 (t, J= 9.2 Hz ,2H, OCH2), 4.34 (d, J= 12.8 Hz, 1H, CH), 4.42 (d, J= 12.8 Hz, 1H,

CH), 4.68 (d, J=10.4 Hz, 1H, CH), 7.72 (s, 1H, ArH).

Molecular ion peak in the EI-MS spectrum was observed at m/z 692 which was in agreement

with the calculated molecular weight for molecular formula C38H60O11 (Figure 4.3). The 1H


50

NMR spectra of the synthesized surfactant shows a singlet peak at δ 7.72 ppm for aromatic

proton. At δ 0.85 ppm, a triplet peak of 6H is observed for the terminal six protons of the

aliphatic tail. All the remaining protons of the aliphatic hydrocarbon side chains give multiplet

peak of 32 protons at δ 1.23-1.32 ppm. Similarly, for the methoxy protons of the aromatic ring, a

singlet peak is observed at δ 3.86 ppm (Figure 4.4). The reaction resulted in 90% yield of the

product.

Figure 4. 3: Mass spectra of “BRG-LRC-12”


51

Figure 4. 4: 1HNMR spectra of “BRG-LRC-12”


52

4.1.3 BRG-BRM-11

EI-MS, m/z (%); 636.0 (100%)

1H NMR (400 MHz, CDCl3): δ 0.84 (t, J= 6, 6.8 Hz, 6H), 1.23-1.33 (m, 32H), 1.72 (m, 4H),

3.56-3.64 (m, 2H), 4.76 (dd, J= 9.5; 10.0 Hz, 1H), 3.64 (dd, J=9.5; 8.5 Hz, 1H), 3.78 (s, 3H,

OCH3), 3.90 (m, 2H), 4.02 (t, J= 6,6.4 Hz, 4H), 4.75 (d, J=10Hz, 1H), 7.29 (s, 1H).

Molecular ion peak for the synthesized surfactant in EIMS spectra was observed at m/z 636.4.

The fragmentation pattern of the compound is also matching with the theoretical data. As loss of

the hydroxyl substituted ring gave peak at 362 as shown in Figure 4.5 while losing one aliphatic

side chain, the fragment peak is observed at m/z 482.1. As demonstrated in Figure 4.6, the titled

compound show singlet peak at δ 7.29 for one aromatic proton in 1H NMR spectra. Similarly, at

δ 3.78 ppm a singlet peak is observed for 3 protons of OCH3 group and a triplet peak at 0.84 ppm

for terminal 6 methyl H of the aliphatic side chains were observed. A multiplet peak at δ 1.23-

1.33 ppm for all the remaining 32 protons of the aliphatic side chains was observed. These data

confirms the successful synthesis of the surfactant BRG-BRM-11.


53

Figure 4. 5: Mass spectra of “BRG-BRM-11”


54

Figure 4. 6: 1HNMR spectra of “BRG-BRM-11”

4.1.4 BRG-BRD-10

Yield (91 %), 1H NMR (CDCl3, 400 MHz) δ: 0.85 (t, 6H, CH3, J12 = 2.0 Hz, J13= 6.8 Hz), 1.25

(m, 24H, CH2), 1.41 (m, 4H, CH2), 1.77 (m, 4H, CH2), 3.35 (m, 4H, CH2), 3.58 (s, 2H, CH), 3.87

(s, 3H, OCH3), 3.95 (m, 4H, OCH2), 3.99 (m, 1H, CH), 4.70 (m, 2H, CH2-OH), 7.34 (s, 1H,

ArH). EI-MS m/z: 608.0,

The EI-MS spectra of the compound show its molecular ion peak at 608 (Figure 4.7). Similarly,

the 1H NMR spectra shows triplet for terminal aliphatic protons at 0.85 ppm and CH2 proton of


55

the aliphatic chain appears at 1.25 ppm as multiplet. The characteristic peak for OCH3 of

Bergenin and aromatic proton appeared at 3.87 and 7.34 ppm as singlet respectively (Figure 4.8).

Figure 4. 7: Mass spectra of “BRG-BRD-10”


56

Figure 4. 8: 1HNMR spectra of “BRG-BRD-10”

4.1.5 BRG-BRN-9

Yield 90 %, EIMS, m/z = 580.2, 1H NMR (CDCl3): δ 7.41 (s, 1H, aromatic), 4.74 (d, J = 12.0

Hz, 1H), 3.98 (m, 5H), 3.90 (s, 3H, OMe); 3.64 (dd, J = 12.0 Hz, 2H); 1.82 (m, 14H); 1.43 (d, J

= 8.0 Hz, 3H), 1.25 (m, 24H), 0.86 (t, J = 6, 6.8 Hz, 6H, H-110).

The theoretical calculated mass of the synthesized product is 580.36, and the experimental

observed mass is 580.2 as shown in Figure 4.9. Similarly, the proton NMR spectra of the

synthesized compound shows characteristic peaks for terminal aliphatic tail’s six protons as

triplet at 0.86 ppm, a singlet for the aromatic one proton is observed at 7.4 ppm, while a singlet


57

of 3 protons for methoxy group attached to the aromatic ring of Bergenine is observed at 3.9

ppm. All other peaks for respective protons are in their respective chemical shifts as shown in

Figure 4.10.

Figure 4. 9: Mass spectra of “BRG-BRN-9”


58

Figure 4. 10: 1HNMR spectra of “BRG-BRN-9”


59

4.2 Biocompatibility studies

4.2.1 In-vitro cells cytotoxicity studies

All the synthesized nonionic surfactants were screened for their in-vitro cytotoxicity against 3T3

and HeLa cancer cell lines. They were tested in a concentration range of 62.5-1000 µg/mL.

BRG-BTD-14 exhibited 78.65 ±3.00 and 72.66 ±2.54% cell viability against 3T3 cells at 1000

µg/mL after 24 and 48 h incubation respectively. Tween 80 revealed 64.29 ±4.57 and 37.30

±5.93% cell viability against the same cells at 1000 µg/mL after 24 and 48 h incubation

respectively as shown in Figure 4.11A and 4.11B. Results confirm that BRG-BTD-14 is less

cytotoxic against 3T3 cells as compared to Tween 80. Similarly, BRG-BTD-14 showed 73.88

±1.65 and 66.56 ±2.50% cell viability against HeLa cancer cells at 1000 µg/mL after 24 and 48 h

incubation respectively. Reference standard Tween 80 exhibited 58.31 ± 2.39, 49.76 ±3.70% cell

viability against HeLa cancer cells at 1000 µg/mL after 24 and 48 h incubation respectively as

shown in Figure 4.11C and 4.11D. BRG-BTD-14 demonstrated to be less cytotoxic against HeLa

cancer cells as compared to Tween 80, but when compared to 3T3 cells, an increase in its

cytotoxicity was observed.


60

Figure 4. 11: In-vitro cytotoxicity of BRG-BTD-14 against 3T3 cells after (A) 24 and (B) 48 h

where (C) and (D) show its cytotoxicty against HeLa cells after 24 and 48 h respectively

BRG-LRC-12 was also investigated against 3T3 and HeLa cell lines in 62.5-1000 µg/mL

concentration range. BRG-LRC-12 revealed cell viability in a dose dependent manner and did

not exhibit cytotoxicty against both cell lines even at the highest concentration as compared to

reference standard Tween 80. It showed higher cell viability in comparison with Tween 80 in

both the cases, thus confirming to be nontoxic. When tested against 3T3 cells, its cell viability

was as high as 90.94 ±2.46% and 88.19 ±3.35% at 1000 µg/mL concentration after 24 and 48 h

respectively as compared to 66.69 ±3.78% and 38.52 ±5.93% respectively using Tween 80


61

(Figure 4.12A and 4.12B). BRG-LRC-12 demonstrated to be nontoxic against HeLa cells. It

revealed higher cell viability of 78.82 ±3.29 and 74.96 ±3.07% at 1000 µg/mL concentration

after 24 and 48 h respectively as compared to 65.91 ±4.67% and 49.76 ±3.07% respectively

using Tween 80 (Figure 4.12C and 4.12D).

Figure 4. 12: In-vitro cytotoxicity of BRG-LRC-12 against 3T3 cells after (A) 24 and (B) 48 h


Results of BRG-BRM-11 cytotoxicity are given in Figure 4.13. Results confirm its

comparatively nontoxic nature in 62.5-1000 µg/mL concentration range. Its cell viability against

3T3 cells was found to be 90.77 ±3.15% and 86.86 ±3.02% at 1000 µg/mL concentration after 24


62

and 48 h respectively as compared to 64.29±4.47% and 37.30±5.93% respectively using Tween

80 (Figure 4.13A and 4.13B). Moreover, when tested against HeLa cells, BRG-BRM-11

revealed 79.21 ±2.54% and 76.42 ±1.87% at 1000 µg/mL concentration after 24 and 48 h

respectively as compared to 65.56 ±3.22% and 47.56 ±3.89% respectively using Tween 80

(Figure 4.13C and 4.13D).

