Open Access Repository of Indian Theses

M., MADHUMALA

Abstract 
 
 
ABSTRACT 
Lysozymes are hydrolases that are divided into 6 different families, based on their 
distribution and function. They are c-type, g-type, i-type, plant, phage and bacterial. C-type 
lysozymes are predominantly expressed among a wide variety of species. Most recently, 
lysozyme like (LYZL) proteins that belong to the c-type lysozyme family were identified, 
which include the six members namely LYZL1, SLLP1/LYZL3, LYZL4, LYZL5, LYZL6 
and LYZL7. Recently, the importance of mouse SLLP1 as a receptor for glycoprotein 
interaction during fertilization was reported. However, the exact role of LYZL proteins in 
general physiology and in male reproductive function in particular has not been studied. 
Hence, we attempted to characterize the rat LYZL proteins with specific reference to male 
reproductive tract function.  
 
Our results indicate that the rat LYZL proteins are encoded by separate genes and are not 
isoforms. Phylogenetic analysis showed that they are highly conserved and are distributed in 
various organisms. In silico analysis of the primary, secondary and tertiary structures of 
LYZL proteins revealed that they are homologous to chicken lysozyme, suggesting that Lyzl 
genes would have arose from a common ancestor. Among the LYZL proteins characterized, 
only LYZL1 and LYZL6 have conserved the lysozyme active site residues. Lyzl mRNA 
transcripts seem to be expressed predominantly in male reproductive tract. Their expression 
was not developmentally regulated and androgen independent. Immunostaining revealed the 
presence of these proteins in male reproductive tract tissues and on spermatozoa. LYZL1 and 
6 display peptidoglycan binding property and they possess muramidase, isopeptidase and 
antibacterial activities, whereas the remaining proteins did not. The role of LYZL proteins in 
sperm function was evaluated by incubating spermatozoa with LYZL6 antibodies. In vitro 
LYZL6 neutralization seems to inhibit calcium influx and acrosome reaction suggesting that 
Abstract 
 
 
they have important role in sperm function. In vivo studies using rats immunized with LYZL6 
displayed spermatozoa with reduced hypermotility and decreased fertility. Taken together, 
these results suggests that LYZL6 may have a role in sperm function. 


English

SYNTHESIS AND CHARACTERIZATION OF NOVEL SOLVENT - RESISTANT POLYMERIC BARRIERS AND THEIR APPLICATION IN MEMBRANE CONTACTOR SYSTEMS

GHULE, NAMDEV VITTHALRAO

XIV 
 
Pharmacokinetic Implications of Intestinal P-glycoprotein 
in Diabetic Complications 
Abstract 
This study was designed with an objective to understand the effect of intestinal P-
glycoprotein on pharmacokinetic profile of its substrates (verapamil and atorvastatin) in 
diabetes with hyperlipidemia. Mechanistically, the change in intestinal P-glycoprotein 
expression and activity with progression of diabetes and its effect on intestinal uptake as 
well as pharmacokinetics of its substrates was evaluated. In this study, the mechanism of 
action of epigallocatechin-3-gallate (EGCG) which is reported as a potential P-glycoprotein 
inhibitor was also elucidated. Initially, the animal model for diabetes with hyperlipidemia 
was developed. This was followed by intestinal mucosal protein content analysis which 
inferred that total protein increased from duodenum towards the ileum and with the 
progression of diabetes. Thereafter, single pass intestinal perfusion study was performed in 
normal and diabetic rats. It was observed that effective permeability of P-glycoprotein 
substrates viz. verapamil and atorvastatin decreased from duodenum to ileum and also with 
progression of disease. Co-perfusion of EGCG with verapamil or atorvastatin significantly 
improved their intestinal uptake. Thereafter, pharmacokinetic studies were done in normal 
and diabetic rats to determine the presence of any change in the oral bioavailability of P-
glycoprotein substrates with the progression of disease. The oral bioavailability of P-
glycoprotein substrates decreased significantly with the progression of diabetes; however, 
co-administration of EGCG with P-glycoprotein substrates significantly improved their 
bioavailability in normal as well as in diabetic animals. In order to have a mechanistic 
insight, expression and activity of P-glycoprotein in intestinal segment of normal and 
diabetic animals were studied. The expression level of mdr1a and mdr1b genes that encoded 
for P-glycoprotein were determined by RT-PCR analysis whereas activity of P-glycoprotein 
was determined by quantifying the Na+-K+-ATPase enzyme activity in intestinal mucosal 
samples of normal and diabetic animals. It was observed that both expression and activity of 
P-glycoprotein was significantly higher in diabetic animals as compared to normal animals 
XV 
 
and also increased with the progression of disease. It was also observed that intestinal 
oxidative stress and advanced glycation end-products (AGEs) levels, which are responsible 
for modulating expression of P-glycoprotein, was higher in diabetic animals as compared to 
normal. Treatment with EGCG significantly reduced the expression and activity of intestinal 
P-glycoprotein as well as intestinal oxidative stress and AGEs level in both normal and 
diabetic animals. It can be inferred from the obtained results that increased intestinal 
oxidative stress and AGEs level triggered the expression and activity of P-glycoprotein. 
This condition subsequently decreased the intestinal uptake and oral bioavailability of its 
substrates. Treatment with EGCG improved the intestinal uptake and oral bioavailability of 
P-glycoprotein substrates, by reducing the expression and activity of intestinal P-
glycoprotein as well as intestinal oxidative stress and AGEs level. This study gave a 
mechanistic insight about the bioenhancement potential of EGCG. Hence, monitoring the 
enhanced expression and activity of P-glycoprotein in diabetes will be helpful in effective 
therapeutic drug monitoring and EGCG may be opted as an adjuvant therapy for 
overcoming P-glycoprotein mediated drug efflux. 
 


