Final Thesis Henna

M.
Phar
m.
Thesi CO-DELIVERY OF RIVASTIGMINE AND GRAPHENE
s
CO-DELIVERY
QUANTUM DOTS FOR ENHANCED BLOOD-BRAIN BARRIER
OF PERMEABILITY AND TREATMENT OF ALZHEIMER’S
RIVASTIGMINE
AND GRAPHENE
DISEASE
QUANTUM DOTS
FOR ENHANCED
BLOOD-BRAIN
BARRIER
PERMEABILITY
AND
TREATMENT
OF
ALZHEIMER’S
DISEASE-
HENNA.T.TK BY
HENNA T. K
(Reg. No. 172760078)
Thesis submitted to the Kerala University of Health Sciences in

partial fulfillment of the requirements for the award of the Degree of
MASTER OF PHARMACY
IN
PHARMACEUTICS
Under the guidance of
Dr. PRAMOD K
Assistant Professor
Dept. of Pharmaceutics
College of Pharmaceutical Sciences
Govt. Medical College
Kozhikode – 673008
FACULTY OF PHARMACY
KERALA UNIVERSITY OF HEALTH SCIENCES
THRISSUR – 680596
JULY 2019
Declaration
I hereby declare that this dissertation entitled “Co-delivery of rivastigmine

and graphene quantum dots for enhanced blood-brain barrier
permeability and treatment of Alzheimer’s disease” herewith submitted in
partial fulfillment of the requirements for the award of the Degree of Master of
Pharmacy in Pharmaceutics in the Faculty of Pharmacy, Kerala University of
Health Sciences, Thrissur - 680596, is a bonafide research work carried out by
me under the guidance of Dr. Pramod K, Assistant Professor, College of
Pharmaceutical Sciences, Govt. Medical College, Kozhikode.
Henna. T.K
Kozhikode
24h July, 2019
KUH
S
2019
COLLEGE OF PHARMACEUTICAL
SCIENCES
Govt. Medical
College,
Calicut, Kerala
India 673008
copsclt@gmail.com
------------------------------------------------------------------------------------------------------------
Certificate
This is to certify that the dissertation entitled “Co-delivery of rivastigmine and

graphene quantum dots for enhanced blood-brain barrier permeability and
treatment of Alzheimer’s disease” herewith submitted by Mrs. Henna.TK, in
partial fulfillment of the requirements for the award of the Degree of Master
of Pharmacy in Pharmaceutics in the Faculty of Pharmacy, Kerala University of
Health Sciences, Thrissur - 680596, is a bonafide research work done by her
under my guidance and supervision. It is also certified that the candidate has
satisfactorily presented two seminars on relevant topic during the period of
thesis work. The thesis is submitted for evaluation.
Dr. PRAMOD K
Assistant professor
Kozhikode College of Pharmaceutical Sciences
24th July, 2019 Govt. Medical college
Kozhikode-673008
CHAPTER 1
INTRODUCTION |4
COLLEGE OF PHARMACEUTICAL
SCIENCES
Govt. Medical
College,
Calicut, Kerala
India 673008
copsclt@gmail.com
------------------------------------------------------------------------------------------------------------
Certificate
This is to certify that the dissertation entitled “Co-delivery of rivastigmine and

graphene quantum dots for enhanced blood-brain barrier permeability and
treatment of Alzheimer’s disease” herewith submitted by Mrs. Henna. T.K, in
partial fulfillment of the requirements for the award of the Degree of Master
of Pharmacy in Pharmaceutics in the Faculty of Pharmacy, Kerala University of
Health Sciences, Thrissur - 680596, is a bonafide research work done by her
under the guidance of Dr. Pramod K, Assistant Professor, College of
Pharmaceutical Sciences, Govt. Medical College, Kozhikode.
Dr. VINAYA OG
Professor and Head of
Kozhikode College of Pharmaceutical Sciences
24th July, 2019 Govt. Medical college
Kozhikode-673008
COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

CHAPTER 1
INTRODUCTION |5

ACKNOWLEDGEMENTS
The endless thanks goes to Lord Almighty for all the blessings he has
showered onto me, which enabled me to complete my thesis work. I am
blessed by almighty with some extraordinary people who have spun a
web of support around me. Words can never be enough in expressing
how grateful I am to those incredible people in my life who made this
thesis possible.
I am deeply indebted to my guide Dr. Pramod K, Assistant professor,

College of Pharmaceutical Sciences, Govt. Medical College, Kozhikode,
who was with me till the completion of the work, providing me with
invaluable suggestions and necessary information which encourage me a
lot in my academic life. His knowledge and experience provided me an
opportunity to study many new things that I have never learned. Apart
from educational knowledge he also helped me to build personality and
raise my confidence. From him I learned to think critically, to select
problems, to solve them and to present their solutions. He has given me
the best which I never expected in my life. He helped me in furthering my
knowledge in many areas. This work would not have been possible
without his guidance, ideas and support. I am glad to express my feeling
that I am very lucky to have such a guide for my work. I don’t have words
to express my gratitude to him for his support and positive energy he
passed to me while facing problems in different situations. It was a great
pleasure for me to have the opportunity to work under his guidance.
Words can never be enough to express my gratitude towards him.
I respect and thank Smt. Vinaya O.G Professor and Head, College of
Pharmaceutical Sciences, Govt. Medical College, Kozhikode for
providing all approvals for my work and giving us all support which
made me complete the project duly.
CHAPTER 1
INTRODUCTION |8
I heartily thank Dr. H.V Gangadharappa, and Dr. K.L Krishna Assistant
Professors, JSS college of pharmacy for spending their valuable time for
us. I also owe thanks to Miss. Nandhini, and Mr. Maruthi Reddy, Mr.
Chandan, Ph.D. Research Scholars, and Safvana Fava, post graduate
student, JSS college of pharmacy, for their extensive help during in
vivo.studies.
I remember the help from Dr. Sandhya Rani, head of department, School
of Nano Science and Technology, National Institute of Technology (NIT),
Calicut. And Dr.Ramani Thekkathu, and Mr Sajeesh. P, who helped me
to work in NIT and their valuable instructions were more helpful to
accomplish my work.
I also like to convey my thanks to Mr. Shinto, and Mr. Jithin, ph.D
Research scholars, National Institute of Technology, Calicut.
I express my sincere thanks to Dr. Jose John Mallikasseri, Head of the

chemistry department, St.Joseph’s College, Devagiri, Calicut, Dr.Tania
Francis and Dr Aneesh, department of chemistry, St.Joseph college,
Devagiri, and Mrs. Meril , Miss Annie Stephie , Anju and Miss
Theertha, Ph.D reaserch scholars, St. Joseph’s college, who helped me
to do Thermogravimetric analysis, IR spectroscopy and freez drying.
I am grateful and fortunate enough to get constant encouragement,

valuable advice, support and guidance from my respected teachers Smt.
Amina Ali, Sri. Raveendran K.C, Sri. Manoj K, Smt. Anju P, and Smt.
Jisha Mohanan, Assistant Professors, College of Pharmaceutical
Sciences, Govt. Medical College, Kozhikode, during my M.Pharm course.
I profusely thank Mr. Shaji and Ms. Thara, Lab assistants, College of
Pharmaceutical Sciences, Govt. Medical College, Kozhikode, for their
immense help and support.

CHAPTER 1
INTRODUCTION |9
I also thank my PG mates Riya Raphey. V and Nivitha.K.P, for their

help and co-operation during my work.
I also remember my friends Junaid Abdul Majeed. MP, Lubna. T.P,

Anusree. V, Aswathy. K, Riya.T.Raju, Shafeena and all my juniors their
help and support created a suitable surrounding for my work.
The warm thanks to my husband Abdussalam. M who supported me in

each and every way, and inspired me in my all dimensions of life
I can’t conclude my words without thanking my parents and other family

members who gave me moral and emotional support in my life.
I am also grateful to others, whose name I didn’t mentioned here who

have supported and helped me along the way.
Finally, I understand the word ‘thank you’ is not enough to express my

feelings to all those with me to complete these work with their support,
help, advice, co-operation and trust on me.
Thanks for all your encouragement.
Henna. T.K
Date: 24-7-2019

Graphical abstract
ABSTRACT
Alzheimer’s is an age related neurodegenerative disorder characterised by dementia,

due to the deposition of amyloid β plaques in the brain, leads to neuronal death and
decreased level of acetylcholine. Several acetylcholine esterase inhibitors are
formulated and delivered into the brain as a treatment of Alzheimer’s disease. But,
many of these delivery systems fail to cross blood-brain barrier (BBB). In this
scenario, we introduce a biocompatible, non-toxic nanocarrier, graphene quantum
dots (GQDs), which can easily cross the blood-brain barrier. Moreover, it is reported
that, GQDs itself has a capacity to inhibit amyloid β peptide aggregation by degrading
peptides into small fragments. Herein, we performed co-delivery of rivastigmine
tartarate (RT) and GQD (GQD-RT), which may cause adsorption of RT on GQD
surface and it may deliver RT across BBB. Thus, a concomitant suppression of
amyloid β aggregation and acetylcholine esterase may be expected. GQD were
synthesised from L-glutamic acid (LGA) and it was mixed with RT in 3;1 ratio. And
the BBB permeability of GQD-RT was evaluated by in-vivo studies on Wistar rats,
and the concentration of GQD and RT in brain and blood were calculated using high
pressure liquid chromatography (HPLC).
The data show that, GQD can penetrate BBB, but the combination of GQD-RT
couldn’t deliver RT into brain. Thus, more research studies are required to develop
such a system for the delivery of RT across BBB using GQD. Detailed chemical
conjugation reactions are needed for the successful conjugation of RT on GQD, and
which can offer a synergistic benefit in treatment of Alzheimer’s disease.
Key words: Graphene quantum dot; Rivastigmine tartarate, Alzheimer’s disease;

Amyloid beta; Blood-brain barrier; High pressure liquid chromatography
DEDICATED TO
MY DEAR PARENTS, HUSBAND,
TEACHERS
AND FRIENDS
CONTENT
Page
No.
ABBREVIATIONS
1 INTRODUCTION 1
1.1 Alzheimer’s disease
1.2 Blood-Brain Barrier 1
1.2.1 Mechanism of BBB crossing 3
1.2.2 Factors influencing BBB crossing 5
1.2.3 Approaches for BBB crossing 5
1.3 Carbon nanostructures 6
1.3.1 Graphene nanofamily 7
1.3.2 Graphene Quantum Dots 7
1.3.3 Biomedical Applications of Graphene Quantum Dots 9
1.4 Rivastigmine tartarate 13
2 REVIEW OF LITERATURE 15
2.1 Graphene quantum dot 15
2.1.1 Synthesis of graphene quantum dots 15
2.1.2 Graphene quantum dots as nanocarrier 18
2.2 Carbon nanostructures in brain disorders 25
2.2.1 Graphene quantum dots in neurodegenerative disorders 28
2.3 Drug adsorption on graphene quantum dot and graphene
30
derivatives
2.4 Biocompatibility study of GQD and graphene derivatives 30
2.4.1 In-vitro biocompatibility studies of GQD 30
2.4.2 In-vitro biocompatibility studies of graphene derivatives 31
2.4.3 In-vivo biocompatibility studies of GQD 33
2.4.4 In-vivo biocompatibility studies of graphene derivatives 35
3 AIM AND PLAN OF WORK 36

3.1 Aim of study 36
3.2 Objectives of the study 36
3.3 Rationale of the study 36
3.3.1 Rationale for choosing Alzheimer’s disease 36
3.3.2 Rationale for graphene quantum dots 36
3.3.3 Rationale for rivastigmine tartarate 37
3.4 Plan of work 37
4 MATERIALS AND METHODS 38

4.1 Materials 38
4.2 Analysis of rivastigmine tartarate 40
4.2.1 Appearance 40
4.2.2 IR spectroscopy 40
4.2.3 Determination of λmax of rivastigminr tartarate 40
4.2.4 Preparation of calibration curve of rivastigmine tartarate 40
CONTENT
Page
No.
4.3 Synthesis of graphene quantum dot 41
4.4 Characterization graphene quantum dot 41
4.4.1 Product yield 41
4.4.2 Fluorescence 41
4.4.3 SEM 41
4.4.4 HR-TEM 42
4.4.5 Particle size distribution and polydispersity index 42
4.4.6 Zeta potential 42
4.4.7 UV-Visible spectroscopy 42
4.4.8 Fluorescence spectroscopy 42
4.4.10 X-ray diffraction 42
4.5 Preparation of GQD-RT 43
4.5.1 Preparation of 54µg/ml rivastigmine tartarte 43
4.5.2 Preparation of 162µg/ml GQD 43
4.6 Characterization of GQD-RT 43
4.6.2 Thermogravimetric analysis 43
4.6.3 Diffusion study 43
4.7 In-vivo evaluation of BBB permeability 45
4.7.1 High pressure liquid chromatography (HPLC) 45
4.7.2 Development of standard chromatogram of RT by HPLC 46
4.7.3 Development of standard chromatogram of GQD by HPLC 46
4.7.4 Analysis of blood samples by HPLC 47
4.7.5 Analysis of brain samples by HPLC 47
5 RESULTS AND DISCUSSION 48