Figure 4. 13: In-vitro cytotoxicity of BRG-BRM-11 against 3T3 cells after (A) 24 and (B) 48 h


BRG-BRD-10 in-vitro cytotoxicity was investigated by exposing them to 3T3 and HeLa cells for

24 and 48 h. The synthesized nonionic surfactant displayed negligible toxicity against both types


63

of cells even after prolonged exposure and at the highest concentration. After 24 and 48 h, it

showed 84.55 ±1.77 and 80.92 ±2.87% cell viability respectively against 3T3 cells at 1000

µg/mL surfactant concentration, much higher than the reference standard Tween 80 (Figure

4.14A and 4.14B). Similarly, it showed 80.44 ±1.83 and 79.77 ±2.11% cell viability against

HeLa cells after 24 and 48 h respectively at 1000 µg/mL, comparatively higher than Tween 80

(Figure 4.14C and 4.14D).

Figure 4. 14: In-vitro cytotoxicity of BRG-BRD-10 against 3T3 cells after (A) 24 and (B) 48 h



64

The synthesized nonionic surfactant BRG-BRN-9 revealed negligible cytotoxicity against both

the cell cultures at all tested concentrations. Against 3T3 cells, it revealed 77.56 ±2.53 and 83.88

±2.44% cell viability at 1000 µg/mL after 48 and 24 h respectively. This was much higher than

Tween 80 that exhibited 39.66 ±3.60 and 68.21 ±2.54% cells viability against the same cell line

at 1000 µg/mL after 48 and 24 h respectively (Figure 4.15A and 4.15B). Similarly, BRG-BRN-9

revealed 79.77 ±2.19 and 78.33 ±2.44% cells viability against HeLa cells at 1000 µg/mL after 48

and 24 h respectively. It demonstrated to be much less cytotoxic than Tween 80 that showed

65.56 ±3.22 and 47.56 ±3.89% cells viability against the same cell line at 1000 µg/mL after 24

and 48 h exposure respectively (Figure 4.15C and 4.15D).


65

Figure 4. 15: In-vitro cytotoxicity of BRG-BRN-9 against 3T3 cells after (A) 24 and (B) 48 h


Before investigating the toxicity profile of an expected biomaterial in animal model, estimation

of its in-vitro cytotoxicity against cell lines is necessary [130]. HeLa and 3T3 are cancer cell

lines that are well-known and are most widely used for investigating the cytotoxicity of

substances intended for drug delivery applications. They are preferred for investigation of

toxicity due to higher reproducibility of results [131]. All the synthesized nonionic surfactants

were investigated for their in-vitro cytotoxicity using 3T3 and HeLa cancerous cells. They were

non-toxic to both types of the cells even after their prolonged incubation with cells and at their

highest concentrations (1000 µg/mL). The most probable reason of their nontoxic nature can be


66

the saturation in their structures as hypothesized by the previous studies [132]. Recently

surfactants derived from lysine have been reported with cytotoxicity profile like our surfactants

and have been successfully employed as drug carriers [97]. Moreover, the nonionic nature of

these nonionic surfactants can also be linked to their lower cytotoxicity. The appropriate alkyl or

acyl chain length of these synthesized nonionic surfactants can also be taken into consideration

for the lower or negligible in-vitro cytotoxicity. BRG-BTD-14 was found to be more cytotoxic

among all the nonionic surfactants. This may be due to its comparatively longer alkyl chains that

show comparatively higher disruptive effect on cell membranes [133].

4.2.2 Blood hemolysis assay

All the synthesized nonionic surfactants were screened for their hemolytic activity in a

concentration range of 62.5-1000 µg/mL. They were incubated with suspension of RBCs for 4 h

and the amount of hemoglobin released from blood cells was quantified spectrophotometrically.

Tween 80 was used as reference standard for comparison. The first nonionic surfactant BRG-

BTD-14 caused 1.46 ±0.11 and 12.43 ±0.61% hemoylsis in RBCs at 62.5 and 1000 µg/mL

concentrations respectively. This was too low as compared to 4.39 ±0.30 and 28.43 ±2.51%

hemolysis caused by Tween 80 at 62.5 and 1000 µg/mL concentrations respectively as shown in

Figure 4.16A. Similarly, BRG-LRC-12 was found to cause 0.91 ±0.18 and 4.53 ±1.51%

hemoylsis in RBCs at 62.5 and 1000 µg/mL concentrations respectively. In comparison,

reference standard Tween 80 caused 3.28 ±0.97 and 27.78 ±3.11% hemolysis at 62.5 and 1000

µg/mL concentrations respectively as shown in Figure 4.16B.


67

Figure 4. 16: Hemolysis activity of (A) BRG-BTD-14 and (B) BRG-LRC-12 at various

concentrations.

Surfactant BRG-BRM-11 revealed negligible hemolytic activity, releasing 5.49± 1.62% and

0.92± 0.15% hemoglobin from RBCs for 1000 and 62.5 µg/mL concentrations respectively. This

hemolytic effect was significantly lower than Tween 80 surfactant as shown in Figure 4.17A.

Figure 4.17B summarizes the hemolytic activity results of synthesized nonionic surfactant BRG-

BRD-10. It caused 0.78 ±0.31 and 5.77 ±1.34% hemolysis in fresh human blood at 62.5 and

1000 mg/mL concentrations respectively. The hemolytic activity of the synthesized nonionic

surfactant was significantly lower than that of Tween 80 which caused 3.61 ±0.65% and 21.45

±2.63% hemolysis at 62.5 and 1000 µg/mL concentrations respectively. BRG-BRN-9 revealed

negligible release of hemoglobin from the RBCs as compared to the Tween 80 tested as

reference standard. It caused 11.33 ±0.50% hemolysis at 1000 µg/mL that was much lower than

that of Tween 80 (27.78 ±3.11%) at the same concentration (Figure 4.17C).


68

Figure 4. 17: Hemolysis activity of (A) BRG-BRM-11, (B) BRG-BRD-10 and (C) BRG-BRN-9

at various concentrations

Materials intended for use in drug delivery must not cause detrimental effects to blood

components (e.g. RBC). For this reason, all the synthesized nonionic surfactants were

investigated for their hemolytic activity. Hemolytic tests have been widely used for predicting

surfactant toxicity in terms of hemoglobin release from RBCs [134]. Surfactants are generally

reported for their hemolytic activities, but there are only a few publications reporting on hemo-

toxicity of sugar based surfactants [94]. Hemolysis occurs when the surfactant gets partitioned

into the outer cell membrane. This is followed by lipid-surfactant mixed micelles formation,

resulting in leaking of the cell components. The structures of the surfactants always play an


69

imperative role in release of hemoglobin from cells [117]. All the synthesized nonionic

surfactants demonstrated to be non-hemolytic and non-disruptive towards RBCs, thus confirming

them highly hemo-compatible. The non-hemolytic nature of these nonionic surfactants may be

due to their appropriate lipophilicity that they gain due to presence of two alkyl or acyl chains on

both sides of the hydrophilic head groups. Moreover, glycosidic origin of these synthesized

nonionic surfactants can also be a major contributing factor to their non-hemolytic nature [94].

4.2.3 In-vivo acute toxicity

In-vivo toxicity was conducted for all synthesized nonionic surfactants in order to evaluate their

safety in animals. Mice were injected with test samples by i.p route and were then monitored

continuously for mortality or morbidity (e.g. muscles paralysis, abdominal cramps, etc.) up to 24

h. The acute toxicity study was carried out in two different stages for all the test samples. In first

stage, acute toxicity was investigated at three different doses (100, 500 and 1000 mg/kg body

weight). No lethal effects were shown for all the samples in the first stage acute toxicity study at

all tested doses. Similarly, the test doses of all the samples were increased to 1500 and 2000

mg/kg body weight in second stage. All the animals remained alive and without any muscles

paralysis and abdominal cramps. Thus acute toxicity study confirms that all the synthesized

nonionic surfactants were safe and did not show any lethality and side effects up to 2000 mg/kg

body weight in animals.

Surfactants are widely used for the solubiliization of various drugs, but their use has been

restricted in such applications due to their known toxicities in animals and humans. Drug

delivery systems must be subjected to in-vivo acute toxicity studies in proper animals for

validating their safety in animals and humans. This study ascertains the validity of in-vitro


70

toxicity strategies carried out through cancer cells and blood hemolysis assays [11]. All the

synthesized nonionic surfactants were investigated for acute toxicity in animals. Results confirm

them well tolerated in animals and did not cause any morbidity and mortality up to 2000 mg/kg

body weight dose.

4.3 Ciprofloxacin loaded BRG-BTD-14 niosomal formulation

4.3.1 Characterization

4.3.1.1 Surface morphology, Size, PDI and zeta potential

AFM was used for studying the surface morphology of Ciprofloxacin loaded BRG-BTD-14

niosomal formulation. These microscopic studies reveal the loaded vesicles are spherical in

shape as shown in Figure 4.18. The drug loaded vesicles were also studied for their size, PDI and

zeta potential. Drug loaded vesicles were found smaller in size, revealing an average diameter of

125.2 ±4.63 nm as shown in Table 4.1. They also showed a PDI of 0.31 ±0.04 (Table 4.1). The

PDI indicates that these vesicles are almost similar in size. The surface charge study shows that

these drug loaded vesicles are negatively charged having a net zeta potential of -25.8 ±3.20.