FABRICTION OF SOFT MATERIAL VIA SELF ASSEMBLY OF NOVEL AMPHIPHILE AND DEVELOPMENT OF NEW SENSORS

Karisetty, Bhanu Chandra

Abstract
Differential Equations constrained by algebraic equations and inequalities on the states
occur naturally in the modeling of many engineering systems such as simulation and
control of multibody dynamical systems and electrical circuits, in robotic path planning
and sliding mode control systems, in guidance design for aerospace vehicles and in chem-
ical reactor systems. These systems are often known as path constrained Differential
Algebraic Equation (DAE) systems. In this work we investigate the numerical methods
for (1) simulation of path constrained DAEs with high differential index, (2) computa-
tional solution of optimal control problems with high index constraints and (3) dynamic
optimization problem with disjunctive algebraic constraints.
At first, this thesis develops the generalized-α method and analyzes it in detail. The
generalized-α method is shown to have regularization property which becomes a key
advantage in solving high index path constrained dynamical systems.
We show that the α-method is a good simulator for high index DAEs due to the
regularization property involved in the discretization. This solves effectively the index 3
Euler-Lagrange equations, Kane’s equations formulation and other higher index DAEs
occurring naturally.
We study a direct method known as transcription method, a numerical approach,
equipped with the generalized-α discretization to solve an Optimal Control Problem
(OCP). State-constrained path constraints in an OCP pose numerical challenges because
(1) along the (active) constraints a high index DAE is often formed and (2) the number
and interval of active inequality constraints (even if not state constrained) is not known in
priori. It is shown that the regularization property of the generalized-α method is useful
iv
vin these situations. The proposed method does not perturb the inequality constraints
and hence the physics of the problem is not diluted. The convergence analysis of the
discretized dynamic optimization problem to the continuous OCP is proved for a wide
range of control problems by assuming low continuity conditions on the controls.
Also this thesis solves OCPs having either-or (disjunctive) constraints by considering
an equivalent simple convex hull of the disjunctive constraints. This idea is implemented
to a control problem in vehicle dynamics to stabilize the rollover of a road vehicle un-
dergoing severe maneuver. From a practical point of view we obtain the control forces
as sensory output.
This thesis also focuses on the computation of the radial hedgehog solution, solution
of a Singular Boundary Value Problem (SBVP) arising from the energy minimization of
nematic liquid crystal, by solving an equivalent boundary value DAE. The equivalent
DAE, which is index 3 at the point of singularity and index 1 in the rest of the simulation
interval, is solved by the α-method. It is shown that the point of singularity can be
removed by the DAE approach. Advantages of the DAE approach over classical solvers
for SBVP and transcription method for the radial hedgehog problem are also shown.
Some computational findings of the radial hedgehog solution completes the thesis.
Overall, the thesis shows that the singularity and ill-conditioning inherent in path
constrained DAEs can be dealt with numerically in a consistent and correct way by a
discretization with regularization property.


Sex difference in behavioral and molecular neural response to chronic stress in mouse models of depression and related disorders

G., Sriramya

xviii 
 
ABSTRACT 
Lymphatic filariasis (LF) is an infectious tropical disease caused by nematode parasites 
Wuchereria bancrofti, which is transmitted by Culex quinquefasciatus. There is a need 
to develop effective vector control strategies to break the disease cycle as traditional 
insecticides impose the problem of resistance in mosquitoes and environmental hazard. 
Eupatorium odoratum, has been traditionally known for its insect repellent property. In 
the current study we chose to investigate if chemical compounds derived from this 
plant are effective on mosquito, C. quinquefasciatus. 
Plant extract of E. odoratum has shown mosquito larvicidal activity. The extract was 
subjected to fractionation and the bioactive fractions were further fractionated. The 
chemical nature and the structures of the compounds in the bioactive fraction were 
identified using NMR and Mass spectrometry. Three flavonoid compounds were 
identified from the bioactive fraction 1. All the compounds have shown larvicidal 
activity with acetylcholinesterase inhibitory effect. They were also found to have 
minimal toxic effect on mammalian cell lines.  
Phytosterols and alkanols were identified from the bioactive fraction 2 of the plant 
extract. Stigmasterol and hexacosanol were identified to be the chief orchestrators of 
larvicidal activity and their mode of action has been observed to be neurotoxicity. At a 
molecular level both stigmasterol and hexacosanol were found to be inhibiting 
acetylcholinesterase activity in Culex & Aedes. The Neurotoxic effects of these 
compounds were confirmed in vitro using recombinant acetylcholinesterase. 
Electrophysiological studies using Electroantennography confirmed the activities  
 Neurotoxic effect of stigmasterol and hexacosanol was responsible for the mortality of 
C. quinquefasciatus larvae. The acetylcholinesterase inhibition was also observed in 
xix 
 