5.1 Analysis of rivastigmine tartarate 48
5.1.1 Appearance 48
5.1.3 Determination of λmax of rivastigminr tartarate 49
5.1.4 Preparation of calibration curve of rivastigmine tartarate 49
5.2 Synthesis of GQD 50
5.3 Characterization of GQD 51
5.3.1 Product yield 51
5.3.2 Fluorescence 51
5.3.3 SEM 52
5.3.4 TEM 53
5.3.5 Particle size distribution and polydispersity index 53
5.3.6 Zeta potential 54
5.3.7 UV-Visible spectroscopy 55
5.3.8 Fluorescence spectroscopy 55
5.3.10 X-ray diffraction 57
5.4 Characterization of GQD-RT 58

CONTENT
Page
No.
5.4.2 Thermogravimetric analysis 58
5.4.3 Diffusion study 59
5.5 In-vivo evaluation of BBB permeability 60
5.5.1 Standard chromatogram of RT by HPLC 61
5.5.2 Standard chromatogram of GQD by HPLC 61
5.5.3 Analysis of blood samples 61
5.5.4 Analysis of brain samples 69
6 SUMMARY AND CONCLUSION

76
BIBLIOGRAPHY 77
ANNEXURE 95

LIST OF FIGURES
Figure Page
No No
1.1. Representation of formation of amyloid beta fibrils and 2
development of Alzheimer’s disease.
1.2. Representation of different pathways for BBB crossing of some 4
molecules and nanoparticles
1.3. Structure of Rivastigmine Tartarate 13
2.1. Illustration of receptor-mediated endocytosis of targeting ligand- 19
conjugated GQDs loaded with an anticancer drug into a tumor cell
and drug release inside the cell
2.2. Release of DOX from GQDs-DOX-FA and its distribution into 23
HeLa cells after incubating at 37º (33.7g/mL concentration
2.3. Representation of size changeable nanoaircraft aggregation in the 24
acidic tumor environment, NIR induced disassembly and
penetration of GQDs/DOX into tumor tissue.
2.4. Diagrammatic representation of applications of GQDs as a drug in 29
neurodegenerative disorders such as Alzheimer’s and Parkinsons
disease.
2.5. SEM images of RBCs exposed to different concentrations of 32
GQDs
2.6. Schematic representation of evaluation GQD toxicity in male 34
mouse reproduction and offspring health
4.1 Diffusion study set up for RT and GQD-RT 44
5.1 Rivastigmine tartarate powder 48
5.2 ATR-IR spectrum of rivastigmine tartarate 48
5.3 UV spectrum of rivastigmine tartarate 49
5.4 Calibration curve of RT in 7.4 pH phosphate buffer 50
5.5 Synthesis of GQD from L-glutamic acid 50
5.6 GQD under visible light and UV lamp 51
5.7 L-glutamic acid under UV lamp 52
5.8 SEM image of GQD 52
5.9 TEM images of GQD 53
5.10 Particle size distribution of GQD 54
5.11 Zeta potential distribution plot of GQD 54
5.12 UV-Visible spectrum of GQD 55
5.13 Excitation spectrum of GQD 56
5.14 Emission spectrum of GQD 56
5.15 Comparison ATR-IR spectrum of GQD and LGA 57
5.16 XRD pattern of GQD 57
5.17 ATR-IR spectrum of GQ-RT 58
5.18 Thermogram of GQD and GQD-RT 58
5.19 Calibration plot of RT in 7.4 pH phosphate buffer 59
5.20 Chromatogram of 10µg/ml RT 60
5.21 Chromatogram of 54µg/mlGQD 61
5.22 Plasma collected at different time intervals after RT injection 61
5.23 HPLC chromatogram of RT in blood sample after 1 h 62
5.24 HPLC chromatogram of RT in blood sample after 4 h 62
5.25 HPLC chromatogram of GQD in blood sample after 0.5h 62
5.26 HPLC chromatogram of GQD in blood sample after 1h 63
5.29 HPLC chromatogram of GQD-RT in blood sample after 0.5h 65
5.30 HPLC chromatogram of GQD-RT in blood sample after 1h 67
5.33 Separated brain of Wistar rat 69
5.34 Crushed brain samples and their supernatant solutions 69
5.35 HPLC chromatogram of RT in brain sample 70
5.36 HPLC chromatogram of GQD in brain sample after 0.5h 70
5.37 HPLC chromatogram of GQD in brain sample after 1h 71
5.40 HPLC chromatogram of GQD-RT in brain sample after 0.5h 73
5.41 HPLC chromatogram of GQD-RT in brain sample after 1h 74

LIST OF TABLES
Table No. Page No.
2.1. Different sources and methods for synthesis of GQDs 16
2.2. The summary of GQD based nanocarriers with their drug 22
and target
4.1. List of materials 38
4.2. List of equipments and softwares used 39
4.3. Outline of in-vivo study 45
5.1 Calibration curve data of rivastigmine tartarate 49
5.2 Calibration curve data of rivastigmine tartarate 59
5.3 Concentration of RT in blood of RT treated rats at different 63
time intervals
5.4 Concentration of GQD in blood of GQD treated rats at 68
different time intervals
5.5 Concentration of GQD and RT in GQD-RT treated rats at 65
5.6 Concentration of GQD in brain of GQD treated rats at 72
5.7 Concentration of GQD in brain of GQD-RT treated rats at 75
ABBREVIATIONS
GQD Graphene Quantum dot

RT Rivastigmine tartarate
CNT Carbon nanotube
ND Nanodiamond
BBB Blood-brain barrier
ZO Zonula occludens
AP Alkaline phosphatise
γ-GTP γ-glutamyl transpeptidase
BMVEC brain microvascular endothelial cells
CNS Central nervous system
PBCA poly(butylcyanoacrylate)
GO Graphene oxide
rGO Reduced graphene oxide
QDs Quantum dots
LGA L-Glutamic acid
Hiapp Human islet amyloid polypeptide
DOX Doxorubicin
EMR Electromagnetic radiation
ROS Reactive oxygen species

DMF Dimethyl formamide
FRET Fluorescence resonance energy transfer
HER Herceptin
MTX Methotrexate
MWNT Multiwalled carbon nanotube
SOD Superoxide dismutase
NMDA N-methyl-D- aspartate
AMPA α-amino-3- hydroxyl-5-methyl-4-isoxazole propionate
Aβ Amyloid beta
PLGA poly (lactic-co-glycolicAcid
IGF-1 Insulin-like growth factor 1
BACE-1 Beta-site Amyloid precursor protein Cleaving Enzyme-1
SEM Scanning electron microscopy
TEM Transmission electron microscopy
XRD X-ray diffraction
HPLC High pressure liquid chromatography
HR-TEM High resolution transmission electron microscopy
ATR-IR Attenuated total reflectance infra red spectroscopy
DNA Deoxy ribonucleic acid
TGA Thermogravimetric analysis
CHAPTER 1 I N T R O D U C T I O N | 20
PDA Photo diode array

CHAPTER 1
INTRODUCTION
CHAPTER 1 INTRODUCTION |1
1.1 Alzheimer’s disease

Alzheimer’s is a chronic progressive neurodegenerative disorder characterised by
dementia and cognitive impairment, which develops due to the reduced level of
neurotransmitter acetyl choline in brain neurons. It is observed in people over 60
years age may span 8 to 12 years. Alzheimer’s was first reported in1906 by a German
psychiatrist, Alois Alzheimer’s; he observed the symptoms in a 55 years old woman
and published his findings in 1907. The symptoms of Alzheimer’s include loss of
memory, impaired language skill, problems in problem solving and inability to
perform everyday activities (1).
Pathologically it is characterized by intracellular neurofibrillary tangles and

extracellular amyloidal protein deposits contributing to senile plaques. Amyloid β
fibrillation in the brain, which is formed by the action of enzyme beta-secretase 1 or
Beta-site Amyloid precursor protein Cleaving Enzyme-1 (BACE1), the formation of
amyloid beta fibrils and development of Alzheimer’s is represented in Figure 1.1.
The neuropathological features of Alzheimer’s disease are recognized but the
intricacies of the mechanism have not been clearly defined. This lack of
understanding regarding the pathogenic process may be the likely reason for the non-
availability of effective treatment which can prevent onset and progression of the
disease (2,3).
1.2 Blood Brain Barrier
Blood-brain barrier (BBB) is the selectively permeable protective layer surrounding

the brain; it prevents the entry of toxic substances into the brain and supplies nutrients
such as glucose, oxygen etc to the brain tissues. The structural components of BBB
include microvascular endothelium , astrocytes, basement membrane, pericytes and
neurons in the endothelium, collectively called neurovascular unit of BBB (4,5). The
endothelial cell in the brain is usually called brain microvascular endothelial cells
(BMVEC) and is composed of tight junctions, adherent junctions and junctional
adhesion molecules. Tight junction is composed of tight junction proteins such as
occludins and claudins and some cytoplasmic accessory proteins like zonula
occludens (ZO) ZO-1, ZO-2, ZO-3 and cingulin. The ZO-1 and ZO-2 connects
membrane proteins to actin, which is responsible for integrity of endothelium.
Cadherins, catenins, vinculin, and actinin form the adherens junction. Large

molecules such as insulin, leptin, and iron transferring can cross BBB vai receptor
mediated endocytosis. Claudins are phosphoproteins with 22-kDa and caludin-claudin
linkages make primary seal of tight junction. Occludins are 65-kDa phosphoprotein
and are larger than claudins. The paracellular spaces of two adjacent cells contain
extracellular loops of occluding and claudin, and these loops form the paracellular
barrier of tight junctions (4,6). Rather than these membrane proteins, some enzymatic
barriers are also present on brain endothelium, including γ-glutamyl transpeptidase (γ-
GTP), alkaline phosphatase (AP), and aromatic acid decarboxylase. γ-GTP and AP
are present on luminal (apical) membrane surface of endothelium and it also contain
efflux transporters such as P-glycoprotein, BCRP and MRP. The abluminal
(basolateral) surface contains Na+-K+ ATPase and sodium dependent neutral amino
acid transporter. Glucose transporter GLUT-1 is present in both luminal and
abluminal surface in the ratio 1:3 (4).
Figure 1.1. Representation of formation of amyloid beta fibrils and development of

Alzheimer’s disease. Reproduced with permission from (3). Copyright 2019, Elsevier.

Brain endothelial cells have large number of mitochondria to meet the energy
requirements for active transport of nutrients. Transcellular flux is limited across BBB
due to small mumber of endocytic vesicles. Paracellular flux is also reduced due to
high electrical resistance offered by tight junction. The absence of fenestrations makes
BBB endothelium different from any other endothelium and thus, transport of
molecules become difficult. BMVEC can act as a barrier and also permits some
selective transport of micromolecules and some macro molecules like albumin and
low-density lipoprotein into brain and also can regulate osmolarity (7). Astrocytes are
glial cells and an astrocyte-BMVEC interaction is essential for neuronal function.
Along with BMVEC, astrocytes ensure BBB integrity and tightness. Pericytes are flat,
connective tissue cells seen around the capillary wall and they are essential for
endothelial cell proliferation, survival, migration, differentiation, and vascular
branching. Some portions of pericytes may contain macrophages and they can
phagocyte exogenous proteins and antigens (4,6).
1.2.1 Mechanism of BBB crossing
Nutrients and ions can cross BBB via transcellular pathway and paracellular pathway.
Figure 1.2 represents different transport mechanisms to cross BBB. Paracellular
transport is the concentration dependent passive diffusion of lipophilic or low
molecular weight solute molecules between the cells and finally enters BBB. It has
less importance in brain targeted drug delivery. Transcellular pathway is a major
transport system in brain, and it may be energy dependent or independent.
Transcellular pathway includes transcellular diffusion, receptor mediated endocytosis,
efflux transport system, and carrier mediated transport (8). Transcellular diffusion is
the diffusion of molecules across luminal and abluminal membrane of brain
endothelial cell (9). In receptor mediated transcytosis, the ligand molecules interact
with receptors on brain endothelial surface, and form an endocytic vesicle, which is
taken into cells. Then the receptor dissociates from ligand and the contents are
released out from the endosome by exocytosis. Hormones or high molecular weight
proteins such as insulin, leptin, low density lipoproteins, transferrin and IGF are
transported by this method. This a saturable active transport system with high
specificity and it is the major transport mechanism of CNS drugs, because it is not
size dependent and lipophilicity dependent (9). In efflux transporter, the substances

moves from brain back into systemic circulation to prevent accumulation of

substances in brain. Because of this efflux pump some drugs can’t attain therapeutic
concentration. ATP binding cassette efflux transporter P-gp has a high concentration
in brain capillaries and it helps to prevent accumulation of drugs and toxins in brain
(10). Carrier mediated transport may be energy-dependent or independent saturable
transport system, located on both luminal and abluminal surfaces. Different carriers
are expressed on brain endothelial cells and they specifically take the molecules and
takes into the cell. The transport of glucose is achieved by glucose transporter GLUT-
1 according to concentration gradient. There are different types of transporters for
amino acids according to their nature and properties. Large, neutral and aromatic
amino acids are uptaken by the N- or L- transport system, neutral amino acids taken
by A-system and sulphur containing amino acids especially cysteines are taken by
ASC system (7,11).
Figure 1.2. Representation of different pathways for BBB crossing of some

molecules and nanoparticles. Reproduced with permission from (12). Copyright
2018, Elsevier.