The BRG-BTD-14 drug loaded vesicles revealed to be highly smaller in size. Moreover, they

were well homogenous regarding their size distribution. The nonionic surfactants based niosomal

vesicles size has been an imperative indicator for their fate in the in-vivo biological systems.

Moreover, it also predicts the physical stability of the drug loaded formulations. Size of vesicles

has also a considerable impact on therapeutic efficiency, toxicity, their phagocytic uptake, drug

targeting, and membrane permeability and formulation stability [135-137]. Moreover, vesicles

with narrow size distribution and smaller size have been reported for increased absorption of


71

their entrapped drugs. They are also capable of protecting drugs from enzymatic degradation or

inactivation in a highly efficient way. Similarly, they release the entrapped drugs in sustained

manner in the physiological environment and prevent the drugs associated toxicities [138-140].

Results of the current study confirm BRG-BTD-14 drug loaded vesicles in nano-range with

lower PDI. These findings suggest the vesicles to be of greater importance for enhancing in-vivo

performance and oral bioavailability of the encapsulated drug.

When particles are in dispersed state, they gain charge on their surfaces and the estimate of that

charge is known as zeta potential. Zeta potential measurement is a vital parameter as it predicts

the stability of colloidal dispersions during storage. When the zeta potential of the particles is

high, there are fewer chances for their aggregation. The estimation of zeta potential also helps in

designing dosage forms with decreased reticulo-endothelial system uptake, hence the drug

clearance from the body is decreased and its mean residence time increases in the biological

system [141]. The BRG-BTD-14 based drug loaded niosomal formulation showed higher

negative surface charge, thus confirming its higher physical stability and expected better in-vivo

performance.


72

Figure 4. 18: AFM images of Ciprofloxacin loaded BRG-BTD-14 niosomal vesicles.

4.3.1.2 Drug entrapment efficiency

BRG-BTD-14 based niosomal formulation was studied for its Ciprofloxacin entrapping

efficiency using the HPLC developed method as discussed earlier. The results are shown in

Table 4.1. The vesicles were able to entrap about 79.34 ±4.81% of the total drug used in

formulation. The results show that each 1 mg of the vesicles (lipid phase) was able to entrap

about 0.2 mg of Ciprofloxacin.

From pharmaceutical perspective, high drug entrapment efficiency in niosomal formulation is

required as the entrapped proportion of the drug contributes to sustained release behavior and

increased bioavailability. Drug entrapment efficiency has been one of the important

characteristics for drug delivery systems. The drugs release from nano-carriers is regulated by

the drug entrapment efficiency. Increased drug entrapment efficiency of the vesicular drug


73

delivery systems is also important for the sustained release of the drug form formulation [142].

Moreover, increased drug entrapment efficiency of vesicles ensures enhanced bioavailability of

the drugs and leads to reduction in doses frequency thus eliminates the side effects associated

with frequent dosing of the therapy. The niosomal vesicles drug entrapment efficiency depends

upon the quantity and nature of the surfactants used. It also depends upon the amount of

cholesterol in the vesicles bilayers [143].

Increased drug entrapment efficiency was observed for BRG-BTD-14 based niosomal

formulation. This can be due to various factors. The chains present on both sides of sugar moiety

of the nonionic surfactants cause its high lipophilicity, hence solubilization of hydrophobic drugs

is enhanced [144]. This may also be linked to the increased cholesterol content (almost 33% of

the total lipid phase in formulation of the current study) as supported by reported literature that

increasing the concentration of cholesterol enhances the drug entrapment efficiency of niosomes

[67, 145]. Similarly, cholesterol prevents the drug leakage from niosomes through its ability of

cementing the leaking spaces in bilayers membranes, enabling the vesicles to retain maximum

amount of drug [146]. Structure of surfactant also plays important role in drug entrapment

efficiency. Usually, nonionic surfactants having long acyl or alkyl lipophilic chains reveal

elevated phase transition temperatures and increased drug entrapment efficiency [147]. The

aforementioned trend of increased drug entrapment efficiency is fulfilled here well by virtue of

the long lipophilic chains on both sides of glycosidic head group of the nonionic surfactant.


74

Table 4. 1: Characterization of Ciprofloxacin loaded BRG-BTD-14 niosomes for zeta potential,

size and PDI and encapsulation efficiency

Sample

Composition

BRG-BTD-

14/Cholesterol/Drug

(w/w/w)

Drug

entrapment

efficiency (%)

Average vesicle

size (nm)

Polydispersity

Index (PDI)

Zeta

Potential

(mV)

Ciprofloxacin

loaded BRG-

BTD-14 vesicles

3 : 1 : 1 79.34 ±4.81 125.2 ±4.63 0.31 ±0.04 -25.8

±3.20

4.3.1.3 In-vitro drug release study

BRG-BTD-14 based ciprofloxacin loaded niosomes were investigated for in-vitro drug for 24 h

at various pH i.e 1.2, 4, 6.8 7.4. Ciprofloxacin released in medium at different time intervals was

quantified spectrophotometrically at 278 nm. Results of are summarized in Figure 4.19. BRG-

BTD-14 niosomal formulation showed an organized release of the entrapped drug. The drug

release seemed to be pH dependent but no abrupt release was observed at any stage of the study

or any pH. When the acidity of the medium was increased, the drug release from the vesicles was

decreased. The formulation showed 50% of drug release almost at 6th h of the study for pH 7.4,

6.8 and 4. In case of pH 1.2, 50% drug release reached at 7th h of the study. Maximum drug

release for pH 7.4 (86.00%) and 6.8 (79.32%) was achieved at 10th h of the study. In case of pH

4 and 1.2, the maximum drug release (73.00 and 67.50% respectively) was achieved at 9th h of

the study period.


75

Figure 4. 19: In-vitro drug release study of Ciprofloxacin loaded BRG-BTD-14 niosomal

formulation.

4.3.2 Stability studies

4.3.2.1 Storage stability

Ciprofloxacin loaded in BRG-BTD-14 based niosomal vesicles were investigated for storage

stability for a time period of 30 days. The formulation stability was determined in term of its

ability of retaining the entrapped drug during storage period. The amount of Ciprofloxacin

released was detected at 278 nm spectrophotometrically and then quantified. The formulation

showed negligible amount of drug release up to 20th day of the storage period. It retained about

98.43 ±0.27, 94.78 ±1.52 and 89.66 ±2.43% of the entrapped drug at 1st, 10th and 20th day of the


76

storage period respectively. There formulation lost of about 17% (retaining 83% of drug) of the

entrapped drug after 30 days of storage.

Storage stability is carried out in order to determine the leaching of the entrapped drug from the

vesicles upon storage for extended period of time. BRG-BTD-14 niosomal formulation revealed

to retain maximum amount of the entrapped drug upon its storage for a period of one month,

confirming increased stability upon storage. The higher stability of these vesicles can be

attributed to the presence of increased negative charge on the surfaces of drug loaded vesicles.

The negative surface charge causes electrostatic repulsion among the vesicles, thus prevents their

aggregation, fusion and leaking of the entrapped drug [148]. Similarly, cholesterol used as

additive in these vesicles also play a vital role in drug leaking from the vesicles as it cements the

leaking spaces of the vesicles.

4.3.2.2 Stability against simulated gastric fluid

The Ciprofloxacin loaded BRG-BTD-14 based niosomal vesicles were studied for their stability

against SGF. The amount of drug retained in the vesicles after their incubation with SGF for 10,

20, 40 and 60 min was quantified through HPLC developed method. BRG-BTD-14 based

niosomal vesicles were highly stable against SGF up to 60 min. They retained about 96.85 ±1.07,

94.35 ±0.65, 91.76 ±2.56 and 86.04 ±1.87% of the loaded drug after 10, 20, 40 and 60 min

respectively. Gastric pH has been reported for destabilization of vesicular drug delivery systems.

Formulations designed for oral delivery of drugs must be investigated for their stability in

digestive fluids [149]. BRG-BTD-14 niosomal formulation was quite stable upon its incubation

with SGF, thus confirming its technical suitability for oral drug delivery purposes.


77

4.3.3 In-vivo bioavailability studies

Ciprofloxacin was administered to rabbits at 7 mg/kg body weight in the form of BRG-BTD-14

loaded niosomal vesicles, commercial suspension or free solution and its mean plasma

concentrations at different time intervals are depicted in Figure 4.20. Various pharmacokinetics

were determined form individual blood plasma time curves and are presented in Table 4.2. The

novel nonionic surfactant based vesicles were able to cause the elevated plasma level of

Ciprofloxacin as compared to its suspension and free solution. For niosomal formulation of

Ciprofloxacin, maximum plasma concentration (Cmax) was found to be 2.67 ±0.43 µg/mL, higher

than its suspension (1.56 ±0.21 µg/mL) and free solution (1.32 ±0.37 µg/mL). Niosomal

encapsulated Ciprofloxacin revealed a Tmax of 2.5 h as compared to its suspension and free

solution (1 h). Similarly, the mean residence time (MRT) of Ciprofloxacin was higher (9.1 ±0.23

h) when given orally in the form of niosomal formulation as compared to its suspension (7.67

±0.45 h) and free solution (6.90 ±0.38 h). More importantly, the niosomal loaded suspension

exhibited a decreased clearance (Cl, 0.25 ±0.02 L/h.kg) from the body as compared to its

suspension and free solution showing 0.48 ±0.03 and 0.71 ±0.03 L/h.kg Cl respectively.