Aedes aegypti & Chironomus riparius but in HEK293 cells the inhibition was 
negligible. These compounds were also observed to be toxic to the adult stages of 
mosquitoes.  EAG studies confirmed the enhanced neuro-transmission of these 
compounds was responsible for larvicidal activity. We believe these could be potential 
larvicidal compounds, for the vector control. Field trials are pending in addition to 
molecular analysis to take this further. 
 


Isolation, chemical characterization & biological evaluation of novel mosquito larvicidal compounds from Eupatorium odoratum

MUKHERJEE, SUDIP

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Synopsis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SYNOPSIS	  
xii	  
 
CHAPTER 1: Introduction & review of literature 
 
The topic of the thesis is broadly related to the areas of nanomedicine research that 
includes the design and development of nanoparticles based drug delivery for cancer 
therapeutics and anti-bacterial activity. Over the past 10 years, nanomedicine has 
become an emerging field of nanotechnology applications to health. Recently, several 
groups including ours have extensively investigated for the development of metal 
nanoparticles based theranostics approaches for the treatment of several diseases 
including cancer, antibacterial activity and cardiovascular related diseases etc. Among 
several nanoparticles, gold, silver and platinum nanoparticles have been taken great 
attentions due to several advantages such as easy to synthesize, control their shape and 
size, easy to characterize, biocompatibility and easy to surface functionalization for drug 
delivery. Hence, design, development, fabrication and synthesis of gold, silver and other 
novel and smart nanomaterials/nanocomplexes through a simple, efficient and 
economically cheap method is very important area for nanobiotechnology research. In 
this context, we designed and fabricated metal nanoparticles (gold, silver, platinum, 
inorganic complex) based drug delivery systems using FDA approved anti-cancer drugs 
for cancer therapeutics and graphene oxides for angiogenesis study. 
 The introductory Chapter 1 will address the scope and purpose of the present 
nanomedicine research. A brief overview of (i) the background and history of cancer, 
(ii) conventional treatment strategies and its limitations, (iii) alternative therapy: 
nanotechnology & nanomedicine, (iv) cancer nanotechnology, (v) passive targeting and 
active targeting, (vi) synthesis of inorganic nanoparticles & nanoconjugates (chemical & 
biological approach), (vii) advantages and applications of nanotechnology in drug 
delivery, (viii) FDA approved nanoparticles based medicine and nanomedicine in 
clinical trials, (ix) nanoparticles as antibacterial agent, (x) nanoparticles in angiogenesis 
& anti-angiogenesis, (xi) future challenges with nanomedicine: cost effectiveness, 
toxicity, immunogenicity, biodegradability and clearance from body will be elaborately 
explained with relevant references.  
Considering these facts, the present research work was undertaken with the following 
objectives.  
Ø Fabrication and characterization of novel inorganic 
nanoparticles/nanoconjugates (chemical synthesized and biosynthesized) based 
drug delivery systems (DDS). 
Ø Thorough characterization of these nanoconjugates by several physico-chemical 
techniques. 
 
SYNOPSIS	  
xiii	  
 
Ø Detailed In vitro and in vivo toxicity studies of as prepared biomaterials. 
Functional activity of these nanoparticles/nanoconjugates for several in vitro 
and in vivo assays towards cancer theranostics, antibacterial activities and 
angiogenesis/anti-angiogenesis. 
CHAPTER 2: Multifunctional theranostics applications of bio-synthesized gold 
and silver nanoparticles and their toxicity studies 
The green synthesis approach for the synthesis of metal nanoparticles have several 
advantages over conventional chemical methods namely it is (i) a very simple, fast, 
clean & efficient method, (ii) eco-friendly & economically cheap method; and (iii) 
occurred at ambient temperature and pressure. Chapter-2 will focus on the synthesis and 
fabrication of bio-synthesized gold (AuNPs) and silver nanoparticles (AgNPs) using 
plant extract (e.g. Eclipta alba, Olax scandens, Lantana montevidensis, Peltophorum 
pterocarpum etc.) and their applications towards multifunctional activities (anti-cancer, 
anti-bacterial, fluorescence property etc.)[Mukherjee et al, Nanotechnology, 2012 
(Highlighted in Nature India, Global Medical Discovery Alert)]; [Mukherjee et al, RSC 
Advances, 2013, (Highlighted in Hindu, Early times)]; [Mukherjee et al, Theranostics, 
2014 (Highlighted in Nature India)] [Mukherjee, Sau and Madhuri et al, Journal of 
Biomedical Nanotechnology, 2015]; [Mukherjee et al, J Mater B, 2015]; [Patra and 
Mukherjee et al, Material Science and Engineering C, 2015]. Detailed in vitro and in 
vivo toxicity studies of green synthesized nanoparticles and their detailed mechanistic 
studies for anti-cancer and anti-bacterial activities will be demonstrated. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Scheme-1: Overall Scheme regarding the synthesis of biosynthesized silver nanoparticles and its multi-
functional applications towards cancer theranostics and anti-bacterial activity. 
SYNOPSIS	  
xiv	  
 