1.2.2 Factors influencing BBB crossing
The molecular weight greatly influences the diffusion of molecules across the blood
brain barrier. If the molecular weight of a drug exceeds 500 Da, it will not be
transported across the blood brain barrier. The CNS drugs are having the molecular
weights lesser than any other therapeutics as they can undergo significant passive
lipid mediated transport across the blood brain barrier (13). The hydrogen bonding is
having an inverse relation with the blood brain barrier penetration. It is evident that
the compounds having high hydrogen bond forming capacity such as peptides
containing amide groups possess minimal distribution ability across the blood brain
barrier (14). The Lipinski’s rule of five predicts that the drug may develop poor
absorption and permeability across the blood brain barrier when the number of
hydrogen bond donors and hydrogen bond acceptors exceeds the number 5 and 10
respectively. For each pair of H-bonds there is a decrease of one log of magnitude in
the BBB permeability of a drug (13). Lipophilicity and molecular weight are the two
major defining factors for permeability across the blood brain barrier. The lipophilic
drugs cross the blood brain barrier either by passive diffusion or by getting solubilised
in the lipid bilayer of the endothelial cell membrane.
1.2.3 Approaches for BBB crossing
Penetration of BBB becomes a critical factor in the treatment of glioma and CNS
disorders such as stroke, Alzheimer’s disease, Parkinson’s disease etc. In past, the
treatment of these disorders was a great challenge as the drug molecules can’t
penetrate BBB. In those days, the drugs were administered into brain via invasive
methods including surgical procedures, intracranial and intracerebral injections and
provide only a temporary increase in the BBB permeability. These painful techniques
were inconvenient for patient and reduce the patient compliance. Thus, certain non-
invasive techniques were developed later. Nasal administration of drug can reach
brain via olfactory bulb and different formulations are currently available in the
market. But the difficulty of drugs to pass through nasal mucosa and mucociliary cells
makes the nasal delivery less acceptable. The intracerebral implants with
biodegradable polymeric matrix containing the therapeautic agent, has found to be the
most traumatic drug delivery approach to the brain. The intraventricular, intrathecal
and the interstitial delivery systems were accepted as the most direct way for

circumventing the blood brain barrier, however, catheter obstruction, CNS infection
and the inadequate drug distribution are certain disadvantages of these delivery
systems (15). Moreover inducing the opening of BBB by the temporary disruption of
tight junctions using osmotic pressure and ultrasounds may cause uncontrolled inflow
of medicines and other unwanted molecules into the brain during the opening of tight
junctions (13).
The advancement in nanotechnology could solve these kinds of problems by

introducing different types of nanocarriers capable of crossing BBB. In non-invasive
approach the drug molecules are modified by colloidal, physiological and chemical
carrier system approach. Nanoparticles with small size (≤ 200nm) can successfully
penetrate BBB and their biocompatibility, non-toxicity also offers additional benefits.
This nanoparticle mediated brain targeting is emerging as an effective and non
invasive approach. Poly(lactic-co-glycolic acid), poly(butylcyanoacrylate) (PBCA)
and poly(lactic acid) NPs, liposomes and inorganic composites such as silver, gold
and zinc oxide NPs are mainly applied polymeric nanoparticles to cross the blood
brain barrier. Numerous BBB impermeable drugs such as fluorophores,
chemotherapeautic agents and molecular imaging contrasting agents are transported
across the BBB using PBCA nanoparticles. Among the inorganic metal based
nanoparticles, the customised surface and the flexibility in size makes the gold
nanoparticles the most promising one (12). The intranasal targeting of olanzapine to
the brain using the nanocubic vesicles formulated with the nonionic copolymers
(poloxamer 188 or 407) was found to be a promising approach (13). The cell
penetrating peptides are having brain targeting efficiency as vehicles with different
therapeutic agents. The viral vectors such as adeno-associated virus have shown the
potential to target to CNS cells on bypassing the BBB (16).
1.3 Carbon nanostructures
Carbon exists in different electronic orbital features like SP, SP2, SP3, and thus there
are many unique structures and properties for carbon. Carbon can form different
allotropes in solid state, and their properties changes according to the type of
hybridization. New research and developments in nanotechnology increases the
applications of carbon based nanoparticles in biomedical field. They have good
mechanical, electrical and optical properties and the properties depend on the atomic

structures. Carbon nanofamily consists of different carbon based structures including

zero dimensional fullerene, one directional carbon nanotube, two dimensional
graphene and graphene based structures, nanodiamonds, etc. Different biomedical
applications of carbon based nanostructures are reported, which include, drug
delivery, gene delivery, cellular imaging, biosensing, tissue engineering, contrast
agent etc. The brain targeted drug delivery using carbon based nanostructures is
getting more interest in recent days. Brain targeted drug delivery becomes a
challengeable one due to the presence of two barriers; blood-brain barrier and blood
cerebrospinal fluid barrier. Only the drug molecules with extremely small size, high
lipophilicity and partition coefficient can cross these barriers and enter the brain.
1.3.1Graphene nanofamily
Graphene is an allotrope of carbon, discovered by Geim and Novoselov in 2004, from

the exfoliation of graphite. It is a 2D single layer of carbon atoms in honeycomb like
lattice and each atom is connected with three SP2 hybridized carbon atoms. The
graphene family of nanomaterials includes; few-layer graphene, ultrathin graphite,
graphene oxide, reduced graphene, reduced graphene oxide (rGO), graphene
nanoribbon, graphene oxide nanoribbon, graphene quantum dot, etc. Less toxicity and
flexible physicochemical properties of graphenenano family render their different
biomedical applications, especially in drug delivery. Drug molecules can be
conjugated on the graphene surface through π-π stacking, and the conjugation doesn’t
need surface functionalization. But, if functionalization is done, conjugation of
molecules becomes easier. The solubility problems of pristine graphene limit its
biomedical applications. Thus, several graphene derivatives are introduced which
possess superior properties than pristine graphene. (17).
1.3.2 Graphene quantum dots
Quantum dots (QDs) are material of interest in nanotechnology and were discovered
in the 1980s by Alexie Ekimov. They are artificial semiconductor nanoparticles below
100 nm with excitons confined in all three spatial dimensions. Fluorescence is their
remarkable property; they have 20 times brighter fluorescence than ordinary
fluorescent materials. They are good candidates for drug delivery, diagnosis,
bioimaging, and sensing applications. The emission wavelength of QDs can be
adjusted from 450 to 1800 nm by altering their size, shape, and composition. Carbon

quantum dots, selenium quantum dots, silver quantum dots, silicon quantum dots,
gold quantum dots, etc are some among the known quantum dots. Many studies have
been reported on graphene-family nanomaterials such as graphene oxide, reduced
graphene, etc. These studies finally resulted in the development of GQDs in 2008 by
Ponomarenko and Geim, which marked a beginning for tremendous biomedical
applications. Then researchers focused their interest on GQDs and found that they are
more suitable for biological applications than any other quantum dots (18,19).
GQDs are carbon-based zero-dimensional fluorescent nanomaterials with a graphene

lattice inside. They show better solubility than carbon nanotubes. They are very small,
having a usual size less than 20 nm and with a reported maximum size of 60 nm, and
can easily penetrate through biological membranes (20). GQD suspension is very
stable in a high electrolyte concentration and lower pH. The luminescence properties
of GQDs are well studied. They show fluorescence in solution and film and they have
higher quantum yield than carbon dots. It is very interesting to note that, their
photoluminescence depends on their size and sometimes on shape. Furthermore, the
photoluminescence is tunable by changing edge functional groups (21).
GQDs can be synthesized in two forms, levo and dextro GQD. Levo and
dextrorotatory GQDs can be prepared from L/D-cysteine by covalently attaching them
on edges of GQD. The carboxylic acid group on GQD reacts with the amine group of
cysteine by carbodiimide/N-hydroxysuccinimide cross-linking. Dextro GQD has a
high affinity towards lipid bilayer than levo GQD. Their biocompatibility was studied
in Hep2G2 liver cells and found biocompatible. Chiral GQDs open new opportunities
in drug delivery, phototherapy, and optoelectronic applications (22).
GQDs are widely applied in different biomedical applications; they can be employed
as a nanocarrier for targted drug delivery, bioimaging and sensing agent and moreover
they can have a wide variety of biological activities. Different nanoparticles have
been used in biomedical applications, but GQDs have a prime position among those
nanoparticles. Different in-vivo and in-vitro studies conducted on toxicity of GQDs
concluded that, they are more biocompatible and non-toxic. Their small size enables
easy blood-brain barrier permeability and this helps to targted delivery of drugs to
brain. Their luminescence property is widely expolited for bioimaging applications.
Moreover, they can be easily synthesised from different organic molecules. GQDs

have a superior properties than other nanoparticles and it enables somany biomedical
applications (21,22).
1.3.3 Biomedical applications of graphene quantum dots
GQD as a drug
Some special characteristics and activities of GQDs are utilized in medical

applications. We are familiar with the drug carrying capacity of the GQDs and other
carbon nanostructures. Beyond a carrier, they also act as a good therapeutic agent in
some disease conditions. Low cytotoxicity and good biocompatibility make them
suitable for biomedical applications. This opens a new window in therapeutics, and a
synergistic effect can be expected in some cases, where the GQD simultaneously acts
as a therapeutic agent and a carrier for the drug.
GQD as an anti-diabetic agent
Diabetes mellitus can be classified to Type 1 and Type 2. The role of GQD in
alleviating Type 2 diabetes is recently identified. Type 2 Diabetes is sometimes
characterized by islet amyloidosis. It is the formation of amyloid peptide aggregates
by a neuro pancreatic hormone, human islet amyloid polypeptide (hIAPP) that leads
to β cell dysfunction and death. Fluorinated GQDs contain highly electronegative
fluorine atoms. It is noticed that fluorinated GQD can inhibit the peptide aggregation
by converting long fibril hIAPP aggregates into short thin fibrils. Interestingly,
fluorinated GQD alone possesses this property and.not.by.GQD.and.N-GQD.(23,24).
GQD in hepatitis and wound healing
Immune-mediated fulminant hepatitis is the impairment of hepatic functions due to

the death of hepatocyte by the activation of immune cells and cytokine secretion.
Concanavalin A can be injected into mice for inducing immune mediated fulminant
hepatitis. Since large GQDs are eliminated through liver, an intravenous injection of
large GQD is effective against immune-mediated fulminant hepatitis and it acts by
decreasing liver transaminase, lipid peroxidation in liver, apoptosis, and autophagy.
GQD attenuates genes involved in autophagy such as Atg4b, Atg7, Atg12 and beclin-
1 and thus liver autophagy can be decreased (25,26). Self-assembled GQDs are non-
toxic and have an important role in wound healing. They facilitate fast wound closure
and they can easily enter into the cell nucleus and induce cell proliferation (27).

Antimicrobial activity of GQDs
One of the interesting features of GQD is its antimicrobial activity. Gaussian-

curvature match helps in the association of GQDs on the bacterial cell surface and it
disrupts the bacterial cell envelop and thus providing anti-bacterial effect (28). The
mechanism of antibacterial activity is through the generation of reactive oxygen
species (ROS) followed by oxidative stress. The photoexcitation of quantum dots at
470 nm leads to ROS generation and they are capable of killing Escherichia coli and
Staphylococcus aureus. In a study performed on Escherichia coli using ZnO/GQD
nanocomposites, the generation of ROS was found to be higher by UV irradiation
than ambient light. They are useful in water and surface disinfection (29,30). Now,
obviously, it would be interesting to know the effect of the size of GQD on their
antibacterial activity. A study on Escherichia coli by using small GQD and large
GQD has unveiled that small GQD possesses higher killing rate than large GQD. The
oxidative stress and small size of GQD will help in easy bacterial membrane
permeability (31).
Anti-bacterial activity of GQD is utilized for developing band-aids for wound

disinfection by incorporating a small dose of hydrogen peroxide (H 2O2). GQDs can
catalyze the degradation of H2O2 into -OH and has good antibacterial activity. Thus,
band-aids developed using GQD with a mild dose of H 2O2 possess excellent wound
healing property by killing both Gram-positive and Gram-negative bacteria (32). A
synergistic anti-bacterial effect was observed from PEGylated silver-GQD
nanocomposites. The cytotoxicity of GQD can be minimized by PEGylation. They are
used for coating surgical instruments and wound dressings (33). Nitrogen-doped
GQDs can be used as a photosensitizing agent; it can produce a high number of ROS
and thus it has application in photodynamic antimicrobial therapy (34).
Besides the antibacterial effect, GQD has antiprotozoal activity too. Malaria, a
protozoal infection is caused by mainly Plasmodium falciparum through its vector
female Anopheles stephensi mosquito. GQDs are found to be effective against P.
falciparum, P.berghei, and larvae of malaria mosquitos. The predation efficacy of
mosquito predators such as fish Gambusia affinis, dragonfly Anax immaculiforns, and
tadpoles of Asian bullfrog Hoplobatrtrachus tigerrinus is increased by small doses of
GQDs (35).