The drug loaded niosomal formulation was investigated for enhancing the oral bioavailability of

the entrapped Ciprofloxacin. These vesicles were highly able to enhance the oral bioavailability

of the entrapped Ciprofloxacin. An elevated Cmax was observed for drug when it was delivered in

the synthesized nonionic surfactant based niosomal vesicles in comparison with its respective

commercial formulation or free solution. Interestingly, the delivery of the selected drug in the

niosomal formulations almost changed its Tmax, indicating a sustained and delayed type in-vivo

release of the drug from the formulation. This sustained and delayed release of the entrapped


78

drugs from this formulation is also evident from the increased MRT value as compared to those

of the respective commercial formulation or free solution.

Another important pharmacokinetic parameter of a drug is its Cl from the biological system of

the body. The Cl of Ciprofloxacin got decreased upon its entrapment in these niosomal vesicles.

This clearly indicates that the drug remains in the biological system for an extended period of

time upon its oral delivery in such niosomal vesicles. This would ultimately lead to prolonged

therapeutic effects of the drug even at same doses. Moreover, the drug was present in plasma in a

good quantity even up to last sampling interval (24 h) after its oral administration in the form of

niosomal formulation. In comparison, its commercial formulation or free solution cleared from

the body very earlier. Results show that Ciprofloxacin oral bioavailability was increased when it

was delivered in the niosomal formulation based on BRG-BTD-14 synthesized nonionic

surfactant.

Various factors can be considered for enhancing the oral bioavailability of Ciprofloxacin through

its niosomal formulation. The niosomal spherical morphology and nano-size play imperative role

in gastric absorption mechanism, causing increased bioavailability of the drug. Size of the drug

delivery systems plays always a vital role in the increased absorption of drugs from GIT [150],

thus it can be a possible reason for the increased drugs permeation through the epithelial cells

and enhanced the drugs oral bioavailability [138]. Similarly, lipophilic and the nonionic nature of

the niosomal vesicles also greatly affect the gastrointestinal membrane permeability, increasing

drug bioavailability [63, 74]. Surface charge of the niosomal vesicles is also an important

parameter for enhancing the oral bioavailability of the entrapped drugs. The drugs enhanced oral

bioavailability when delivered with this niosomal formulation can also be attributed to its

negatively charged surfaces as reported elsewhere [63].


79

The observed sustained release behavior of our novel surfactant based formulation can be better

understood by the fact that niosomes act as controlled release formulations and carry drug

through the mucosal layers, where the entrapped drug is gradually released [151]. Moreover, the

hydrophobicity of the surfactant makes the vesicles more stable and reduces their permeability,

causing the vesicles to release the drug in the body for a longer time [67]. Another possible

mechanism for achieving the sustained release behavior is that niosomal vesicles act as sustain

release vehicle. Niosomes are capable of carrying the drugs deeper into the mucosal layers

through the epithelium and then release the loaded drugs slowly and gradually [151]. This

sustain release effect of niosomal formulation may be due to the presence of cholesterol.

Cholesterol performs as membrane stabilizer and thus modulates the drug release from niosomal

vesicles. Similarly, cholesterol fills the gaps in the niosomal bilayers and prevents the gel to

liquid transition, thus making the niosomal vesicles less leaky with ultimate sustain release of the

encapsulated drug contents [152, 153].


80

Figure 4. 20: Plasma drug concentration of Ciprofloxacin loaded in BRG-BTD-14, commercial

suspension and solution at various time intervals.


81

Table 4. 2: Pharmacokinetic parameters of Ciprofloxacin loaded in BRG-BTD-14 based

niosomal formulation, commercial suspension and solution. P values<0.05 was considered

significant

Pharmacokinetic

Parameters

BRG-BTD-14 based

niosomal formulation

Commercial

suspension Free drug solution

Dose (mg/kg body

weight) 7 7 7

Cmax (µg/mL) 2.67 ±0.43* 1.56 ±0.21 1.32 ±0.37

Tmax (h) 2.5 1 1

AUC0-24 (µg.h/mL) 27.84 ±0.93*** 14.66 ±0.51 9.80 ±0.66

Cl (L/h.kg) 0.25 ±0.02*** 0.48 ±0.03 0.71 ±0.03

MRT (h) 9.1 ±0.23ns 7.67 ±0.45 6.90 ±0.38

AUMC0-24(µg.h2/ml) 255.37 ±1.75*** 112.50 ±1.43 67.70 ±0.94

4.4 Cefixime loaded BRG-LRC-12 niosomal formulation


4.4.1.1 Surface morphology, PDI, size and zeta potential

AFM was used to directly visualize the morphology of Cefixime-loaded BRG-LRC-12 vesicles.

Smooth sphere morphology was observed for Cefixime-loaded vesicles as shown in Figure 4.21.

Using DLS, the size of the drug loaded vesicles was found to be 159.76 ±6.54 nm with 0.32

±0.01 PDI as shown in Table 4.3. The PDI value shown by our surfactant based niosomes is


82

technically excellent as it falls near 0.3, indicating narrow size distribution and homogeneous

formulations. Cefixime-loaded BRG-LRC-12 vesicles revealed a net surface charge of -17.3

±1.83 mV as shown in Table 4.3.

As discussed earlier, smaller vesicles of homogenous nature are important for increasing the in-

vivo performance of their entrapped drug. Smaller drug loaded vesicles enhance the drug

bioavailability through facilitated trans-membrane absorption. Similarly, smaller vesicles result

in the increased physical stability, lower toxicity of carrier system, enhanced membrane

permeability and over all therapeutic efficacy of the entrapped drug [148, 154]. BRG-LRC-12

based Cefixime loaded vesicles were found well within nano-size range with small variations in

their size distribution, confirming their effective utilization for enhancing the therapeutic efficacy

of entrapped Cefixime.

Zeta potential is important parameter for drug delivery systems as it not only predicts the

physical stability but also ensures the enhanced in-vivo therapeutic efficacy of the entrapped

drugs. It also decreases the drug clearance from the body and increases the drug absorption, thus

causing increased oral bioavailability of the entrapped substances. BRG-LRC-12 based Cefixime

loaded vesicles showed increased negative charge on their surfaces, thus confirming their

physical stability and enhanced in-vivo therapeutic efficacy.


83

Figure 4. 21: AFM images of Cefixime loaded BRG-LRC-12 niosomal vesicles.


Cefixime loaded BRG-LRC-12 based niosomal formulation was investigated for it drug

entrapment efficiency through HPLC using the already developed method. The niosomes were

able to entrap 71.39 ±3.52% of the drug as shown in Table 4.3. This means that 0.11 mg of

Cefixime was entrapped per 1 mg of the lipid phase (BRG-LRC-12 and cholesterol).

Drug delivery systems are expected for entrapping increased concentrations of drug substances.

Increased entrapment efficiency results in sustained release of the loaded substances as well as

increase their therapeutic efficacy. Niosomal vesicles with increased entrapment efficiency have

also been reported for delivering the increased concentration of drugs to the target diseased sites.

LRC-BRG-12 based niosomal vesicles entrapped increased amount of Cefixime, anticipating

their effective utilization in oral delivery of the drug. Increased drug entrapment efficiency of

LRC-BRG-12 can be rationalized by proposing the use of cholesterol as additive as well as drug

and surfactant lipophilic nature [155, 156].


84

Table 4. 3: Characterization of Cefixime loaded BRG-LRC-12 niosomes for zeta potential, size,

PDI and drug encapsulation efficiency

Sample Composition

BRG-LRC-12/Cholesterol/Drug

Drug entrapment efficiency

(%)

Average vesicle size (nm)

Polydispersity Index (PDI)

Zeta Potent

ial (mV)

Cefixime

BRG-LRC-12

vesicles

4 : 2 : 1 71.39 ±3.52 159.76 ±6.54 0.32 ±0.01 -17.3

±1.83


In-vitro release behavior of Cefixime loaded BRG-LRC-12 niosomes was studied at 1.2, 4. 6.8

and 7.4 pH. The niosomal vesicles released their loaded drug in a programmed way at all tested

pH. The acidity of the medium affected the drug release from the niosomal vesicles. They

released the loaded drug in different manner at different pH. Niosomal formulation showed

increased drug release for extended period of time at pH 7.4, confirming its enhanced in-vitro

stability and bioavailability. When the pH of the medium was decreased (increasing the acidity

of the medium), the drug release was found to be decreased from the niosomal vesicles as shown

in Figure 4.22. In-vitro release study is performed to investigate the effect of pH on drug release

from niosomal vesicles. Interestingly, no abrupt release of drug from the formulation was

observed at any pH, confirming the stability of the drug-loaded vesicles. The drug release was a

little bit quick during the initial two hours of the study. This may be due to the release of

Cefixime that got loosely bound to vesicles surfaces.