 
CHAPTER 3: Design and fabrication of platinum nanoparticles and their detailed 
characterizations, in vitro and in vivo toxicity studies and development of Pt-
nanoparticles based drug delivery systems (DDS) for cancer therapeutics  
Chapter-3 will demonstrate the synthesis of poly ethylene glycol (PEG) stabilized 
highly stable 5-10 nm colloidal platinum nanoparticles (PtNPs) in aqueous medium at 
room temperature. Detailed characterization of these biocompatible Pt-nanoparticles and 
investigation of in vitro and in vivo toxicity of PtNPs will be discussed. We will 
demonstrate the design and development of platinum nanoparticles based stable drug 
delivery systems (Pt-DOX) using FDA approved anti-cancer drug (Dox: doxorubicin). 
Administration of Pt-DOX significantly inhibits the cancer cell proliferation (in vitro) as 
well as melanoma tumor growth (in vivo) compared to untreated group and group 
treated with pristine DOX. Finally, the detailed mechanistic studies behind the enhanced 
anti-cancer activity using Pt-DOX will be discussed in this chapter. 
CHAPTER 4: Synthesis and fabrication of highly biocompatible prussian blue 
analogues nanoparticles and their application for cancer therapeutics and anti-
bacterial activities  
Prussian blue nanoparticles (PBNPs) have gained huge interests as an antidote for heavy 
metal toxicity. It has got FDA approval as it shows high biocompatibility. PBNPs and 
its analogs have been used for various biomedical applications including drug delivery, 
biomodal imaging, bio-sensing device, cancer theranostics due to unique physico-
chemical properties and porous nature. Chapter-4 will demonstrate the synthesis of 
poly(N-vinyl-2-pyrrolidone) (PVP) stabilized silver analogues of Prussian blue 
nanoparticles (SPBNPs), detailed characterization and biological activity studies for 
cancer therapy and anti-bacterial activities. SPBNPs showed high biocompatibility 
through in vitro and in vivo toxicity studies. Furthermore, it exhibited excellent 
antibacterial and anticancer efficacy towards in vitro and in vivo tumor model. Detailed 
mechanistic studies will be discussed for both anti-cancer and anti-bacterial activities.  
 
CHAPTER 5: Graphene oxides and its role in angiogenesis and anti-angiogenesis 
Chapter-5 will focus on the ROS (reactive oxygen species) dependent switchover role 
between angiogenesis and anti-angiogenesis of graphene oxides (GO) and reduced 
graphene oxides (rGO) and their detailed mechanistic studies [Mukherjee et al, Adv. 
Healthcare Mater, 2015 (Highlighted in Nature India, Chemistry Views)]. 
Angiogenesis, a process resulting in the formation of new capillaries from the pre-
existing vasculature that plays vital role for the development of therapeutic approaches 
for cancer, atherosclerosis, wound healing, and cardiovascular related diseases. In this 
SYNOPSIS	  
xv	  
 
Chapter, the synthesis, characterization, and in vitro and in vivo angiogenic properties of 
GO and rGO will be demonstrated. The formation of intracellular ROS, reactive 	  
nitrogen species (RNS) and activation of phospho-eNOS & phospho-Akt might be the 
plausible mechanisms for GO and rGO induced angiogenesis. The results altogether 
suggest the possibilities for the future development of alternative angiogenic therapeutic 
approach for the treatment of cardiovascular related diseases where angiogenesis play a 
significant role. 
Finally, we will conclude the overall thesis work with highlighting the possible 
outcome of the present thesis work. Also we will discuss the future translational 
directions and challenges of this nanomedicine research for cancer theranostics and 
other biomedical applications. 
	  
 
	  


Design, Fabrication and Characterization of Nanoparticles/Nanoconjugates (Gold, Silver, Platinum, Prussian Blue Analogue, Graphene Oxides) and Their Potential Theranostics Applications in Biomedical Sciences

G., Dayakar

                                                                                                      Abstract 
 
xi 
ABSTRACT 
Due to global depletion of fossil fuels and simultaneous increase of environmental pollution 
there is a great demand for sustainable and renewable energy sources. In this context, 
supercapacitors or ultracapacitors have attracted as the promising alternative energy sources due 
to their high specific power density, long cycle life, fast charge/ discharge processes (within 
seconds) and low maintenance cost. Supercapacitors currently bridge the power gap between 
batteries and traditional solid state and electrolytic capacitors, delivering higher power bursts 
than batteries and storing more energy than capacitors.
 
Owing to these unique properties 
supercapacitors are widely used in various energy storage fields such as energy back-up systems, 
consumer portable devices, electrical/ hybrid electric vehicles and other devices.  
 