GQD in cancer therapy
Rather than conventional chemotherapy, cancer therapy also involves radiotherapy,

photodynamic therapy, and photothermal therapy. A combination of these therapies is
sometimes recommended for synergistic effect and rapid relief. GQDs have an
inevitable role in cancer therapy, and they promise a horizon for oncology
researchers. GQDs in different types of cancer therapy are explained in this section.
Radiotherapy
Radiotherapy in cancer utilizes high-intensity ionizing radiations such as X-

rays and gamma rays. These radiations generate highly reactive free radicals that will
cause damage to the DNA of the cancer cell. GQDs are employed as a new radio-
sensitizer for effective radiotherapy for cancer. Oxidized GQDs produce ROS, the
resulting oxidative stress leads to degeneration of DNA and proteins of carcinoma
cells. This causes increased cell apoptosis and inhibition of cell proliferation.
Moreover, GQDs can increase the sensitivity of cancer cells toward infrared
radiations. The enhanced tissue uptake and cell apoptosis make GQDs an effective
nano radiosensitizer (36–38).
Photodynamic therapy
Photodynamic therapy (PDT) is widely applied for cancer, psoriasis, acne, and
atherosclerosis by using a light source and a photosensitizing agent. Semiconductor
quantum dots gained great attention in PDT as a photosensitizing agent. The
mechanism of photodynamic therapy involves the generation of ROS and resultant
oxidative stress kills targeted cells. Here, GQDs are employed as a photosensitizing
agent and can produce ROS in cancer cells and tumors. Their resistance toward
biological corrosion, stability in different pH and light, and biocompatibility make
them a good photosensitizing agent. With GQD, photodynamic therapy and
simultaneous imaging of cancer cells can be achieved. GQDs can prevent
photobleaching and can produce a high quantum yield of singlet oxygen. Therefore,
GQD stands a little ahead in photodynamic therapy than any other agent. GQDs
induce cancer cell apoptosis and autophagy by oxidative stress. GQD is more
effective in cancer therapy than conventional photodynamic therapy when studied
using HeLa cells (39–42). A combination of chemotherapy and photodynamic therapy
assures a synergistic effect in cancer treatment. PEGylated GQD-silver nanoparticles

loaded with doxorubicin (DOX) shows the synergistic effect in tumor cell apoptosis
by simultaneous photoirradiation and drug delivery (43). Photosensitizing agent rose
bengal coupled with nitrogen-doped GQDs are reported for two-photon photodynamic
therapy (44). Mitochondria-targeted photodynamic therapy was also achieved with
GQDs (45).
Photothermal therapy
Photothermal therapy utilizes electromagnetic radiation (EMR) and the

principle involved is the EMR induced excitation of photosensitizing agent and
release of vibrational energy in the form of heat and all cells that have been reached
by the photosensitizer will be damaged by the heat. Nitrogen and boron dual doped
GQD based photothermal therapy in the near-infrared II region shows a successful
result in cancer cell therapy (46). The incorporation of a drug into GQD with
photothermal therapy enhances treatment effectiveness. GQD-gated hollow
mesoporous carbon nanoplatform loaded with doxorubicin achieves this simultaneous
effect by near-infrared induced controlled drug release (47). A combination of
photothermal and photodynamic therapy is an interesting one. The 808 nm laser
irradiation generates ROS and heats simultaneously and thus forms a multifunctional
GQD for cancer therapy (48).
GQD as a solution for multidrug resistance
One of the promising applications of GQD is their activity against multidrug

resistance. The multidrug resistance is commonly observed in cancer chemotherapy
due to efflux of anticancer drugs by ATP binding cassette such as p-glycoprotein,
multidrug-resistant protein MRP1, and breast cancer protein. GQD have the ability to
down-regulate these transporter genes by interacting with their c-rich regions and thus
promises an efficient solution for multidrug resistance (49).

1.4 Rivastigmine Tartarate (RT)
Rivastigmine tartarate is a reversible acetylcholine esterase inhibitor, used in the

treatment of mild to moderate Alzheimer’s disease. It is used for increasing cognitive
performance of patient. Figure 1.3 shows structure of rivastigmine tartarate.
Figure 1.3.
Structure of Rivastigmine Tartarate
Molecular formula: C14H22N2O2.C4H6O6
IUPAC name: 3-[(1s)-1-(Dimethylamino)ethyl]phenyl ethyl(methyl)carbamate

hydrogen (2R,3R)-2,3-dihydroxybutanedioate.
Molecular weight: 400.43g/mol
Category: Reversible acetylcholine esterase inhibitor
Dose: The initial dose is 1.5mg twice daily which is increased to 3mg twice daily over
a two week interval; 4.5mg twice daily to a maximum and 6mg twice daily. The
effective dose is 3to 6mg daily.
Description: White to off-white fine crystalline powder.

Solibility: Very soluble in water, soluble in ethanol, methanol and acetonitrile,

slightly soluble in n-octanol and very slightly soluble in ethyl acetate.

Identification: Determine by infra red absorption spectrophotometry and compare the

spectrum with that obtained with reference spectrum of rivastigmine tartarate.
Trade names: Exelon, Prometax
Routes of administration: Oral, Transdermal, IV
Medical use: Mild to moderate Alzheimers disease, Parkinson’s disease
Available forms: Capsule, Oral solution
Pharmacology: RT prevents decreases in the amount of acetyl choline and butyryl

choline by inhibiting acetyl choine esterase and butyryl choline esterase. It binds the
esteratic site of the enzymes and dissociation occurs.
Pharmacokinetics: The protein binding is 40%, and apparent volume of distribution

is 1.8 to 2.7 L/Kg. Bioavailability is approximately 36% following a 3mg dose which
may increase when taken with food. RT is metabolised by hydrolysing with esterase
enzymes and eliminated in urine as its conjugates and metabolites. Plasma clearance
is 130L/h (.02mh IV dose). Mean plasma elimination half-life is 1.5-2h, which is
shorter than that of other carbamate derivatives (50,51).

CHAPTER 2
REVIEW OF LITERATURE
CHAPTER 2 R E V I E W O F L I T E R A T U R E | 15
2.1 Graphene Quantum Dots

GQDs are carbon-based zero-dimensional fluorescent nanomaterials with a graphene
lattice inside. They are very small, having a usual size less than 20 nm and with a
reported maximum size of 60 nm, and can easily penetrate through biological
membranes (20). They show bright fluorescence and it depends upon their size and
shape.
2.1.1 Synthesis of Graphene Quantum Dots
They can be synthesised by top-down and bottom-up methods, from a variety of raw
materials. Table 2.1 shows different sources and methods of GQD synthesis.
Top-down methods
Top-down method of synthesis means the synthesis of GQDs from large graphite
materials by cutting down into small fragments. GQDs can be prepared from
materials such as graphene, graphene oxide, graphite powder, coal, carbon nanotubes,
or carbon fiber by physical or chemical means of cutting. Top-down methods are most
widely used because of the availability of raw materials and high product yield.
Hydrothermal cutting
In this method, the precursors are converted into GQDs under high conditions of
temperature and pressure after strong oxidation with suitable chemicals. By this
method, we can synthesize GQDs with different size and tunable fluorescence.
Graphene oxide is oxidized with a mixture of nitric acid and sulphuric acid to
introduce epoxy groups on the carbon lattice as the cleavage sites and then chemical
cutting and deoxidation are carried out using alkali such as sodium hydroxide or
ammonia for around 10 h to obtain fluorescent GQDs. In this method, the temperature
has an important role in GQDs texture. At low temperature, poorly ordered GQDs are
synthesized, and at high temperature, highly crystalline small GQDs are obtained
(52,53). Boron-doped GQDs can be prepared by hydrothermal treatment of glucose in
presence of boric acid, which possesses high electrocatalytic activity than undoped
GQDs.(54).

Table 2.1 Different sources and methods for synthesis of GQDs
Source Method of preparation Ref.

Graphene sheet Hydrothermal cutting (52,53)
Glucose Hydrothermal cutting (54)
Graphene oxide Solvothermal cutting (55)
MWCNT, charcoal, Solvothermal method (56)
carbon fiber, graphite
Citric acid Pyrolysis (57)
L-Glutamic acid Pyrolysis (58)
Glutathione Pyrolysis (59)
Pyrene Hydrothermal reaction (60)
1,5-dinitronaphthalene Hydrothermal reaction (61)
Citric acid Hydrothermal reaction (62)
Carbon nanotube Oxidation (63)
Graphite Oxidation (64)
Graphite Electrochemical exfoliation (65)
Coal Oxidation (66)
Glucose Microwave-assisted (24)
method
Glucose Microwave-assisted (67)
hydrothermal
Cotton Liquid exfoliation (68)
Graphene Liquid exfoliation (69)
Graphene sheet Electrochemical exfoliation (70)
Fullerene Cage opening, oxidation (71,72)

Solvothermal cutting
In this method, organic solvents such as benzene, dimethylsulfoxide,

dimethylformamide (DMF) etc is used instead of water. The size of the prepared
GQDs depends on the solvent selected. Graphene oxide can be used as a precursor. In
a reported procedure, graphene oxide - N-N dimethylformamide suspension was
sonicated and heated to 200ºC in a muffle furnace for 8 h to form GQDs-DMF
suspension. The product was vacuum filtered and DMF was removed by evaporation.
Pure GQDs were obtained following this procedure (55).
Microwave-assisted method
Hydrothermal and solvothermal methods are time-consuming and thus this rapid
method was evolved. Graphene oxide can be cleaved under acid condition by
microwave assisted irradiation to form GQDs with greenish-yellow fluorescence. This
method is rapid and provides high product yield. GQDs with uniform size can be
obtained if homogenous heating is provided. Moreover, the size can be controlled by
varying the reaction time. This method is more suitable for carbohydrates containing
C:H:O ratio 1:2:1 (73).
Oxidative cleavage
The principle of oxidative cleavage is the breaking of carbon-carbon linkage present

in precursors such as graphene, graphene oxide or carbon nanotube by acid treatment.
Sulphuric acid, nitric acid, and other oxidative agents are used for the process. In a
study, small graphene oxide sheet was treated with nitric acid to form small cut
pieces. It was then treated with polyethylene glycol for surface passivation followed
by reduction with hydrazine hydrate to obtain small sized GQDs (74). Treatment with
oxygen leaves oxygenated functional groups such as hydroxyl (−OH), epoxy (−O−),
ether (−OCH3), carbonyl (−(CHO)−), and carboxyl (−COOH) groups on GQD
surface and thus the solubility and optoelectronic properties are enhanced (75).
Electrochemical exfoliation
GQDs are synthesized by electrochemical cleavage of carbon materials such as

carbon nanotubes or graphite rods. In this method, anodic oxidation of water leads to
the formation of hydroxyl and oxygen free radicals, which cleaves the precursors into
GQDs.(76).

Bottom-up methods
Precursor pyrolysis/carbonization
This is the most commonly used and simple method for GQDs synthesis, but the
quantum yield is very low. The method involves carbonization of organic precursors
such as citric acid, amino acids (L-glutamic acid), carbohydrates (glucose, sucrose),
glutathione, acetylacetone, and ethanolamine. GQDs can be synthesized by direct
pyrolysis of citric acid at 200ºC followed by addition of sodium hydroxide solution
(57,77). Similarly, GQDs can be synthesized from L-glutamic acid by heating at
210ºC. On heating, a brown colored liquid is obtained and water is added to get an
aqueous dispersion of GQD (58). Glutathione (a tripeptide containing glutamate,
cysteine, and glycine) is another precursor used. GQDs synthesized from glutathione
is more biocompatible and provides high quantum yield (78).
Catalyzed fullerene cage-opening
Fullerene is used as a precursor for a bottom-up method of GQDs synthesis. GQDs

are prepared from fullerene through oxidation using a mixture of strong sulphuric
acid, sodium nitrate, and potassium permanganate as an oxidizing agent. The
oxidation leads to cage opening and finally, highly fluorescent GQDs are obtained
(71).
In another method, fullerene (C60) molecule is adsorbed on ruthenium crystal. The

interaction between ruthenium and C60 leaves vacancies on ruthenium surface and
thus C60 molecules are embedded in the surface of ruthenium crystal. High
temperature catalyzes the fragmentation of C60 molecules to carbon clusters and then
GQDs were formed by diffusion and aggregation of carbon clusters (72).
2.1.2 Graphene Quantum Dots as nanocarrier
Nanomedicine is the application of nanotechnology in the medical field,

especially in drug delivery. Nanocarrier mediated drug delivery is the most acceptable
and efficient system for targeted delivery of drugs, and often genes are also delivered.
Different nanocarriers such as polymer conjugates, polymeric nanoparticles, lipid-
based carriers, dendrimers, carbon nanotubes, gold nanoparticles, nanogels, magnetic
nanoparticles, nanorods etc have been used as carriers for both drug and gene. They
provide targeted release of the loaded contents; hence enhanced therapeutic effect can

be achieved along with minimum side effects. However, some of these nanocarriers
have their own drawbacks and thus there is an immense need for the development of
advanced.nanocarriers.(79).
Quantum dots are a new class of nanocarriers for drugs which shows good stability in
body fluids, increased duration of action and improved drug stability. Quantum dots
have strong adsorption capacity and high specific surface area. Furthermore, their
luminescence properties enable easy monitoring of drug release if it is paired with
fluorescence resonance energy transfer (FRET) system. Recently, graphene quantum
dot (GQD) was developed which have potential drug delivery and therapeutic
applications. Drugs can be easily loaded or conjugated to GQDs via π-π interactions.
Due to their large surface area, a higher amount of drug can be loaded. Their low
toxicity, good biocompatibility, fluorescence, large surface area, and easy
functionalization make them suitable for a variety of biological applications (80).
Table 2.2 gives a summary of GQD based nanocarrier.
Figure 2.1 Illustration of receptor-mediated endocytosis of

targeting ligand- conjugated

GQDs loaded with an anticancer drug into a tumor cell and drug release inside the
cell.