85

Figure 4. 22: In-vitro drug release study of Cefixime loaded BRG-LRC-12 niosomal

formulation



Cefixime loaded BRG-LRC-12 niosomal vesicles were investigated for their storage stability for

a time period of 30 days. The storage stability of the formulation was determined in term of its

ability of retaining the entrapped drug. The amount of Cefixime leaked from vesicles during the

storage period was detected at 289 nm spectrophotometrically and then quantified. Results show

that very small amount of the entrapped drug leaked out during storage. The formulation retained

about 91.03 ±0.87, 93.88 ±1.43, 97.35 ±0.67 and 99.22 ±0.71% of the entrapped drug at 30th,

20th, 10th and 1st day of the storage period respectively.


86

Storage stability is important for determination of the percent drug retained by the drug delivery

systems. Highly stable niosomal vesicles are able to prevent the pre-mature leaking of the

entrapped drugs, thus ensures the even distribution of drugs in the biological environment [129].

BRG-LRC-12 based niosomal vesicles demonstrated to retain maximum amount of the

entrapped drug inside them for a period of one month. Results suggest the vesicles to be highly

effective in oral delivery of the entrapped Cefixime. Their higher stability can be due to inclusion

of cholesterol in their lipid bilayer membrane and their increased surface negativity.


The Cefixime loaded BRG-LRC-12 niosomal formulation was subjected to SGF stability study.

The formulation was incubated with SGF and the amount of drug released was quantified

through already developed HPLC method and the percent drug retained was calculated

accordingly. The niosoaml formulation was able to retain about 97.58 ±2.81, 93.66 ±0.81, 90.46

±0.45 and 87.65 ±0.76% of the loaded Cefixime after 10, 20, 40 and 60 min respectively.

Niosomal vesicles must not be destabilized upon their contact with SGF after their oral

administration. To achieve higher oral bioavailability and therapeutic efficacy of the entrapped

drugs, niosomal vesicles should be strong enough to retain their maximum loaded contents intact

inside the vesicles [149]. Results show that BRG-LRC-12 based niosomal vesicles can withstand

the harsh gastric fluids and did not release the entrapped drug prematurely. This would

ultimately lead to the desired therapeutic efficacy of the entrapped drug.


Cefixime was administered to rabbits in the form of BRG-LRC-12 niosomal formulation,

commercial capsule and suspension at 6 mg/kg dose and mean plasma drug concentrations at


87

various time intervals are given in Figure 4.23. Plasma drug concentrations at various time

intervals were used for the determination of various pharmacokinetic parameters and are shown

in Table 4.4. BRG-LRC-12 based Cefixime niosomal formulation showed significantly higher

values for Cmax, AUC0-24, MRT and AUMC0-24 as compared to commercial formulations. No

significant difference was found between the values of Tmax for the surfactant based niosomal

and commercial formulations of Cefixime. The entrapment of Cefixime in BRG-LRC-12

niosomal formulation caused Cmax (10.75 ±0.85 µg/mL) twice elevated as compared to

commercial tablets (6.13 ±0.90 µg/mL) and suspension (5.63 ±0.82 µg/mL). Moreover, the drug

entrapment in these vesicles reduced its clearance from the body (0.1 L/h.kg) as compared to

commercial tablets (0.22 L/h.kg) and suspension (0.32 L/h.kg). Another important aspect of

pharmacokinetic parameter is that the entrapment of Cefixime in BRG-LRC-12 niosomal

formulation caused it remained in the body for longer period of time (9.19 h) as compared to

commercial tablets (7.75 h) and suspension (5.93 h).

Results of the current study show that Cefixime plasma level was elevated throughout the study

period when administered orally in the form of BRG-LRC-12 based niosomal formulation.

Similarly, novel niosomal formulation of Cefixime decreased its clearance from the biological

system as compared to its commercial suspension and capsules. This indicates that niosomal

encapsulation causes a sustained release of Cefixime in the blood and maintains its plasma level

for extended period of time. This is also evident from the MRT data of Cefixime, showing its

presence in the plasma for a longer period of the time as compared to its commercial

formulations. The increased oral bioavailability of Cefixime achieved through BRG-LRC-12

based niosomal formulation may be due to synthesized nonionic surfactant BRG-LRC-12 acting

as penetration enhancer. Other factors responsible for enhanced oral bioavailability of Cefixime


88

include increased penetration of mucosa due to lipophilic nature of vesicles, vesicles size in

nano-range and enhanced solubility of Cefixime in vesicles [63, 157].

Figure 4. 23: Plasma drug concentration of Cefixime loaded BRG-LRC-12, Cefiget (Capsule)

and Maxpan (Suspension) at various time intervals.


89

Table 4. 4: Pharmacokinetic parameters of BRG-LRC-12 based Cefixime niosomal formulation,

Maxpan (suspension) and Cefiget (Capsules). P values<0.05 was considered significant

Pharmacokinetic Parameters

BRG-LRC-12 based


Cefiget

(Capsule) Maxpan

(Suspension)

Dose (mg/kg body weight) 6 6 6

Cmax (µg/mL) 10.75 ±0.85*** 6.13 ±0.90 5.63 ±0.82

Tmax (h) 6 6 6

AUC0-24 (µg.h/mL) 119.58 ±0.89*** 54.32 ±1.71 37.36 ±2.26

Cl (L/h.kg) 0.1 ±0.01*** 0.22 ±0.03 0.32 ±0.04

MRT (h) 9.19 ±0.23** 7.75 ±0.66 5.93 ±0.48

AUMC0-24 (µg.h2/mL) 1099.88 ±3.54*** 421.34 ±6.36 221.6 ±4.23

4.5 Cefixime loaded BRG-BRM-11 niosomal formulation


4.5.1.1 Surface morphology, zeta potential, size and PDI

Shape of the drug loaded vesicles was studied through AFM. Images reveal that they are having

spherical with smooth surfaces as shown in Figure 4.24. Similarly, DLS investigation shows that

drug loaded vesicles are having mean hydrodynamic diameter of 198.66 ±8.17 nm as shown in

Table 4.5. The PDI values of the drug loaded was found to be 0.27 ±0.01 as shown in Table 4.5.

The PDI values is less than 0.3, thus reveals that the drug loaded vesicles are having uniform size

distribution and are not polydispersed. The vesicles were found to be negatively charged with a

net zeta potential value of -18.30 ±0.76 mV as shown in Table 4.5.


90

Size, size distribution and surface charge are considered important parameters for niosomal drug

loaded vesicles. They contribute to the overall physical and biological properties of the niosomal

formulations. Smaller size vesicles with narrow size distribution play important role in toxicities

associated with drug delivery systems. Smaller vesicles are less toxic than their larger

counterparts. Similarly, they are able to carry the drug deeper in the mucosal layers, thus

enhancing their oral bioavailability and ultimate therapeutic efficacy. The increased surface

charge of the vesicles performs dual functions of increasing formulation physical stability and

drug absorption [37, 158]. Here in this study, BRG-BRM-11 based niosomal vesicles revealed

smaller size with uniform size distribution and higher surface charge, confirming their technical

suitability for enhancing Cefixime oral bioavailability.

Figure 4. 24: AFM images of Cefixime loaded BRG-BRM-11 niosomal vesicles


Already developed HPLC method was used for investigating the Cefixime entrapment efficiency

of BRG-BRM-11. The niosomal formulation showed the drug entrapment efficiency to be as


91

high as 78.4±0.83% as shown in Table 4.5. Results show that each 1 mg of the lipid niosomes

phase was able to entrap about 0.13 mg of the drug used.

Niosomal formulations are preferred for drug delivery because of their increased drug

entrapment capability. Niosomal vesicles entrapping maximum drug contents are able to deliver

higher concentration of drug into the systemic circulation, thus resulting in higher bioavailability.

BRG-BRM-11 based niosomal formulation entrapped maximum amount of Cefixime, predicting

better in-vivo performance of the entrapped drug. Several factors like cholesterol inclusion as

additive, surfactant structure and lipophilicity of the drug can add to the increased drug

entrapment efficiency of BRG-BRM-11 [159].

Table 4. 5: Characterization of Cefixime loaded BRG-BRM-11 niosomes for zeta potential, size

and PDI and drug encapsulation efficiency.

Sample

Composition BRG-BRM-

11/Cholesterol/Drug (w/w/w)

Drug encapsulation efficiency (%)

Average vesicle size

(nm)


Zeta Potential

(mV)

Cefixime loaded BRG-BRM-11

vesicles

4 : 2 : 1 78.4 ±0.83 198.66

±8.17 0.27 ±0.01

-18.30

±0.76


BRG-BRM-11 based drug loaded niosomes were investigated for their in-vitro drug release

behavior at various pH i.e 1.2, 4, 6.8 and 7.4. The drug release from the vesicles was well

programmed at all tested pH. The increase in acidity of the medium had a great effect on the

drug release from the formulation. The drug release was decreased upon decreasing the pH of the

medium. Maximum drug was released at pH 7.4 in a fair constant way, confirming the enhanced


92

in-vitro stability and bioavailability of the formulation. The drug release from the niosomal

formulation got decreased when the medium acidity was increased as shown in Figure 3.25.

Maximum drug release was achieved for pH 7 (90%) at 10th h of the study. The maximum drug

release for pH 6.8, 4 and 1.2 was 70, 60 and 57% respectively that achieved at 8th h.