Based on the mechanism supercapacitors are classified into two categories: electrochemical 
double-layer capacitors (EDLCs) and pseudocapacitors. Generally, EDLCs based on carbon 
materials provide high cycling stability, but, they show lower specific capacitance and energy 
densities because charge is stored only on the surface area of an active EDLC electrode. 
Whereas, pseudocapacitors show high specific capacitance and energy densities due to charge 
store occurs by rapid redox reactions between the surface and the bulk of the electrodes.
 
Hence, 
pseudocapacitors have been considered as the most attractive materials for energy storage 
applications. Among the reported pseudocapacitor materials, conducting polymer have drawn 
much attention due to their high conductivity, low cost, outstanding electrical and optical 
properties, flexibility. A.J. Heeger, A.G. MacDiarmid and H. Shirakawa were jointly rewarded 
with the Nobel Prize in Chemistry in 2000 for the discovery and development of conducting 
polymers. Among all conducting polymers, polyaniline (PANI) is one of the most significant 
conducting polymers, because of its relatively facile processability, electrical conductivity, 
environmental stability, high specific capacitance, redox activity and unusual doping-dedoping 
chemistry.
 
However, the poor electrochemical stability of PANI due to the shrinkage of 
polymeric chains during redox cycling limits its applications in supercapacitors. Currently, many 
researchers are focused on supercapacitors to improve the energy density while maintaining high 
power density, fast charge/discharge and cycling stability. To achieve these objectives, there is a 
great need to synthesize various nanocomposites of PANI with carbon nanotubes, mesoporous 
carbon,
 
graphene, transition metal oxides, and mixed metal oxides like spinel oxides. The 
                                                                                                      Abstract 
 
xii 
resultant nanocomposites show high cycling stability and specific capacitance due to synergistic 
effect of individual components.  
 
Furthermore, it is reported that with increasing the pH typically at pH ≥4, the emeraldine salt 
form (ES) of PANI gets deprotonated and converts to the electrically non-conducting emeraldine 
base form (EB). This, ES-EB transition greatly limits the use of PANI in biomedical applications 
operating at the physiological pH=7.4. Therefore, the dopants which are biocompatible and can 
maintain electroactivity of PANI at higher pH values have gained much attention.  
 
In this thesis, we are reporting PANI nanocomposites with graphene, spinel copper chromite 
(CuCr2O4) for energy storage applications and carbon sphere as dopants for PANI to retain its 
electroactivity at neutral pH values. With this background the work undertaken in this thesis 
entitled “Synthesis of Electroactive Nanocomposites for High-Performance Energy Storage 
and Beyond” is presented in the form of following four chapters. 
 
Chapter 1: Introduction to Energy Storage devices, Graphene, Spinel Oxides and 
Polyaniline for Supercapacitors and Electroactivity of Polyaniline at Neutral pH 
This chapter is an introductory part that of the thesis which gives an overview on energy storage 
devices, importance and types of supercapacitors. Various materials used for supercapacitors 
such as graphene, spinel oxides, conducting polymers especially polyaniline and various 
polyaniline based nanocomposite materials for supercapacitors. This chapter also explains 
importance of electroactivity of polyaniline at neutral pH. Brief introduction to carbon spheres.  
 
Chapter 2: Synthesis of Polyaniline-β-Cyclodextrin-Graphene Nanosheet Triad and 
Radical Cation Stabilization of Polyaniline: Performance Evaluation for Energy Storage 
Application 
This chapter deals with the synthesis of supramolecular polyaniline-β-cyclodextrin-graphene 
nanosheet (PANI-β-CD-GNS) triad using graphene oxide (GO) and presynthesized PANI-β-CD. 
The microstructure and morphology of the triad are characterized by various techniques. The 
enhanced radical cation stabilization of the triad after conjugating with graphene is confirmed by 
electron paramagnetic resonance (EPR) spectroscopy. Then, the synthesized triad is tested for its 
energy storage application, it showed excellent specific capacitance of 567 F g
-1
 at 2 mV s
-1
, 
                                                                                                      Abstract 
 
xiii 
good long term cycling stability with 88.5% capacitance retention after 1000 consecutive charge-
discharge cycles at  5 A g
-1
. This triad also exhibited high specific energy density of 31.8 Wh kg
-
1
, and
 
high power density of 5061 W kg
-1
.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chapter 3: Copper Chromite-Polyaniline Nanocomposite for Energy Storage Application 
In this chapter, we report a novel copper chromite-polyaniline (CuCr2O4-PANI) 
nanocomposite electrode material for fabrication of high-performance supercapacitor. 
First CuCr2O4 is synthesized via sol-gel driven epoxide method followed by its 
nanocomposite with PANI through in situ chemical oxidative polymerization method. 
The synthesized nanocomposite is characterized by FTIR, EDAX, BET surface area 
measurement, XRD, SEM and XPS. Then, CuCr2O4-PANI nanocomposite is investigated 
for its energy storage application using cyclic voltammetry (CV), electrochemical 
impedance spectroscopy (EIS) and galvanostatic charge-discharge (GCD) tests. CuCr2O4-
PANI nanocomposite exhibited the best capacitance compared to individual components 
                                                                                                      Abstract 
 
xiv 
and it showed the highest specific capacitance of 535 F g
-1 
at 2 mV s
-1
. Further, this 
nanocomposite showed excellent long cycle life with 90.3% retention of its specific 
capacitance at 5 A g
-1
 and showed the highest specific energy density of 13 Wh kg
-1
 at 0.1 
A g
-1
 and high power density of 2926.8 W kg
-1
 at 10 A g
-1
. 
 