Conventional cancer chemotherapy has so many disadvantages like systemic toxicity

and undesired side effects. These drawbacks can be overcome by targeted cancer
therapy using nanocarriers. They deliver drugs into the tumor cell selectively and thus
prevent cytotoxicity to normal cells. GQDs are used as traceable drug delivery system
for targeted delivery of drugs into cancer cells. The pH-sensitive delivery of the
anticancer drug is achieved in the acidic tumor environment. Cancer cell surfaces are
overexpressed with some receptors such as folic acid, biotin, herceptin etc. These
receptors are very helpful in targeting the drug to cancer cells. The fluorescence
property of GQD is utilized for tracing them in the body. Here, GQDs can also act as
a probe for monitoring the drug release. Thus, the drug-carrying capacity and
fluorescence imaging of GQDs can be utilized simultaneously in drug delivery (81).
A schematic diagram showing the combined use of these aspects in tumor-targeted
delivery is shown in Figure 2.1.
The surface of the GQD linked with biotin, a cancer cell targeting ligand molecule,
and loaded with doxorubicin, shows targeted nuclear delivery of doxorubicin. GQDs
were found to be more biocompatible and cell uptake was increased because of the
presence of biotin molecule on the surface. Similarly, folic acid can also be used as a
targeting molecule on the GQD surface and loaded with doxorubicin. This
nanosystem is taken up by HeLa cells via receptor-mediated endocytosis. Figure 2.2
shows releases of DOX (red fluorescence) from GQDs (green fluorescence) and
distribution into HeLa cells at different time intervals (82).
Another targeting molecule is Herceptin (HER), which can target HER-2

overexpressed breast cancer cells. In a study, breast cancer cells were targeted by
labeling HER and β-cyclodextrin on GQD surface. The doxorubicin was loaded into
β-cyclodextrin and HER facilitated targeting. This nanocarrier system shows
enhanced cellular uptake and tumor-targeted release of doxorubicin. Moreover, the
blue fluorescence of GQDs allows diagnosis of breast cancer by fluorescence imaging
(83). In another study, GQDs are functionalized with a targeting molecule, arginine-
glycine-aspartic acid, and loaded anticancer doxorubicin. Arginine-glycine-aspartic
acid enhances the GQD uptake into the pancreatic cancer cell by receptor-mediated
endocytosis. GQDs emit green fluorescence and doxorubicin emits red fluorescence;
cellular uptake, the release of doxorubicin, and the nuclear localization can be easily
visualized (84,85). In another reported study, the surface of GQD was covalently

linked to fluorescent dye Cy with cathepsin D responsive peptide. Then loaded anti-
cancer agent doxorubicin and the drug release was tracked easily (86). GQD modified
with poly-L-lactide and PEG was used for intracellular micro RNA imaging by using
suitable micro RNA probes. Incorporation of gene targeting agents in this system
leads to enhanced cancer cell growth inhibition and apoptosis (87).
GQD-based FRET system can also be employed for drug release monitoring. FRET is
a distance-sensitive ‘smart system’ for drug delivery, biosensing, and bioimaging
applications. In a FRET system, energy is transferred from a donor molecule to
acceptor molecule. The energy transfer reduces the fluorescence intensity of the donor
and increases the fluorescent emission of the acceptor molecule. FRET is used for
measuring the distance between donor and acceptor and it is employed to study
biological interactions. Different nanoparticles such as quantum dots, gold
nanoparticles etc are used to develop a FRET system (88). GQD-based FRET system
was designed for nuclear-targeted drug delivery of doxorubicin. GQDs act as a carrier
for doxorubicin and donor of FRET pairs and it was conjugated with TAT peptide for
nuclear targeting. Doxorubicin release can be monitored in real time using this
system (89).
Size changeable GQD nano aircraft (SCNA) was designed for targeted tumor
penetration and drug delivery. Doxorubicin-loaded GQD nano aircraft having 150 nm
undergo aggregation in acidic tumor environment and the size is enhanced. NIR
irradiation disassembles nano aircraft into small DOX/GQD and it easily penetrates
into tumor cells, and then DOX accumulates in target sites, as shown in Figure 2.3
(90).
Nitrogen-doped GQDs with 10 graphite layers can be prepared from citric acid by
hydrothermal reaction at 180℃ for 10hr and loaded with anticancer methotrexate
(MTX). The GQD-methotrexate interaction occurs via π-π stacking. This nanocarrier
was found to be biocompatible and effective for cancer therapy by releasing
methotrexate into nucleus (91). GQDs can be PEGylated and encapsulated with β
cyclodextrin. The drugs bevacizumab and ranibizumab were incorporated in this
nanocarrier system for managing age-related macular degeneration. The
biocompatibility study was conducted on mice fibroblast L929 cells and it was noted
that the carrier was non-toxic and biocompatible (92).

Table 2.2 The summary of GQD based nanocarriers with their drug and target
Sl. No GQDs-Carrier Drug Targe Ref

1. GQDs-Biotin Doxorubicin Cancer cell (81)
2. GQDs-Folic acid Doxorubicin HeLa cells (82)
3. GQDs- Doxorubicin HER-2 breast cancer (83)
Herceptin-β- cell
cyclodextrin
4. GQDs-arginine- Doxorubicin Pancreatic cancer cell (85)
glycine-aspartic
acid
5. GQDs-TAT- Doxorubicin Nucleus (89)
FRET
6. SCNA-GQDs Doxorubicin Tumor cell (90)
7. N-GQDs Methotrexate Cancer cell (91)
8. PEG-GQDs-β- Bevacizumab Eye (92)
cyclodextrin Ranibizumab
9. GQDs Doxorubicin Glioma cell (93)
Cis-platin
10. Cysteamine HCl- Berberine HCl Cancer cell (94)
GQDs
11. GQDs Curcumin Cancer cell (95)
12. GQDs glycine-proline- Brain (96)
glutamate
13. Mesoprous Rhodamine B HeLa cell (97)
silicaNP-GQDs
14. Chitosan-GQDs Sodium GIT (98)
salicylate
15. SvFvB10-GQDs Cis-Platin breast cancer cells (99)
(MDA-MB-231)

Figure 2.2. Release of DOX from GQDs-DOX-FA and its distribution into HeLa
cells after incubating at 37º (33.7g/mL concentration). A represents images after
0.5h, B(8h) and C (24h). Reproduced with permission from ref (Wang et al., 2014).
Copyright 2014 Elsevier.
The blood-brain barrier crossing capacity of GQD is utilized in some studies for the
development of drug delivery systems into the brain and spinal cord. They are better
taken by glioma cells in the brain than normal brain cells. Thus GQDs are good
carriers for doxorubicin and cisplatin into glioma cells (100).
Milk-derived GQD has good fluorescence property and acts as a good carrier for
drugs. GQDs can be synthesized from milk and functionalized with cysteamine
hydrochloride which acts as a linking agent for drugs. In a study, the anti-cancer drug
berberine hydrochloride was conjugated on the GQD surface with the help of
cysteamine hydrochloride (94). Meanwhile, a synergistic anticancer activity was
achieved with curcumin loaded GQDs. It is reported that the anticancer activity was
increased with GQD than curcumin alone (95).

Figure 2.3. Representation of size changeable nanoaircraft aggregation in the acidic

tumor environment, NIR induced disassembly and penetration of GQDs/DOX into
tumor tissue. Reproduced with permission from ref (90). Copyright 2017, WILEY-
VCH Verlag GmbH & Co. KGaA.
The glycine-proline-glutamate peptide is known for enhancing memory and learning

capacity which are impaired in Alzheimer’s disease. GQDs are conjugated with this
neuroprotective peptide. GQD itself can improve Alzheimer’s by preventing β
amyloid aggregation and the conjugation of neuroprotective peptides on GQD surface
enhances the effectiveness of therapy. It improved learning and memory and it
increased neurogenesis in APP/PS1 transgenic mice (96).

2.2 Carbon nanostructures in brain disorders
Various carbon based nanostructures such as carbon nanotube (CNT), fullerene,

Nanodiamond (ND), Graphene, Graphene oxide (GO), reduced Graphene oxide
(rGO), GQD have different activities in brain and central nervous system.
CNT will become well known for the treatment of ischemic stroke, it is a condition in
which blood vessels to the brain become blocked and as a result, the neurons inside
the brain get damaged. Current treatments for ischemic stroke including
chemotherapy and radiotherapy have many side effects and are painful procedures.
Pre-injection of amine functionalized SWNT into the right lateral ventricle can protect
neurons and decreases the level of inflammatory markers in the blood of the rat stroke
model (101). Similarly, CNT impregnated with subventricular zone neural progenitor
cell can heal the affected neurons after intracranial injection into a cerebral ischemic
rat model. And concluded, CNT has an important role in the treatment of
neurodegenerative disorders (102). Functionalized MWNT conjugated with
gadolinium L2, an amyloid beta targeting agent, can be injected intravenously into
Alzheimer's mice model. It crosses
BBB via receptor-mediated transcytosis and a therapeutic effect is achieved

Alzheimer's (103). Polyurethane and silk fibroin can be associated with functionalized
MWNT by electrospinning technique, and it can act as an electrospun scaffold for
neuronal growth and differentiation. The in-vitro study with Schwann cells reveals
that the scaffold can stimulate their growth and proliferation. It also improves axonal
growth, neural cell sensitivity and neural expression (104).
The neuroprotective activity of fullerene is reported and it gives great hope in the
treatment of neurodegenerative disorders. The neuroprotection is achieved by its
antioxidant activity and oxygen quenching activity and its superoxide dismutase
(SOD) like activity. The combination of antioxidant activity and anti-aggregation
activity of fullerene may be useful for the development of new medicines for
neurodegenerative diseases in future days. When rat embryo brain neurons culture
with polyhydroxylated fullerenes fullerenols, it suppresses the binding of subtypes of
N-methyl-D- aspartate (NMDA) and α-amino-3- hydroxyl-5-methyl-4-isoxazole
propionate (AMPA) receptors and also it reduces the intracellular calcium level.
Carboxy fullerene is synthesized from malonic acid derivatives and which can reduce

the neuronal death resulted by exposure to glutamate receptor agonist like NMDA and
AMPA. It can inhibit excitotoxicity induced death of cortical neurons in NMDA and
AMPA containing culture medium. Intraventricular injection of carboxy fullerene at
low concentration, increases the activity of pyramidal neurons but in higher
concentration, the neuronal activity is suppressed (105–109). Fullerene can be
formulated with PVP and poly (2-alkyl-2oxazoline) (Pox) and both have antioxidant
activity;Pox- fullerene complex is easily taken up by CATH.a neurons and show intra
cellular superoxide scavenging activity but it is not observed in PVP-fullerene
complex (110).
Anti-amyloid action of fullerene is very interesting; fullerene can bind with the
hydrophobic region of amyloid beta peptide and inhibits the peptide precursor
aggregation. Addition of colloidal water solution of fullerene into amyloid beta
peptide fibrils (Aβ ) can inhibit aggregation (105). Fullerene nanoparticle can
25-35
inhibit amyloid beta (16-22) peptide fibrillation. The fullerene-peptide interaction is

due to the strong hydrophobic interaction and aromatic stacking interaction, and this
strong interaction weakens peptide-peptide interaction and thus inhibits peptide
aggregation (111).
Neuro-regenerative action of graphene-based nanomaterial is recently reported.