Figure 4. 25: In-vitro drug release study of Cefixime loaded BRG-BRM-11 niosomal

formulation



BRG-BRM-11 based Cefixime niosomal vesicles were investigated for their storage stability up

to 30 days. The storage stability of the formulation was determined in term of its ability to retain

the entrapped drug. The amount of Cefixime leaked from vesicles during the storage period was

detected at 289 nm spectrophotometrically and then quantified. A small amount of the entrapped


93

drug leaked out during storage, thus showing the vesicles highly stable. The vesicles were found

able to retain about 85.19 ±02.44, 89.80 ±3.71, 94.53 ±1.75 and 97.69 ±2.41% of the entrapped

drug at 30th, 20th, 10th and 1st day of the storage period respectively.

Leaking of drugs from drug delivery systems upon storage has been a matter of great concern for

formulation scientists. The premature leaking of the loaded contents form the formulation

decreases the success of treatment strategy as minimum amount of the drug reaches the systemic

circulation. BRG-BRM-11 based niosomal formulation was found highly stable upon storage for

one month. It exhibited minimum leaking of the drug, thus authenticating its applications for oral

drug delivery of Cefixime.


The Cefixime loaded BRG-BRM-11 niosomal formulation was subjected to SGF stability study.

The formulation was incubated with SGF and the amount of drug released was quantified

through already developed HPLC method and the percent drug retained was calculated

accordingly. The niosomal formulation was able to retain about 95.38 ±0.34, 92.56 ±2.04, 86.78

±1.48 and 82.00 ±2.32% of the loaded Cefixime after 10, 20, 40 and 60 min respectively. Results

show that leaking out of the drug increases as the incubation time increased.

Drug delivery systems intended for oral administration face the harsh gastric environment due to

lower pH and presence of enzymes. Such systems must not release the loaded drugs and should

remain stable upon their contact with harsh gastric fluids of stomach. If they do not withstand the

harsh gastric environment, then the loaded drugs will get released and will be ultimately

deactivated by the stomach enzymes. This enzymatic deactivation of drug substances results in

lower clinical efficacy of the drugs. BRG-BRM-11 based Cefixime loaded niosomal vesicles


94

demonstrated to be highly stable after their incubation with SGF, confirming their applications

for Cefixime oral delivery.


Cefixime loaded niosomal formulation of BRG-BRM-11 was administered rabbits orally at 6

mg/kg dose the mean plasma drug concentrations at various time intervals are shown Figure

4.26. Pharmacokinetic parameters were calculated from individual drug plasma concentrations at

various time intervals and given in Table 4.6. Cefixime revealed maximum drug plasma

concentration (Cmax) of 9.69 ±1.22 µg/mL when administered orally in the form of BRM-BG-11

based niosomal formulation. This was almost two times greater than Cmax (5.63 ±0.83 µg/mL) of

Cefixime when administered in the form of its commercially available suspension. Similarly, the

niosomal formulation also showed higher Cmax than that shown by the drug commercial capsules

revealing a Cmax of 6.130 ±090 µg/mL. All the three formulations showed a similar time to reach

maximum drug plasma concentration (Tmax). The entrapment of the drug in such vesicles

decreased its Cl from the body (0.108 ±0.01 L/h.kg) as compared to commercial capsules (0.22

±0.03 L/h.kg) and suspension (0.32 ±0.04 L/h.kg). Moreover, the entrapment of the drug in these

niosomal vesicles also increased its MRT in the body that was 9.20 ±1.00 h as compared to 7.75

±0.66 and 5.93 ±0.48 h shown by commercial capsules and suspension respectively.

Entrapment of Cefixime in BRG-BRM-11 based niosomal vesicles increased its oral

bioavailability in a sustained manner as compared with its commercial formulations. An elevated

Cmax was observed for Cefixime when given orally in the form of niosomal formulation.

Moreover, its administration in the form of niosomal formulation maintained its plasma level

much higher than its commercial formulations given at the same oral dose. The amount of drug


95

cleared from the biological system was increased for commercial formulations as compared to

the niosomal formulations, confirming the sustained release of Cefixime from niosomal

formulation. Furthermore, Cefixime retained in the body for longer time when administration in

the form of niosomal formulation. This indicates that the novel niosomal formulation will keep

the minimum therapeutic level of the drug for extended period of time at the same dose as

compared to its commercial formulations. Enhanced oral bioavailability of Cefixime can be

attributed to multiple factors like niosomes nano-size range, their enhanced permeability through

biological membranes, increased surface negativity and their sustained release ability of the drug.

Figure 4. 26: Plasma drug concentration of Cefixime loaded BRG-BRM-11, Cefiget (Capsule)

and Maxpan (Suspension) at various time intervals.


96

Table 4. 6: Pharmacokinetic parameters of BRG-BRM-11 based Cefixime niosomal suspension,

Maxpan (suspension) and Cefiget (Capsules). P values<0.05 was considered significant


BRG-BRM-11 based


Cefiget

(Capsule) Maxpan

(Suspension)

Dose (mg/kg body weight)

6 6 6

Cmax (µg/mL) 9.69 ±1.22** 6.13 ±0.90 5.63 ±0.82

Tmax (h) 6 6 6

AUC0-24 (µg.h/mL) 110.55 ±2.07*** 54.32 ±1.71 37.36 ±2.26

Cl (L/h.kg) 0.108 ±0.01*** 0.22 ±0.03 0.32 ±0.04

MRT (h) 9.20 ±1.00** 7.75 ±0.66 5.93 ±0.48

AUMC0-24 (µg.h2/mL) 1017 ±6.80*** 421.34 ±6.34 221.6 ±4.23

4.6 Levofloxacin loaded BRG-BRD-10 niosomal formulation


4.6.1.1 Surface morphology, Size, PDI and zeta potential

Drug loaded niosomal vesciles were studied through AFM for their shape. The vesicles revealed

a spherical shape (Figure 4.27). DLS zetasizer was used for the determination of vesciles mean

diameter and PDI and results are given in Table 4.7. The mean diameters of drug loaded vesicles

were found to be 190.31 ±4.51 respectively. The PDI of the drug loaded vesicles was found to be


97

0.29, revealing that the drug loaded vesicles population was relatively homogenous (i.e. size

distribution was relatively narrow). The drug loaded vesicles showed -36.24 ±1.36 mV zeta

potential, confirming the formulation stability.

Vesicles size and zeta potential are highly important for their stability and drug increased

absorption. Vesicles of smaller size exhibit lower toxicity as compared to larger ones prepared

from the same nonionic surfactants. Smaller size vesicles are capable of efficiently crossing the

biological membranes, thus increasing the oral bioavailability of entrapped drugs [160, 161].

Levofloxacin loaded niosomal vesicles of BRG-BRD-10 were found to be smaller in size,

confirming their use for enhancing oral bioavailability of Levofloxacin. Zeta potential is also

important characteristic of the niosomal vesicles that predicts their stability upon storage. It also

helps in decreasing the drug clearance from the body, thus increases the drug circulation time in

the body for a longer period [141]. Levofloxacin loaded niosomal vesicles of BRG-BRD-10

revealed increased negative surface charge, thus predicting their higher physical stability and in-

vivo performance.


98

Figure 4. 27: AFM images of Levofloxacin loaded BRG-BRD-10 niosomal vesicles.


Entrapment efficiency was investigated using the developed HPLC method for detection and

quantification of Levofloxacin. Results show that BRG-BRD-10 niosomal vesicles entrapped

68.28 ±3.45% of the drug (Table 4.7). This shows that each mg of the lipid phase (surfactant plus

cholesterol) has entrapped 0.15 mg of the drug.

Increased drug entrapment is vital for designing effective drug delivery systems. Increased oral

bioavailability in a sustained and controlled manner has been the function of increased drug

entrapment of the drug delivery systems as discussed earlier. BRG-BRD-10 entrapped increased

concentration of Levofloxacin, showing its applicability for niosomal drug delivery. This can be

due BRG-BRD-10 lipophilicity, cholesterol presence in the bilayer of the vesicles as well as drug

hydrophobic nature as discussed earlier.


99

Table 4. 7: Characterization of drug loaded BRG-BRD-10 niosomes for zeta potential, size and

PDI and drug encapsulation efficiency.

Sample

Composition BRG-BRD-

10/Cholesterol/Drug (w/w/w)

Drug encapsulation efficiency (%)

Average vesicle

size (nm)


Zeta potential

(mV)

Levofloxacin loaded BRG-

BRD-10 vesicles

3 : 1.5 : 1 68.28 ±3.45 190.31

±4.51 0.29 ±0.03

-36.24

±1.36


Levofloxacin in-vitro release from BRG-BRD-10 based niosomal formulation was studied by

employing the dialysis method at pH 7.4, 6.8, 4 and 1.2. Drug released in the medium was

detected at 298 nm spectrophotometrically and then quantified accordingly. Release of the drug

from formulation was found to be well controlled and programmed at all pH. However the

proportions of drug released were different using different pH system, indicating the effect of

medium acidity on the drug release from the niosomal formulation. Maximum drug release was

obtained at the same time for all media studied (i.e. 9 h) and the highest was at pH 7.4 (84.23%)

and lowest at pH 1.2 (59.76%). At pH 7.4, the drug release from the vesicles was increased in a

fair sustained manner, validating the increased in-vitro bioavailability and stability of the drug

loaded formulation. Decreasing pH caused a decrease in the percentage of drug released from the

vesicles, but no abrupt release of drug at any stage of the study at any pH was observed (Figure

4.28).