Chapter 4: Carbon Sphere-Polyaniline Nanocomposite: Retention of Electroactivity at 
Neutral pH  
In this chapter, we have demonstrated a novel strategy to retain electroactivity of polyaniline 
(PANI) using inherently fluorescent carbon spheres (IF-CSP) as a doping agent by making 
nanocomposite (IF-CSP@PANI) through in situ chemical oxidative polymerization of aniline in 
presence of IF-CSP. The synthesized composite was characterized by FTIR, UV-Vis, FE-SEM, 
EDX, XPS, XRD, TGA and zeta potential studies. IF-CSP@PANI showed excellent 
electrochemical activity than PANI in neutral media at pH 7.4 with high cycling stability which 
was confirmed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). 
Additionally, we have investigated biocompatibility of the nanocomposite using MTT assay and 
due to intrinsically fluorescent property of carbon spheres in the nanocomposite, cellular uptake 
study also carried out using confocal laser scanning microscopy (CLSM).  
 
Future scope of the work is discussed briefly at the end of the thesis. 
 
 


Synthesis of Electroactive Nanocomposites for High-Performance Energy Storage and Beyond

KOMMULA, DILEEP

DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF TETRAZOLE CONTAINING PIPERAZINE, QUINOLONE AND QUINAZOLINONE DERIVATIVES & DEVELOPMENT OF NEW SYNTHETIC METHODOLOGIES

PANDA, SUJATARANI ANANTACHARAN

DEVELOPMENT OF ENYNE-ASSISTED ANNULATIONS TOWARDS THE SYNTHESIS OF PYRIDINES, PYRROLES AND MYRMICARIN

Bhunia, Sukanya

Synopsis 
i 
 
Chapter I: Introduction: Cancer is a deadly disease with second highest mortality rate. Existing 
treatment strategies kill not only tumor cells but also non-cancerous healthy body cells leading to severe 
adverse side effects of cancer treatment. Among the modern therapeutic modalities, targeted 
chemotherapy and cancer immunotherapy (the therapeutic modality that exploit body’s own immune 
systems) approaches are emerging out to be the most promising for combating the dreaded disease of 
cancer. 
Targeted chemotherapy is the most widely used current treatment modality for cancer which involves 
selective delivery of anti-cancer chemotherapeutics (potent anti-cancer drug or siRNA) to tumor tissue. 
Distinguishing biomarkers over-expressed on tumor cell-surface are utilized for selective delivery of 
anticancer chemotherapeutics to tumor tissue with minimal harm to other healthy tissue.  
Cancer Immunotherapy exploits body’s own immune cells (e.g., dendritic cells, natural killer cells, T-
cells, etc.) to fight against cancer. There are many types of cancer immunotherapy including monoclonal 
antibodies, oncolytic virus therapy, cancer vaccines, etc. Dendritic cell (DC) based genetic immunization 
(DNA vaccination) has shown lot of promises because of their unique potential to simultaneously induce 
both humoral and cellular immune responses against tumors. In DC-based DNA vaccination, dendritic 
cells (body’s most promising antigen presenting cells) are transfected with plasmid DNA encoding 
distinguishing tumor associated antigen. DCs process the expressed tumor antigen and present the 
processed antigenic fragments on their cell surfaces with the help of major histocompatibility complexes 
MHC-I and MHC-II. MHC bound antigenic fragments on DC-surface are recognized by T cells and the 
activated T-cells thereafter get triggered to start rapid proliferation. The activated T-cells recognize the 
tumor cells expressing the particular antigen and induce intense humoral & cellular immune responses 
against the tumor cells. However, either only targeted chemotherapy or only DC-based Immunotherapy 
hardly results in long-lasting overall survivability of tumor bearing mice. 
Liposomes, the spherical molecular assembly of amphiphiles (often having size within the range 100-200 
nm) with an aqueous interior enclosed by lipid bi-layer, have long been used for targeted delivery of 
bioactives selectively to destined tissue under in vivo settings. In targeted chemotherapy, targeting of 
liposomally entrapped chemotherapeutics to tumor cells are achieved using liposomes made from 
amphiphilic molecule covalently connected with a tumor targeting ligands in their exo-surface regions. 
The liposomes covalently grafted with such a tumor-targeting ligand can target specific receptors over 
expressed in cancerous cells. Such tumor targeting liposomes can encapsulate both hydrophilic 
drug/siRNA (encapsulate inside aqueous compartment) & hydrophobic drug (encapsulate in hydrophobic 
lipid bi-layer) and deliver their therapeutic payloads/cargo (cytotoxic genes/siRNAs, small molecule anti-
cancer drugs, etc.) to the cancerous tissues without affecting normal tissue (Figure 1). However efficient 
and selective targeting of potent cancer therapeutics to tumor cells/tissues remains a difficult challenge. 
            