Incubation of human neuroblastoma cells with graphene seeded medium for 7 days
shows increased neurite outgrowth. When the cells are cultured on glass instead of
graphene, only small neurite outgrowth was observed. This study reveals the
neurodegenerative activity of graphene and related materials (112).
A similar study was also performed on mouse hippocampal cells. Effect of graphene
and polystyrene substrate on neurite sprouting and outgrowth can be compared by
culturing mouse hippocampal cells in these two media. The results show that
graphene can induce neurite sprouting and outgrowth, and enhanced expression of
growth associated protein-43 can be also observed (113).
The graphene oxide incorporated poly (lactic-co-glycolicAcid (PLGA)

electrospunnanofibres containing immobilized Insulin-like growth factor 1(IGF-1) has
resulted in better neural stem cell survival, proliferation and differentiation. The
graphene oxide effectively enhanced the IGF-1 immobilization and these PLGA/GO
nanofibres with IGF-1 incorporation are having great potential in elevating the

neurogenic and neuroprotective effects of nerve implants (114). Recently the ability
of thin graphene oxide sheets(s-GO) to alter specific neuronal synapses has been
reported. The graphene oxide flakes are having the ability to reduce the availability of
glutamate, a main excitatory neurotransmitter and thus to mediate neuronal
development, migration, synaptic maintenance, and transmission. These-GO is having
the potential to be improved as an efficient and specific synaptic transmission
modulator (115).
Since IL-13 is overexpressed in malignant tumors, the chemo-photothermal targeted
therapy of glioma cells has been successfully developed with IL-13 as the targeting
ligand. And GO is used to absorb NIR lasers because of its good heat transfer for
photothermal therapy. It gives a higher effect than single chemotherapy (116).
Alzheimer’s is characterized by amyloid β fibrillation in the brain, which is formed by

the action of enzyme beta-secretase 1 or Beta-site Amyloid precursor protein
Cleaving Enzyme-1 (BACE1), the formation of amyloid beta fibrils and development
of Alzheimer’s. Carbon dots can act as an anti-amyloidogenic agent, which can inhibit
the amyloid β deposition by deactivating BACE1 and oligomer formation. The
hydrophilic surface of the carbon dot is responsible for the inhibitory activity. Human
transferrin conjugated carbon dot can cross BBB and its anti-amyloidogenic activity
can be studied with the help of fluorescence resonance energy transfer (FRET)
system. The substrate for the BACE1 enzyme is conjugated with a fluorescence dye
when the substrate is cleaved by the BACE1, the fluorescence intensity enhances.
When transferring conjugated carbon dot is added to this, the fluorescence intensity
decreases. This study concludes that carbon dot can inhibit the amyloid beta
fibrillation by inactivating BACE1 and the inhibition is concentration dependent. The
more inhibitory activity is observed with a higher concentration of carbon dot (3,116).
Neural stem cells can proliferate and differentiate into astrocytes, neurons, and
oligodendrocytes. This differentiation of neural cells can promise advances in the
treatment of neurodegenerative disorders like Alzheimer's disease, Parkinson's
disease, etc. Nanodiamond can induce neural stem cell adhesion and differentiation.
Monolayers of nanodiamond support rodent neuronal outgrowth. The interaction of
human neural stem cells with ND is investigated. Human neural stem cells can be
incubated with oxygen functionalized and hydrogen functionalized monolayers of
nanodiamonds. Then the neural stem cell adhesion and proliferation are evaluated

after 7 days, and the data shows that higher adhesion and proliferation is observed on
oxygen functionalized nanodiamond than on hydrogen functionalized one (117).
Neurons are usually cultured over proteins of the extracellular matrix; nanodiamond
monolayers can provide similar physicochemical properties of extracellular matrix
protein. Primary neuronal cultures can be seeded on to monodispersed nanodiamond
coated glass, silica, nanocrystalline diamond, and polycrystalline diamond and the
neuronal outgrowth can be compared with uncoated same materials. After incubation,
the coated substrates allow the attachment of murine neurons. The study suggests that
NDs can act as a substrate for neuronal growth and they can promise some future
applications in neurodegenerative brain disorders (118).
Effects of fluorescent nanodiamonds on neurons in the central nervous system (CNS)

and peripheral nervous system (PNS) are investigated in an animal by intracranial
injection. They are found to be nontoxic in both type neurons and intracranial
injection didn’t cause behavioral changes in animals. In in-vitro, studies they are
internalized by primary cortical neurons. These results suggest that NDs can be used
for delivery of drugs into neurons. But they cause a dose-dependent reduction in
neurite length in CNS and PNS neurons due to the spatial hindrance of fluorescent
NDs. This result is contraindicated with previous studies showing ND coated surfaces
induces neurite outgrowth, and this contraindicated results may be due to differences
in,particle.size.of.ND.(119).
2.2.1 Graphene Quantum Dots in neurodegenerative disorders
Alzheimer’s, one of the major neurodegenerative disease characterized by dementia is

caused by the deposition of amyloid β peptides in the brain. Thus, the treatment
strategies of Alzheimer's include the development of agents that are capable of
preventing the aggregation of amyloid β. It is reported that GQDs can prevent the
aggregation of amyloid β peptides and they can protect from the cytotoxicity of
peptides. This activity of GQD is a promise for Alzheimer’s treatment (120).

Figure 2.4. Diagrammatic

representation of applications

of GQDs as a drug in neurodegenerative disorders such as Alzheimer’s and

Parkinsons disease.
Similarly, tramiprosate, a compound that binds to amyloid β peptide has an inhibitory

effect on their aggregation. Tramiprosate when covalently linked to GQDs is proved
as an effective inhibitor of amyloid-β aggregation, and thus their combination gives a
synergistic effect against Alzheimer’s disease (121). Parkinson’s disease is another
degenerative disorder affecting the brain, and its pathophysiology involves the
accumulation of α-synuclein in the midbrain. GQDs are found to be capable of
preventing α-synuclein fibrillization and promote their disaggregation. The blood-
brain barrier permeability of GQDs along with these activities promise an effective
therapy against neuronal disorders (122). The application of GQD in Alzheimer’s and
Parkinsons disease is represented Figure 2.4.

2.3 Drug adsorption on GQD and other carbon nanostructures
Drugs can be easily attached on carbon nanostructures such as MWNT, SWNT, GO,
rGO and GQD by adsorption reaction. Ketotifen, a second generation H 1 anti
histamine drug, can be adsorbed on MWNT, by adding 10ml of Ketotofen (50mg/L)
in to 0.01g of MWNT and stirred for 24h (123). Flutamide anticancer drug can be
adsorbed on SWNT surface by negative van der Waals interaction energy and a high
number of hydrogen bonds between drug and SWNT (124). Doxorubicin can be
adsorbed on covalently functionalized CNT, and it can carry the drug into specific
sites (125). Sodium diclofenac can be physically adsorbed on reduced graphene oxide,
and there is no chemical or structural change on rGO (126). Doxorubicin
hydrochloride can be adsorbed on GO by adding 5 mg GO into 10 mL solution of
DOX concentrations from 300 to 500 mg/L and shaken on a temperature-controlled
waterbath/shaker.(127,128).
2.4 Biocompatibility study of GQD and graphene derivatives
In-vitro and in-vivo biocompatibility and toxicity studies are performed for GQDs.
2.4.1 In-vitro biocompatibility studies of GQD
GQD can be synthesised by the decomposition of graphene oxide in dimethyl

formamide by solvothermal method, and the toxicity of these GQDs can be tested on
prostate cancer cell lines, DU-145 and PC-3. The maximum tolerable toxicity for DU-
145 cells was found to be as 100µg/mL. Below this level, there is no sign of cell
viability and above this level, the cell viability decreases. But, at a concentration of
400µg/mL, the viability still remains 80% and similar results are observed in PC-3
also. Thus GQDs are safe in both cell types (85). The interaction of GQDs with blood
cells such as monocytes and granulocytes can be studied. GQDs are taken into the
cells by caveolae endocytosis and accumulate in endoplasmic reticulum and nucleus.
Finally the study concluded that, GQDs doesn’t interfere the cell proliferation (100).
GQD can be synthesised from salicylic acid by free radical polymerization under
ultraviolet irradiation, and that can be used for in-vivo and in-vitro bio imaging.
OCM-1 cell line can be used to evaluate its in-vitro cytotoxicity. GQD concentration
from 0-500 µg mL-1 is treated with OCM-1 cell lines and the metabolic activity was
examined. After 24 hr incubation, cell viability was not affected even at higher

concentration (500 µg mL-1) (129). GQDs can be prepared by the exfoliation of

carbon fiber and it can be carboxylated by treating with citric acid. And the toxicity of
this carboxylated GQDs can be evaluated on different cell lines including KB, MDA-
MB231, A549 cancer cells, and MDCK normal cell line. Carboxylated GQDs are
easily taken by all the cells and no toxicity or morphological differences were
observed (130).
Blue fluorescence emitting GQDs can be synthesised from L-glutamic by its pyrolysis
at 220ºC. They are easily uptaken by KB cells without any toxicity, and are also non-
toxic in MDCK cells. Their haemolytic activity is tested by keeping different
concentrations of GQDs in red blood cells for 2h. Figure 2.5 shows SEM images of
red blood cells exposed to different concentrations of GQDs; the RBC remains as
such in control, and GQD exposed samples show cell membrane damage and cell-cell
fusion (131).
The membrane permeability of different sized GQDs can be evaluated; 3nm and 12
nm GQDs are used for the same. Small GQDs penetrate into Madin-Darby canine
kidney cells by lipid raft-mediated transcytosis (Wang et al., 2015). Similarly, the
penetration of GQD into human stem cells can be studied without affecting cell
viability and proliferation (76). In-vitro cytotoxicity of different concentrations of (0
to 500 µg/mL) selinium doped GQDs can be tested on HeLa cells. After 24 hr
incubation, the cell viability was evaluated, and there is no significant reduction in
cell viability (133).
2.4.2 In-vitro biocompatibility studies of graphene derivatives
The interaction of graphene nanoparticles with red blood cells is very important one;
the haemolytic activity is to be evaluated to ensure the safety. GO and graphene
sheets exhibits haemolytic activity in red blood cells but, GO is more potent than
graphene sheets. This is due to the electrostatic interaction between positively charged
phosphotidylcholine on RBC surface and negatively charged groups on GO surface.
Since graphene sheets have lower oxygen groups, it doesn’t induce haemolysis, but
leads to haemagglutinin. To reduce the haemolytic effect of GO, some techniques
have been developed; biocompatible chitosan coated GO can reduce the electrostatic
interaction and thus, haemolysis can be reduced (134).

Figure 2.5 SEM images of RBCs exposed to different concentrations of GQDs.

Reproduced with permission from (131). Copyright 2018, Elsevier.
Different concentrations of GO can be prepared and its biocompatibility is evaluated

on human fibroblast cells, and it induces dose dependent apoptosis (135). GO can be
synthesised by modified Hummer method, and the prepared GO suspension can be
centrifuged to collect both supernatant and sediment. In-vitro toxicity study of the
above prepared three samples of GO can be performed with A549 cell lines. The
study states that GO can increase ROS levels even at low concentrations without
affecting cell viability. Thus it can be concluded that, GO can induce oxidative stress
and leads to cell toxicity (136). Biocompatibility and cellular toxicity of GO in
intraocular level was performed and study reports that, there is no significant effect on
human retinal pigment epithelial cell morphology, viability, membrane integrity, and
apoptosis and thus GO have good intraocular biocompatibility (137).

GO and carboxylated graphene nanoplatelets can be treated with HepG2 cell lines, the
particles penetrates phospholipid bilayer of cell membrane and enter into the cell then
exhibits dose and time dependent ROS production (138). The use of reducing agents
such as hydrazine hydrate and hydroquinone for the synthesis of rGO leads to toxicity
(139). Toxicity of GO and rGO can be compared by culturing with human glioma cell
lines U87 and U118 and result states that, rGO is more toxic than GO (140). The
toxicity of N-GQD and graphene oxide on RBCs can be compared. The haemolytic
activity, morphological changes and ATP content of RBCs after the exposure can be
evaluated. Graphene oxide reduces membrane integrity due to lipid bilayer extraction
and leads to haemolysis, but N-GQDs show only structural changes on RBC
membrane and are found as aberrant shaped RBCs on microscopic examination
(Wang et al., 2015).
2.4.3 In-vivo biocompatibility studies of GQD
In-vivo studies are real animal study, which gives more accurate results than cell line
studies. Here, the biocompatibility studies are tested on mice, zebra fish, some
nematodes etc.
The toxicity of GQDs in rat lungs after intravenous administration can be evaluated
by histological examination of the lung tissue. The tissues can be examined for any
damages, pathological changes, inflammation or necrosis due to GQD, and no gross
changes or abnormalities were observed in lower concentration. At higher GQD
concentration, alveolar septa were thickened (142). Zebra fish Danio rerio can be
used for in-vivo toxicity studies of graphene. Polylactic acid and fluorescein o-
methacrylate functionalized graphene can be microinjected unto zebra fish embryo
and exhibits good bio distribution and there is no detectable toxicity or abnormalities
observed (143). The effect of prolonged exposure of N-doped GQDs on a nematode
starin Caenorhabditis elegans and its progeny can be evaluated. The locomotion
behaviour and number of offsprings are taken as evaluation parameters, and there is
no effect for the nematode locomotion after prolonged exposure and also non-toxic to
offsprings (144). Long term intravenous administration of carboxylated GQDs into
mice leads to accumulation in liver, lung, kidney, spleen and biochemical evaluation
of treated mice serum shows no significant toxicity (130).