100

Figure 4. 28: In-vitro drug release study of Levofloxacin loaded BRG-BRD-10 niosomal

formulation.



Levofloxacin loaded BRG-BRD-10 niosomal vesicles were investigated for their storage stability

up to 30 days. The storage stability of the formulation was determined in term of its ability to

retain the entrapped drug. The amount of Levofloxacin leaked from vesicles during the storage

period was detected at 298 nm spectrophotometrically and then quantified. Results show that a

small amount of the entrapped drug leaked out during storage. The formulation retained about

83.51 ±2.90, 87.61 ±0.75, 92.47 ±2.62 and 96.89 ±1.33% of the entrapped drug at 30th, 20th, 10th

and 1st day of the storage period respectively.


101

Storage stability is one of the important requirements for drug delivery systems. Pharmaceutical

formulations should not release the loaded contents during their storage at specific temperature

conditions. Formulations that release their loaded contents during storage period result in inferior

therapeutic efficacy of the treatment strategies. BRG-BRD-10 niosomal vesicles were stable up

to one month and retained the maximum amount of Levofloxacin, confirming their higher

storage stability.


The Levofloxacin loaded BRM-BRD-10 niosomal formulation was investigated for its stability

against SGF. The formulation was incubated with SGF and the amount of drug released was

quantified through already developed HPLC method and the percent drug retained was

calculated accordingly. The niosoaml formulation was able to retain about 95.57 ±0.78, 91.84

±2.87, 87.39 ±0.1.69 and 82.54 ±2.26% of the loaded Levofloxacin after 10, 20, 40 and 60 min

respectively.

Enzymatic deactivation of drugs in gastric environment is one of the factors that lead to poor

clinical outcomes of the drugs therapy. Pharmaceutical formulations should be enough strong to

withstand the harsh gastric juices and enzymes. It should not be destabilized within stomach and

should not release the loaded drugs so to avoid their enzymatic deactivation. BRG-BRD-10

niosomal vesicles were investigated for their stability against incubation with SGF for a time

period of one hour. They were found to be highly stable and retain about 82.54 ±2.26% of the

loaded Levofloxacin at the end of the study. This confirms that BRD-10 niosomal vesicles are

capable of protecting Levofloxacin from enzymatic deactivation in the stomach and would

ultimately lead to its increased oral bioavailability.


102


Levofloxacin was orally administered to rabbits at 10 mg/kg single dose in the form of BRM-

BRD-10 niosomal formulation, commercial tablets (suspended powder) and its solution. The

mean plasma drug at different time intervals is shown in Figure 4.29. Pharmacokinetic

parameters for Levofloxacin after its BRM-BRD-10 niosomal formulation, tablets and solution

administration were determined from individual mean drug plasma values at various time

intervals and are shown in Table 4.8. Levofloxacin when administered in BRM-BRD-10

niosomal formulation showed significantly higher Cmax, AUC0-24, MRT and AUMC0-24 as

compared to the drug in tablets or solution. Increased oral bioavailability of Levofloxacin was

achieved with this novel niosomal formulation having 3.42 ±0.45 µg/mL Cmax as compared to the

tablets and solution revealing 2.39 ±0.38 and 2.13 ±0.51 µg/mL Cmax respectively. Sustained

release effect of this novel surfactant based niosomal formulation can be predicted from an

increase in MRT (9.71 ±0.42 h) and AUC0-24 (41.19 ±0.53 µg.h/mL) and decrease in Cl (0.24

±0.03 L/h.kg) when compared with the tablets and drug solution. The sustained release of the

niosomal formulation can also be confirmed from the increased Tmax (4 h) as compared to that of

drug in solution (2 h).

Drugs show their therapeutic efficacy when they reach systemic circulation. The amount of drug

that becomes available in the systemic circulation is called its bioavailability. Poor

bioavailability of drugs results in their poor clinical outcomes. Enhancing drugs oral

bioavailability is one of the important strategies for achieving their effective clinical outcomes.

BRG-BRD-10 based niosomal vesicles increased the oral bioavailability of Levofloxacin in a

sustained and controlled manner. It caused an elevated Cmax of the drug as compared to its


103

commercial tablets and free solution. The niosomal formulation was capable of maintaining the

Levofloxacin plasma level elevated during the study period as compared to its commercial

formulation or drug free solution. This was evident from the increased MRT and decreased Cl

values of Levofloxacin when delivered in the form of niosomal formulation. Surface negativity,

nano-range size and higher permeability of the niosomal vesicles are among the possible factors

resulting in enhanced oral bioavailability of Levofloxacin.

Figure 4. 29: Plasma drug concentration of Levofloxacin loaded in BRG-BRD-10 nioosmal

formulation, Tablets and solution at various time intervals.


104

Table 4. 8: Pharmacokinetic parameters of Levofloxacin loaded in BRG-BRD-10 based

niosomal formulation, Tablets and solution. P values<0.05 was considered significant


BRG-BRD-10 based

niosomal formulation Tablets Levofloxacin

solution

Dose (mg/kg body weight) 10 10 10

Cmax (µg/mL) 3.42 ±0.45*** 2.39 ±0.38 2.13 ±0.51

Tmax (h) 4 4 2

AUC0-24 (µg.h/mL) 41.19 ±0.53*** 25.37 ±0.75 17.32 ±1.08

Cl (L/h.kg) 0.24 ±0.03*** 0.39 ±0.02 0.58 ±0.04

MRT (h) 9.71 ±0.42*** 8.44 ±0.55 6.23 ±0.85

AUMC0-24 (µg.h2/mL) 400.01 ±1.65*** 214.17 ±1.74 107.92 ±2.18

4.7 Moxifloxacin loaded BRG-BRN-9 niosomal formulation


4.7.1.1 Surface morphology, zeta potential, size and PDI

The AFM study reveals a spherical morphology for the Moxifloxacin loaded niosomal vesicles

as shown in Figure 4.30. The DLS analysis reveals that drug loaded vesicles have mean

hydrodynamic diameter of 221.63 ±7.45 nm as depicted in Table 4.9. The PDI of the

Moxifloxacin loaded vesicles was found to be 0.302 ±0.027. Thus, the drug loaded niosomal

vesicles have their populations with homogenous size distribution. When investigated for

determination of surface charge, the drug loaded vesicles revealed -13.61 ±2.43 mV zeta

potential as shown in Table 4.9.


105

Niosomal vesicles are preferred as drug delivery systems because of their certain advantageous

characteristics. Surface charge and nano-range size are two important characteristics that predict

the in-vivo performance and physical stability of niosomal vesicles. Smaller vesicles with higher

surface charge are able to remain stable and absorbed well through biological membranes, thus

leads to better clinical outcomes of the therapy. BRG-BRN-9 based drug loaded niosomal

vesicles revealed smaller size with increased surface negativity. Both these characteristics predict

better in-vivo performance and higher physical stability of the BRG-BRN-9 based drug loaded

niosomal vesicles.

Figure 4. 30: AFM images of Moxifloxacin loaded BRG-BRN-9 niosomal vesicles


The developed HPLC method was used for the detection and quantification of Moxifloxacin

loaded in BRG-BRN-9 niosomal formulation. The vesicles were able to entrap 54.73 ±2.56% of

Moxifloxacin. About 0.145 mg of the drug got encapsulated in 1 mg lipid phase (surfactant and

cholesterol) as shown in Table 4.9.


106

Higher drug entrapment efficiency is required for niosomal vesicles. Enhanced oral

bioavailability and therapeutic efficacy have been the functions of increased drug entrapment

efficiency of the niosomal vesicles. BRG-BRN-9 based niosomal vesicles were found to entrap

moderate amount (about 55%) of Moxifloxacin. But this was relatively lower entrapment

efficiency as compared to other nonionic surfactants used in this research work. Hydrophilic

nature of the drug and relatively lower lipophilicity of the BRG-BRN-9 can be the possible

reasons for a relative lower entrapment efficiency of these niosomal vesicles.

Table 4. 9: Characterization of Moxifloxacin loaded BRG-BRN-9 niosomes for zeta potential,

size and PDI and encapsulation efficiency.

Sample

Composition BRG-BRN-9:Chol:Moxi

(w/w/w)

Drug entrapment

efficiency (%)

Mean hydrodynamic diameter (nm)

Polydispersit

y index (PDI)

Zeta Potential

(mV)

Moxifloxacin

loaded BRG-

BRN-9 vesicles

2:1.5:1

54.73 ±2.56

221.63 ±7.45

0.302 ±0.027

-13.61

±2.43


The Moxifloxacin release from the novel nonionic surfactant based vesicles was monitored for

24 h using dialysis membrane method and was quantified through UV spectrophotometer.

Moxifloxacin release was obtained in a fair controlled way. There was no premature or abrupt

release of the drug at any stage of the study. Initially, at 2nd h of the study, a little higher

Moxifloxacin (16.86 and 13.49%) was found to be released at pH 7.4 and 6.8 respectively.