 
 
 
Figure 1: Cartoon for Tumor Targeting Liposomes. 
In the present thesis, I report on the development and bio-activity evaluation of two new liposomal 
drug delivery systems for delivering potent cancer therapeutics to neuroblastoma and glioblastoma in 
mouse tumor models. 
Synopsis 
ii 
 
Chapter II: Development of CDC20siRNA and Paclitaxel co-loaded new liposomal nanocarrier 
for combating neuroblastoma 
Despite significant recent progresses in neuroblastoma treatment, accomplishing markedly enhanced 
overall survivability remains an arduous task mostly due to tumor relapse and/or drug-resistant 
tumors. CDC20, a key cell cycle regulator protein required to complete mitosis, is over expressed in 
many cancer cells and therapeutic strategies based on selective delivery of CDC20siRNA to tumor & 
tumor vasculatures hold promise for combating cancer. Prior reports demonstrate that human 
neuroblastoma cells IMR-32 express functional GABAa receptor on their surface and nipecotic acid 
is a GABA related compound widely used as an inhibitor of both glial and neuronal GABA uptake. 
Chapter II of my doctoral thesis reports on the development of a novel GABAa receptor targeting 
circulation stable liposomal system prepared from nipecotic acid-derived cationic amphiphile 
(NACA) for combating neuroblastoma (Figure 2). I have demonstrated that liposomes of NACA can 
efficiently encapsulate siRNAs inside its inner hydrophilic core and protects them from attack by 
RNase. Findings in the cellular uptake study demonstrated that liposome of NACA delivers siRNA to 
human neuroblastoma IMR-32 cells via GABAa receptor. CDC20siRNA-loaded liposome of NACA 
silenced CDC20 gene  expression in IMR-32 cell.  Prior studies reported that silencing of CDC20 
gene sensitizes cancer cells to paclitaxel (PTX). I have exploited this prior findings in my doctoral 
thesis work.  Importantly, I have demonstrated that administration of NACA-liposomes containing 
co-encapsulated CDC20siRNA & PTX induces significant tumor growth inhibition in athymic nude 
mice bearing xenografted human neuroblastoma (Figure 2, taken from S. Bhunia, R.Vegesna and A. 
Chaudhuri, manuscript accepeted in Nanoscale, 2016, DOI: 10.1039/C6NR07532K, Accepted 
manuscript on line published on December 6, 2016).  
. 
 
 
 
 
 
 
 
Figure 2: Intravenous administration of CDC20 siRNA and paclitaxel (PTX) co-encapsulated within 
liposomes of NACA significantly inhibits growth of human neuroblastoma xenografted in athymic nude mice. 
 
Chapter III: Combating orthotopically established mouse glioblastoma using targeted 
Chemotherapy in combination with in vivo dendritic cell targeted cancer immunotherapy  
 
Despite remarkable advances in glioblastoma treatments during the past decade, accomplishing 
significantly enhanced overall survivability (OS) remains a formidable challenge. STAT3, a key 
signal transduction protein, is activated in numerous cancer cells including glioblastoma. Inhibition 
of STAT3 leads to apoptosis in tumor cells and enhanced DC-maturation (through up regulated 
expressions of MHC class II, co-stimulatory molecules CD80, CD86, etc.). Major obstacle for 
systemic chemotherapy of glioblastoma is the presence of Blood Brain barrier (BBB) which limits 
entry of chemotherapeutics to brain tumor cells. Recent approaches for delivering potent 
Synopsis 
iii 
 
chemotherapeutics selectively to brain tumors are exploiting some receptor and transporter molecule 
over expressed on the brain capillary endothelial cells (BCECs) of BBB. Large Amino Acid 
Transporter type -1 (LAT-1) is abundantly and selectively expressed not only on BCECs of BBB but 
also in many types of cancers including glioblastoma multiforme. Chapter III of the present thesis 
delineates development of a novel BBB-crossing liposomal drug carrier for targeting potent 
chemotherapeutics selectively to orthotopically established mouse glioblastoma.  The liposomes were 
prepared using a novel amphiphilic molecule (Amphi-DOPA) containing the well known BBB-
crossing L-3,4-dihydroxyphenyl alanine (L-DOPA, a potent drug for treatment of Parkinson’s 
disease that crosses BBB via LAT-1) moiety covalently grafted in its polar head-group region and 
two n-hexadecyl hydrophobic tails in its non-polar region. I have demonstrated that liposomes of 
Amphi-DOPA containing encapsulated WP1066 (a commercially available small molecule STAT3 
inhibitor) significantly inhibit growth of orthotopic glioblastoma in BL6J/C57 mice by down 
regulating p-STAT3 under in vivo conditions. Furthermore, I have shown that targeted chemotherapy 
using WP1066-loaded liposomes of Amphi-DOPA, when used in combination with in vivo dendritic 
cell targeted genetic immunization (with a recently reported in vivo DC-targeting liposomes from our 
group in complexation with survivin encoded DNA vaccine), elicits significantly enhanced overall 
survivability of orthotopic glioblastoma bearing mice 
 