The effect of GQD toxicity in male mouse reproduction and offspring health can be
investigated by oral gavage or intravenous administration. Then mouse housed with
female mouse and studied its reproductive health by evaluating testosterone level,
sperm motility, α-glucosidase activity in seminal plasma etc. After gestational period,
the mouse gave birth to healthy pups in three subsequent litters. Thus, GQD show less
toxicity to reproductive cells and no effects in reproductive system. Figure 2.6 gives a
schematic representation of the experiments and evaluation on the mouse (145).
Figure 2.6 Schematic representation of evaluation GQD toxicity in male mouse

reproduction and offspring health. (a) Administration of GQD and evaluation of
mouse sexual behaviours and offspring health. (b) Toxicity studies of GQD in male
mouse germ cells. Reproduced with permission from (145). Copyright 2019, Elsevier.

2.4.4 In-vivo biocompatibility studies of graphene derivatives
Administration of GO into mice leads to pulmonary toxicity, pulmonary edema

fibrosis, granulomatous lesions, and inflammatory cell infiltration, and all these
effects depends on the concentration of GO (146,147). Different concentrations of GO
can be administered into mice to evaluate its in-vivo toxicity. The results conclude
that, GO induces lung granuloma in mice and is found that toxicity is dose dependent
(135). GO induces certain cardio vascular problems, the study was performed in zebra
fish embryo. A t a concentration of 0.4-1 mg/ml, GO causes cardiotoxicity, increased
heart beat and some other cardiovascular defects (148). Intravenous administration of
GO into mice cause pulmonary thrombo embolism due to thrombogenic nature of GO
and also shows aggregation of human platelets (149). To study the intraocular
biocompatibility and cytotoxicity of GO, it is given into rabbit eye as intravitreal
injection. The injection didn’t cause any changes on eyeball appearance (137). Effect
of graphite nanoplatelets on nematode Caenorhabditis elegans was studied by taking
longevity and reproductive capacity as toxicity parameters. Different concentrations
of graphite nanoplatelets didn’t cause nematode death and then concluded that, there
is no acute toxicity (150). Toxicity of rGO can be evaluated in mice after single
intravenous administration; rGO didn’t show toxicity in mice hippocampus (151).
PEG functionalized rGO shows toxic effects in brain astrocytes and endothelial cells
in rat. PEG causes down regulation of some brain components and the production of
reactive oxygen species, that induces oxidative stress (152). An in-vivo comparative
toxicity study of GO and rGO can be performed and the results concluded that, both
rGO and GO can reduce the tumor volume and weight (140). The effects of GO
toxicity can also be tested on fresh water zooplankton Ceriodaphnia dubia. Both
acute and chronic exposure of GO affects their survival, and inhibits feeding and
reproduction, and GO accumulates in gut tract (153).

CHAPTER 3
AIM AND PLAN OF WORK

CHAPTER 3 A I M A N D P L A N O F W O R K | 36
3.1 Aim of the study
To develop a system for co-delivery of Graphene Quantum Dot and Rivastigmine

Tartarate (GQD-RT) and to evaluate its blood-brain barrier permeability.
3.2 Objectives of the study
 To prepare and evaluate GQD

 To prepare and evaluate GQD-RT
 To evaluate the blood brain barrier crossing of GQD-RT
3.3 Rationale of the study
3.3.1. Rationale for choosing Alzheimer’s disease
• Around 1.6 million people in India are suffering from Alzheimer’s. It is expected to
triple in number by 2050.
• According to reports of 2012, 40 million people are affected with Alzheimer’s in
world wide. And it is estimated that 81.1 million people will be affected by 2040
(154).
• The existing treatments are only symptomatic, and don’t treat the real cause of
disease and blood brain barrier crossing of the medication is difficult (154).
3.3.2. Rationale for Graphene Quantum Dots
• Graphene Quantum Dots are nano-sized, highly soluble, have the high surface area,
low cost and they can cross Blood Brain Barrier (BBB) (21,96).
• GQD itself has the capacity to prevent Amyloid β aggregation and they have low
cytotoxicity and great biocompatibility (Liu et al., 2013).
• GQDs can disaggregate α-synuclein in amygdala Lewy body (Kim et al., 2018).
• GQDs are highly fluorescent in nature and thus their presence inside the body can be
traceable (85).

3.3.3. Rationale for rivastigmine tartarate
• Acetylcholine esterase inhibitors such as rivastigmine can improve cognition and
dementia by inhibiting the degradation of Acetylcholine esterase (155).
3.4 Plan of work
The study is an attempt to develop and evaluate GQD-RT and to ensure its BBB
crossing and drug release into the brain. The research work was carried out in
following steps.
 Preparation and characterization of graphene quantum dots

 Transmission electron microscopy (TEM)
 Scanning electron microscopy (SEM)
 IR spectroscopy
 Zeta potential
 UV-Visible spectroscopy
 Fluorescence spectroscopy
 X-Ray Diffraction (XRD)
 Particle size distribution
 Characterization of RT
 Physical examination
 IR spectroscopy
 Preparation and Evaluation of GQD-RT
 FT-IR Spectroscopy
 TGA analysis
 HPLC
 In-vivo study

CHAPTER 4
MATERIALS AND METHODS

CHAPTER 4 M A T E R I A L S A N D M E T H O D S | 38
4.1 MATERIALS
A list of materials used for the research work is given in Table 4.1
Table 4.1 List of materials
Sl. No Materials Manufactures/Suppliers
1. L-glutamic acid Chemind Chemi Calicut

2. Rivastigmine tartarate Kerala State Drugs and
Pharmaceuticals Ltd, Alappuzha
3. Potassium dihydrogen Universal Chemical & Scientific
orthophosphate Industries Harippad
4. Sodium hydroxide Nice Chemicals Pvt.Ltd Cochin
5. Sodium citrate Nice Chemicals Pvt.Ltd Cochin
6. Ammonium acetate Nice Chemicals Pvt.Ltd Cochin
7. Acetonitrile Central Drugs House Pvt.Ltd New
Delhi

List of equipments and softwares used for the research work is given in Table 4.2.
Table 4.2 List of equipments and softwares used
Sl.No Equipment/Software Manufacturer/Supplier
1. UV Spectrophotometer SHIMADZU UV Spectrophotometer UV

1800
2. IR Spectrophotometer PerkinElmer Frontier FT-IR Spectrometer
3. X-ray diffractometer RIGAKU miniflux diffractometer
4. Fluorescence spectrometer PerkinElmer LS 45 Fluorescence
spectrometer
5. Freez drier SCANVAC CoolsafeTM
6. Thermogravimetric analyser PerkinElmer STA 6000
7. UFLC LC-20AD SHIMADZU
8. Refrigerated Centrifuge Laby Industry Instruments, Ambala,
Haryana
9. UV Lamp VILBER LOURMART SERIAL N
10. Electronic balance SHIMADZU AY 120, JAPAN
11. Muffle furnace KEMI FOURTECH
12. pH Meter EUTECH Instruments pH Tester 30
13. Magnetic stirrer REMI Laboratory instruments, Mumbai,
India
14. Zeta Sizer Malvern Instruments Ltd, Worcestreshire,
United kingdom
15. TEM Jeol-1011, Jeol India Pvt. Ltd,Mumbai,
India
16. Origin Pro 8 Origin Lab Corporation

4.2 ANALYSIS OF RIVASTIGMINE TARTARATE
4.2.1 Appearance
The drug was visually inspected for its colour, crystallinity and other physical
characteristics.
4.2.2 IR spectroscopy
ATR-IR Spectroscopy was measured using PerkinElmer Frontier FT-IR Spectrometer

and the spectrum obtained was compared with that obtained with reference spectrum
of RT (Bhatt et.,al 2016).
4.2.3 Determination of λmax of rivastigmine tartarate
λmax of RT was determined by scanning in pH 7.4 phosphate buffer at the wavelength

range of 200-400nm on a UV-visible spectrophotometer (SHIMADZU UV
Spectrophotometer UV 1800). The wavelength correspond to the maximum peak was
noted.
4.2.4 Preparation of calibration curve of rivastigmine tartarate
Preparation of pH 7.4 phosphate buffer
Dissolved 6.804g of potassium dihydrogen ortho phosphate and 1.564g of sodium

hydroxide in sufficient distilled water to 1000ml.
Preparation of stock solution
16 mg of RT equivalent to 10mg of rivastigmine was dissolved into 100ml pH 7.4

phosphate buffer. From this stock solution, 10ml was pipette and transferred into
another 100ml standard flask and volume was made upto 100ml to get a stock
solution of concentration 10,00µ/ml. From this solution, again 10ml was pipette and
transferred into another standard flask and made up the volume upto 100ml with pH
7.4 phosphate buffer to obtain a stock solution of concentration 100 µ/ml.
Preparation of RT standard plot in pH 7.4 phosphate buffer
For the preparation of calibration plot, 2, 4, 6 and 8ml of the final stock solution was
transferred into a series of 100ml standard flask and volume made upto 100ml with
pH 7.4 phosphate buffer to result solutions of concentration 20, 40, 60, 80 and

100µ/ml respectively. The absorbance of these samples was measured

spectrophotometrically at 263nm using UV spectrophotometer VILBER LOURMAT
GeNeiTM SERIAL N model.
4.3 SYNTHESIS OF GQDs
GQDs were synthesised from L-glutamic acid (LGA) by pyrolysis. 2g of L-glutamic

acid was weighed and transferred into a crucible and heated on a muffle furnace at
210℃. After 10-15 minutes, white crystalline powder of L-glutamic acid started
melting and then changed into brown viscous liquid, this indicates the formation of
GQDs. Then added 10ml of millipore water into the solution and stirred for 30
minutes. After cooling to room temperature, the yellowish solution was centrifuged at
13,000 rpm for 30 minutes and collected supernatant solution (157).
4.4 CHARACTERIZATION OF GQDS
4.4.1 Product yield
The prepared GQD solution was freez dried under -110℃ in SCANVAC CoolsafeTM
freez drier. And weight of the powder obtained was noted. Then calculated the yield
obtained from the following equation.
Weight of powder obtained after freez drying

Yield GQD ¿ * 100
Weight of L−glutamic acid taken
4.4.2 Fluorescence
The GQD solution was kept in front of the UV lamp VL-6.LC GeNei TM at 365nm and
254nm, and then observed for fluorescence.
4.4.3 Scanning Electron Microscopy (SEM)
The Scanning Elelctron Microscopic (SEM) images of GQDs were taken. The
obtained SEM image was compared with reported data.
4.4.4 High Resolution Transmission Electron Microscopy (HR-TEM)
The samples were sonicated for 15-20 min at room temperature; taken 10µl of GQD
and put over 400 mesh carbon coated grids and incubated for 3-5 min at room
temperature. Stained the grid with 2% UA and drained off excess of stain immediately
with the help of Whatman filter paper. The grids are observed under Jeol-1011

transmission electron microscope at 80KV at different magnification and images were

captured with SIS OLYMPUS MEGAVIEW G2CCD camera attached with the
microscope.The obtained TEM image was compared with reported data.
4.4.5 Particle size distribution and poly dispersity index
The particle size distribution and poly dispersity index (PDI) of GQDs were measured
by photon correlation spectroscopy, using Malvern Zeta sizer version 7.13.
4.4.6 Zeta potential
The zeta potential of prepared GQDs was measured after suitable dilution with
distilled water using Malvern zeta sizer version 7.13 at 25℃.
4.4.7 UV-Visible spectroscopy
The GQD solution was scanned between the wavelengths of 200-400nm

(SHIMADZU UV Spectrophotometer UV 1800) to determine the absorption maxima.
The recorded spectrum was compared with reported data.
4.4.8 Fluorescence spectroscopy
The fluorescence spectra, both the excitation and emission spectra of prepared GQD
were recorded with PerkinElmer LS 45 Fluorescence spectrometer.
4.4.9 IR-spectroscopy
ATR-IR spectroscopy of GQD was taken from PerkinElmer Frontier FT-IR

Spectrometer and compared the obtained spectrum with standard GQD spectrum.
4.4.10 X-Ray Diffraction (XRD)
The freez dried white crystalline powder of GQD was subjected to XRD. And the data
were analyzed.