Results of the study show that the medium acidity greatly affects release of drug form the


107

formulation. For pH 1.2 and 4, drug release was decreased and 50% Moxifloxacin was achieved

earlier (at 8th h) as compared to drug release in medium of pH 6.8 and 7.4 (almost 50% drug

release was achieved at 6th and 5th h respectively). Similarly, maximum drug release was

achieved at 9th h of the study for almost all pH. But this was higher for pH 7.4 and 6.8 (82.45 and

75.67% respectively) as compared to pH 1.2 and 4 (66.38 and 71.62% respectively) as shown in

Figure 4.31.

Figure 4. 31: In-vitro drug release study of Moxifloxacin loaded BRG-BRN-9 niosomal

formulation.



Moxifloxacin loaded in BRG-BRN-9 niosomal vesicles were investigated for their storage

stability for a time period of 30 days. The storage stability of the formulation was determined in


108

term of its ability to retaining the entrapped drug. The amount of Moxifloxacin leaked from

vesicles during the storage period was detected at 294 nm spectrophotometrically and then

quantified. The BRG-BRN-9 niosomal vesicles loaded with Moxifloxacin were highly stable

upon storage. The niosomal vesicles were found to retain about 89.46 ±0.66, 93.81 ±1.61, 95.71

±0.39 and 99.03 ±0.52% of the encapsulated drug at 30th, 20th, 10th, and 1st day respectively.

Results of storage stability studies confirm that BRG-BRN-9 based niosomal vesicles are highly

stable and can retain the loaded contents for a period of one month. The stability of the vesicles

can be attributed to many factors like smaller particles size and inclusion of cholesterol in the

niosomal vesicles. Cholesterol cements the leaking spaces in the vesicles bilayer and thus

prevents the drug leaching. Surface negativity also plays important role as it prevents the vesicles

fusion and destabilization.


Moxifloxacin loaded novel niosomal vesicles demonstrated to be highly stable after their

incubation with SGF. The amount of drug released was quantified through already developed

HPLC method and the percent drug retained was calculated accordingly. Negligible amount of

the loaded drug leaked out from niosomal vesicles upon their contact with SGF for 1 h time

period. They were found to retain 98.43 ±3.64, 96.81 ±1.87, 92.51 ±0.45 and 87.93 ±2.60% of

the loaded drug after 10, 20, 40 and 60 min respectively.

Gastric fluids contain enzymes that develop a harsh biological environment. Drugs taken orally

are often deactivated by these enzymes and thus lead to lower efficacy of the drugs. Drug

delivery systems are designed in order to protect the drugs from such harsh enzymatic activities.

BRG-BRN-9 based Moxifloxacin loaded niosomal vesicles were subjected to their stability study


109

by incubating them with SGF for a time period of one hour. They were found stable and able to

retain maximum amount of the entrapped drug, confirming their utilization for effective oral

delivery of Moxifloxacin.


Moxifloxacin was administered to rabbits at 8 mg/kg body weight in the form of NB-BG loaded

niosomal vesicles, tablets or free solution and its mean plasma concentrations at various time

intervals are shown in Figure 4.32. Various pharmacokinetics were determined form individual

blood plasma time curves and are presented in Table 4.10. The novel nonionic surfactant based

vesicles were able to cause the elevated plasma level of Moxifloxacin as compared to its tablets

and free solution. For niosomal formulation of Moxifloxacin, maximum plasma concentration

(Cmax) was 3.39 ±0.24 µg/mL, higher than its tablets (2.880 ±0.49 µg/mL) and free solution (2.39

±0.33 µg/mL). Niosomal encapsulated Moxifloxacin revealed a Tmax of 3 h as compared to its

tablets and free solution (2.5 and 2 h respectively). Similarly, the mean residence time (MRT) of

Moxifloxacin was higher (8.03 ±1.37 h) when given orally in the form of niosoaml formulation

as compared to its tablets (7.36 ±0.63 h) and free solution (6.54 ±0.71 h). More importantly, the

niosomal loaded Moxifloxacin exhibited a decreased Cl (0.22 ±0.01 L/h.kg) from the body as

compared to its tablets and free solution showing 0.34 ±0.05 and 0.43 ±0.04 L/h.kg Cl

respectively. The decreased Cl and increased MRT of Moxifloxacin delivered in niosomal

formulation predict the sustain release behavior of drug loaded NB-BG based niosomal vesicles.

Niosomal nano-vesicles are intended for enhancing oral bioavailability of drugs. BRG-BRN-9

based niosomal vesicles were used for enhancing oral bioavailability of Moxifloxacin. They

caused a moderate elevation in the drug plasma concentration as compared to its tablets and free


110

solution. They vesicles were able to maintain the plasma level of the drug much higher than the

reference standards during study period, showing a controlled release of Moxifloxacin from

vesicles in biological system. The controlled release of the drug from vesicles in biological

system is further supported by its decreased Cl from the body. Moreover, the vesicles maintained

the drug in systemic circulation in a fair way for extended period of time when given orally in

the form of BRG-BRN-9 based niosomal vesicles, thus results in elevated drug plasma

concentration for longer time as compared to its tablets and free solution. Findings of the in-vivo

bioavailability study confirm the effectiveness of BRG-BRN-9 based niosomal vesicles in

enhancing the oral bioavailability of Moxifloxacin in a controlled manner.

Figure 4. 32: Plasma drug concentration of Moxifloxacin loaded in BRG-BRN-9 niosomal

formulation, Tablets and solution at various time intervals.


111

Table 4. 10: Pharmacokinetic parameters of Moxifloxacin loaded in BRG-BRN-9 based

niosomal formulation, tablets and solution. P values<0.05 was considered significant

Pharmacokinetic

Parameters

BRG-BRN-10 based

niosomal formulation Tablets Free drug solution

Dose (mg/kg body weight)

8 8 8

Cmax (µg/mL) 3.39 ±0.24*** 2.880 ±0.49 2.39 ±0.33

Tmax (h) 3.00 2.5 2.5

AUC0-24 (µg.h/mL) 35.59 ±1.49*** 23.23 ±0.48 18.36 ±1.03

Cl (L/h.kg) 0.22 ±0.01*** 0.34 ±0.05 0.43 ±0.04

MRT (h) 8.03 ±1.37 ns 7.36 ±0.63 6.54 ±0.71

AUMC0-24(µg.h2/ml) 285.71 ±2.08*** 171.00 ±2.31 120.19 ±1.47

Conclusion

112

Conclusion

Nonionic surfactants self-assemble in closed bilayered nano-structures called niosomes upon

coming in contact with aqueous medium. These vesicles are able to enclose lipid soluble drugs in

their lipid shells. The water soluble drugs remain dissolved in their inner aqueous pool. Niosomal

nano-vesicular drug carriers have the subject of greater current scientific interests. The

limitations of currently used nonionic surfactants have led the scientists to search for safe and

efficient alternative nonionic surfactants from renewable sources. This study focused the

synthesis of glycoside based biocompatible nonionic surfactants from renewable source. These

surfactants were highly biocompatible and were able to self-assemble in nano-niosomal vesicles.

Their niosomal vesicles entrapped increased amounts of the selected drugs. These vesicles were

highly negatively charged with small variations in their size distribution. These novel niosomal

vesicles enhanced the in-vivo oral bioavailability of their entrapped drugs when delivery orally in

animal model. Findings of this research work suggest these synthesized nonionic surfactants

highly biocompatible and effective novel niosomal nano-carriers for enhancing in-vivo

bioavailability and therapeutic efficacy of drugs. Results also open new avenues for effective

exploitation of these synthesized novel niosomal carriers for targeting drugs to specific sites

through their surface engineering with ligands.

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List of published papers

133

List of published papers

1. Muhammad Imran, Muhammad Raza Shah, Farhat Ullah, Shafi Ullah, Abdelbary

M.A. Elhissi, Waqas Nawaz, Farid Ahmad, Abdul Sadiq, Imdad Ali, “Glycoside-based

niosomal nanocarrier for enhanced in-vivo performance of Cefixime” International

Journal of Pharmaceutics 505 (2016) 122–132

2. Muhammad Imran, Muhammad Raza Shah, Farhat Ullah, Shafi Ullah, Abdelbary M.

A. Elhissi, Waqas Nawaz, Farid Ahmad, Abdul Sadiq, Imdad Ali, “Sugar-based novel

niosomal nanocarrier system for enhanced oral bioavailability of levofloxacin”

Drug Delivery 23 (2016) 3653-3664.

3. Muhammad Imran, Muhammad Raza Shah, Farhat Ullah, Shafi Ullah, Abdul Sadiq,

Imdad Ali, Farid Ahmed, Waqas Nawaz “Double-tailed acyl glycoside niosomal

nanocarrier for enhanced oral bioavailability of Cefixime” Artificial Cells,

Nanomedicine, and Biotechnology 45 (2017) 1440-1451.

SYNTHESIS OF GLYCOSIDE BASED BIOCOMPATIBLE NONIONIC …prr.hec.gov.pk/jspui/bitstream/123456789/9075/1/74... · 2018. 7. 23. · Wadood Ali Shah, Dr. Abdul Sadiq, Dr. Muhammad Ayaz,

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