 
 
 
 
 
 
 
 
Figure 3: Targeted chemotherapy in combination with in vivo dendritic cell targeted DNA 
vaccination leads to significant enhancement of survivability in orthotopic glioblastoma 
bearing mice.  
Conclusion: The present thesis describes design, synthesis and bio-activity evaluation of two novel 
amphiphilic molecules. The liposomes of the first amphiphile, upon intravenous administration, 
showed efficient targeting of their drug payloads selectively to neuroblastoma and causes significant 
tumor growth inhibition. Intravenously administered liposomes of the second amphiphile resulted 
into significant inhibition of orthotopically established mouse glioblastoma. The findings described 
in Chapter III of the present thesis demonstrate that targeted chemotherapy in combination with in 
vivo dendritic cell targeted DNA vaccination leads to significant survivability enhancement in 
orthotopic glioblastoma bearing mice.  


Combating neuroblastoma and glioblastoma with tumor targeting new liposomal drug carriers

Madasu, CHANDRA SHEKHAR

ABSTRACT 
Biodegradation of phthalate esters (PAEs), namely dimethyl phthalate (DMP), diethyl 
phthalate (DEP), dibutyl phthalate (DBP) and their mixture by a bacterial strain (BS1) is 
reported. This bacterium was isolated from soil that was contaminated with plastic wastes and 
shown in laboratory assays to have the potential to utilize PAEs as the sole source of carbon 
and energy. The isolate was identified as Variovorax sp. based on 16S r-DNA sequence 
analysis. The extent of biodegradation of DMP, DEP, DBP and their mixture (300 mg L-1) by 
Variovorax sp. BS1 were: >97%, >99%, >92% and >99%, respectively after 24 h of 
incubation at 30°C. The Variovorax sp. BS1 was able to biodegrade 200 to 600 mg L-1 of 
individual PAEs and their mixture within 30 h of incubation. The specific growth rate of 
Variovorax sp. BS1 increased with increasing concentrations of PAEs up to 200 mg L-1 and 
further increase in concentrations did not change the specific growth rate. The transient 
intermediates and persistent end product formed due to biodegradation of DMP, DEP, and 
DBP by Variovorax sp. BS1 were not detectable by either HPLC or LC-MS analyses in liquid 
culture medium. However, using GC-MS, trace amounts of phthalic acid and its derivatives 
were observed. Total organic carbon (TOC) analysis suggested complete mineralization of 
DMP, DEP, DBP and their mixture by Variovorax sp. BS1. 
In order identify the enzymes involved in the biodegradation of PAEs, cell free crude extracts 
were prepared and assayed for the presence of enzymes using PAEs and para-nitophenyl 
acetate (pNPA) as the substrates. The extent of biodegradation of 1 mM of DMP using cell 
free extracts derived from Variovorax sp. BS1 grown on DMP was observed to be >80% at 
the end of 60 min of incubation at 30°C. Results suggested that the cell-free extracts 
contained the mixture of enzymes responsible for the biodegradation of DMP. Further it was 
observed that the cell free enzymes were more efficient in biodegrading various 
concentrations of DMP (25 to 1000 μM). The enzyme(s) responsible for the biodegradation of 
DMP exhibited highest activity in freshly prepared cell-free crude extracts while the activity 
declined rapidly upon storage. Zinc ions (Zn+2) inhibited the phthalate dioxygenase activity in 
the cell free extracts which resulted in the generation and accumulation of phthalic acid. Cell 
free crude extracts were also prepared from Variovorax sp. BS1 grown on a mixture of DMP, 
DEP and DBP and used for biodegradation of individual PAEs as well as mixture. The extent 
vof removal of 1 mM of DMP or DEP or DBP and mixture of PAEs (0.33 mM of each) were: 
59%, 67%, 65% and 53% after 60 min of incubation using cell free enzyme preparations.  
Variovorax sp. BS1 cells were immobilized in calcium alginate beads and used for the 
biodegradation of PAEs in batch experiments. Degradation efficiency of immobilized cells in 
mineral salts medium (MSM) and growth non supportive medium, de-ionized water was 
compared. Results showed higher degradation rate in MSM as compared to de-ionized water. 
Higher rate of degradation in MSM was due to the growth of cells in/on beads as well as in 
spent medium. Therefore, MSM was replaced with de-ionized water and used for repeat batch 
experiments. The results suggested that the immobilized cells of Variovorax sp. BS1 may be 
re-used upto ten cycles with diminutive loss of DMP degradation activity. The results 
presented suggest the bacterial isolate, Variovorax sp. BS1 has the potential for the 
bioremediation of water contaminated with PAEs. 
Keywords: Biodegradation, Dimethyl phthalate, Diethyl phthalate, Dibutyl phthalate, 
Phthalate esters, Variovorax, Enzyme, Immobilization 


Not a member yet