4.5 PREPARATION OF GQD-RT
The drug : carrier ratio was selected as 1:3, hence 54µg/ml of RT was mixed with
162µg/ml of GQD.
4.5.1 Preparation of 54µg/ml of RT
86.4 mg of RT equivalent to 54 mg of rivastigmine was weighed, and transferred into

100ml standard flask and made up to 100 ml with7.4 pH phosphate buffer. From this
solution, 1ml was pipette out and made up to 10ml to get 54µg/ml of RT.
4.5.2 Preparation of 162µg/ml of GQD
The concentration of freshly prepared GQD was calculated as 1800µg/ml. From this
solution, 0.9ml was pipette out and made up to 10 ml with Millipore water to get
162µg/ml GQD.
Both the solutions were mixed together and shaked thoroughly for 30 minutes on a
magnetic stirrer to get a GQD-RT complex.
4.6 CHARACTERIZATION OF GQD-RT
ATR-IR spectroscopy of GQD-RT was taken from PerkinElmer Frontier FT-IR

Spectrometer and the obtained spectrum was compared with the spectrum of GQD
and RT.
4.6.2 Thermogravimetric analysis
Thermogravimetric analysis (TGA) of GQD and GQD-RT was performed on

PerkinElmer STA 6000. Initial weight of samples were noted and then heated up to
600℃ at a rate of 20℃ per minute. The thermogram of GQD was compared with
thermogram of GQD-RT.
4.6.3 Diffusion study
The conjugation or adsorption of RT on GQD surface can be evaluated by a simple

diffusion study set up. Butter paper was used as diffusion membrane and 7.4 pH
phosphate buffer as diffusion medium (Figure 4.1). RT solution was prepared at a
concentration of 54µg/ml and 2ml of this solution was pipette out into diffusion tube

and kept in beaker containing 20 ml of pH 7.4 phosphate buffer. The diffusion cell
was maintained at small agitation and continued for 1 h. Similar procedure was
followed for GQD-RT; 2ml of GQD-RT solution (162:54µg/ml) was added to
diffusion tube. After 1 h, 5ml of sample was withdrawn from both receptor
compartments and analysed the concentration of RT using UV-Visible spectroscopy
at 263nm.
Figure 4.1 Diffusion study set up for RT and GQD-RT

4.7 IN-VIVO EVALUATION OF BBB PERMEABILITY
The protocol for animal studies was approved (Proposal No. 330/2019) in 36th
Institutional Animal Ethics Committee (IAEC) meeting held on March 7 th at JSS
College of Pharmacy, Mysuru – 570015, Karnadaka. (155/PO/Re/S/99/CPCSEA)
The BBB permeability of GQD-RT can be evaluated on Wistar rats. The animals were
divided into three groups and each group containing 4 rats. The group 1 were given
RT, group 2 were given GQD and group 3 were administered with GQD-RT mixture
(Table 4.3).
Table 4.3 Outline of in-vivo study

GROUPS NO. OF TREATMENT DOSE & ROUTE EVALUATION
ANIMALS
01 04 RT, 0.27 mg/kg, 0.5ml, Tail vein
02 04 GQD, 0.81 mg/kg,0.5ml, Tail vein Brain and Plasma
03 04 GQD-RT , 0.5ml, Tail vein
HPLC
RT was given in dose equivalent to 0.27mg/kg body weight and GQD was at
0.81mg/kg. The samples were administered (0.5ml) intravenously into the tail vein of
Wistar rats according to their body weights. Blood samples (0.5ml) were collected
from tail vein of three animals from each group at 0.5, 1, 2 and 4 h. The blood
samples were mixed with 4µL 0f 3.8% sodium citrate solution and immediately
centrifuged at 4000 rpm for 15 min. The plasma were separated and kept frozen until
analysis. Then the animals were euthanized and separated their brain at 0.5, 1, 2 and 4
h. The brains were weighed and crushed and then suspended in 10ml of phosphate
buffer pH 7.4, and centrifuged at 4000rpm for 15 min and separated the supernatant
solution. RT and GQD content in the plasma and brain at selected time points were
determined using high-performance liquid chromatography (HPLC) (158,159).

4.7.1 High Pressure Liquid Chromatography (HPLC)
The HPLC method for determination of RT and GQD was carried out on a Shimadzu
UFLC (Shimadzu scientific instruments Inc., Kyoto, Japan) using photo diode array
(PDA) detector with column oven. The instrument was controlled by use of LC
solutions software installed with equipment for data collection and acquisition.
Compounds were separated on a Phenomenex C18 column (250 X 4.6 mm, 5μ ID).
4.7.2 Development of standard chromatogram of RT by HPLC
A stock solution of RT was prepared in HPLC grade methanol. From the stock
solution, standard solution of 10µg/ml was prepared. The solution was filtered
through 0.22µm syringe filter. The filtered solution was then filled into sample
container. Then HPLC was performed, and a chromatogram was obtained.
Chromatographic conditions for RT
Column : Phenomenex C18 column (250 X 4.6 mm, 5μ ID)

Flow rate : 1ml/min
Retention time : 4.5 min
Detector : PDA detector
Detection wavelength : 254nm
Injection volume : 10μl
Run time : 10 min
4.7.3 Development of standard chromatogram of GQD by HPLC
A stock solution of GQD was prepared in Millipore water. From the stock solution,
standard solution of 64µg/ml was prepared by dilution. The solution was filtered
through 0.22µm syringe filter. The filtered solution was then filled into sample
container. Then HPLC was performed, and chromatogram was obtained.

Chromatographic conditions for GQD
Column : Phenomenex C18 column (250 X 4.6 mm, 5μ ID)

Flow rate : 1ml/min
Retention time : 2.2 min
Detector : PDA detector
Detection wavelength : 254nm
Injection volume : 10μl
Run time : 10 min
Preparation of mobile phase
Acetonitrile and 20mM Ammonium Acetate (26:74, v/v) was used as mobile
phase.770mg of ammonium acetate was taken in 500mL of Millipore water, then
degassed and passed through a membrane filter to obtain 20mM ammonium acetate.
4.7.4 Analysis of blood samples by HPLC
Blood samples (0.5ml) were collected from each group after 0.5, 1, 2 and 4 th h sample
of injection, and mixed with 4µL 0f 3.8% sodium citrate solution and immediately
centrifuged at 4000 rpm for 15 min. The plasma were separated and subjected to
HLPC analysis and the concentration of GQD and RT in blood were analysed.
4.7.5 Analysis of brain samples by HPLC
Brains were separated at selected time intervals (0.5, 1, 2 and 4h) from each group,
added phosphate buffer and centrifuged and supernatant clear solution were collected.
HPLC of each samples were performed, chromatograms were analysed and calculated
the.concentration.of.GQD.and.RT.

CHAPTER 5 R E S U L T S A N D D I S C U S S I O N | 48
CHAPTER 5
RESULTS AND DISCUSSION

5.1 ANALYSIS OF RIVASTIGMINE TARTARATE
5.1.1 Appearance
RT was found as a hygroscopic white to off-white fine crystalline powder.
Figure 5.1 Rivastigmine

tartarate powder
The IR spectra of pure RT showed characteristic peaks at 3417 (N-H stretching),

3047(C-H stretching), 1702 (C=C stretching) and 1405 (C-O stretching) (Figure 5.2).
The spectrum obtained was in agreement with the reported data (156,160).
Figure 5.2 ATR-IR Spectrum of rivastigmine tartarate

5.1.3 Determination of λmax of rivastigmine tartarate
The UV spectrum of RT in pH 7.4 phosphate buffer shows a characteristic peak at

263nm (Figure 5.3)
Figure 5.3 UV Spectrum of RT
5.1.4 Preparation of calibration curve of RT
Calibration curve for RT in 7.4 pH phosphate buffer by UV-Visible spectroscopy at

263nm is shown in Figure 5.4 and the data is given in Table 5.1
Table 5.1 Calibration curve

data of RT
Concentration Absorbance
(µg/ml)
00 00
20 0.0306
40 0.0586
60 0.0851
80 0.1143
100 0.1361

Figure 5.4 Calibration curve of RT in 7.4 pH phosphate buffer at 263nm
5.2 SYNTHESIS OF GQDS
The brown viscous liquid turned to yellow coloured clear solution on addition of
water. And the solution was then stirred and centrifuged to form GQDs and stored at
room temperature on glass bottle. Figure 5.5 shows synthesis of GQDs.
Figure 5.5 Synthesis of GQDs from L-glutamic acid

5.3 CHARACTERIZATION OF GQDS
5.3.1 Product yield
Weight of GQD powder after freez drying = 1.76g
Weight of L-glutamic acid taken = 2g
Weight of powder obtained after freez drying

Yield of GQD = *100
Weight of L−glutamic acid taken
The yield of GQDs was found to be 88% .
5.3.2 Fluorescence
The prepared GQD solution was kept in front of the UV lamp at 365nm and 254nm, a
bright blue fluorescence was observed at 365nm (Figure 5.6). And the precursor
glutamic acid under UV lamp doesn’t show any fluorescence (Figure 5.7).
Figure 5.6 GQD under visible light and UV lamp

Figure 5.7 L-Glutamic acid solution under UV lamp.
5.3.3 Scanning Electron Microscopy (SEM)
Scanning electron microscopic images of prepared GQDs are shown in Figure 5.8.
Figure 5.8 Scanning Electron Microscopic (SEM) image of GQDs

5.3.4 High Resolution- Transmission Electron Microscopy (HR-TEM)
High Resolution- Transmission Electron Microscopic (HR-TEM) images of prepared

GQDs are shown in Figure 5.9 (157).
Figure 5.9 High Resolution- Transmission Electron Microscopic (HR-TEM) images

of GQDs
5.3.5 Particle size distribution and polydispersity index
The particle size and polydispersity index of GQD was found to be 562.4nm and
0.802 respectively, and the plot is shown in Figure 5.10. Since polydispersity index is
above 0.7, it indicates a very broad distribution of particle sizes. And a higher particle
size might be due to aggregation of GQDs in water.

Figure 5.10 Particle size distribution plot of GQD
5.3.6 Zeta potential
The zeta potential of GQD was found to be 1.08mV and the plot is shown in Figure
5.11.

Figure 5.11 Zeta potential distribution plot of GQD

5.3.7 UV-Visible spectroscopy
The UV-Visible spectrum of GQD in water shows a characteristic peak at 220nm. The
recorded spectrum was similar to reported data (58). Figure 5.12
Figure 5.12 UV Visible spectrum of GQD
5.3.8 Fluorescence spectroscopy
Excitation spectra
The fluorescent excitation spectrum of prepared GQD was recorded. Three peaks
were observed at 248, 282 and 343nm and the intense peak was at 343nm (Figure
5.13). Hence maximum fluorescence emission is occurring at this wavelength region
of excitation. The recorded spectrum was similar to reported data (157). Image was
drawn from Origin Pro 8 Software.

Figure 5.13 Excitation spectrum of GQD
Emission spectra
The emission spectrum of prepared GQD was recorded at excitation wavelength of

343 nm, and maximum fluorescence emission was observed at 430nm (Figure 5.14).
The recorded spectrum was similar to reported data (157). Image was drawn from
Origin Pro 8 Software.
Figure 5.14 Emission spectrum of GQ

ATR-IR spectrum of GQD and L-glutamic acid were obtained. To confirm the
formation of GQD, ATR-IR spectra of GQD was compared with L-glutamic acid
(Figure5.15). The strong peak at 1665cm-1 resulted from the combination of the
carboxylic acid C=O and amide C=O stretches in the GQDs. C=C stretching of
graphite was formed as indicated by the appearance of the 2930 cm -1 peak, which
demonstrated the formation of GQDs. The recorded spectrum was in agreement with
the reported data (157). Image was drawn from Origin Pro 8 Software.
Figure 5.15 Comparison of ATR-IR Spectra of GQD and LGA
5.3.10 X-Ray Diffraction (XRD)
The XRD of lyophilized GQD was performed and the XRD profile shows (Figure
5.16) a peak centered at 22.0 ⁰, which is similar to reported data (58).
Figure 5.16 XRD pattern of GQD

5.4 CHARACTERIZATION OF GQD-RT
ATR-IR spectrum of GQD-RT is shown in Figure 5.17 which shows two peaks at
3312.98 and 1636 cm-1. And those peaks were similar to peaks in GQD and RT.
Figure 5.17 ATR-IR spectrum of GQD-RT
5.4.2 Thermogravimetric analysis
The thermogravimetric analysis of GQD and GQD-RT were carried out to detect any
adsorption or conjugation reaction between GQD and RT. The thermograms of GQD
and GQD-RT (Figure 5.18) were almost similar, and no additional peaks were
observed in GQD-RT, and that might be due to the absence of adsorption of RT on
GQD surface.
Figure 5.18 Thermograms of GQD and GQD-RT

5.4.3 Diffusion study
Calibration curve for RT in 7.4 pH phosphate buffer by UV-Visible spectroscopy at

263nm is shown in Figure 5.19 and the data is given in Table 5.2
Table 5.2 Calibration curve

data of RT
Concentration Absorbance
(µg/ml)
00 00
20 0.0306
40 0.0586
60 0.0851
80 0.1143
100 0.1361
Figure 5.19 Calibration plot of RT in 7.4 pH phosphate buffer
Samples were withdrawn from both compartments after 1 h diffusion and analysed
using UV-Visible spectroscopy at 263nm and concentration was calculated from the
calibration plot.

Absorbance of RT after 1 h in RT receptor compartment= 0.0209
Concentration of RT from calibration plot was found to be =18.9µg/ml
Absorbance of RT after 1 h in GQD-RT receptor compartment= 0.0221
Concentration of RT from calibration plot was found to be = 20.1µg/ml
If RT have adsorbed on GQD surface, the amount of RT diffused out from GQD-RT
filled tube will be small. Both RT and GQD-RT doesn’t show significant variation in
rivastigmine concentration. That might be due to inability of RT to adsorb on GQD
surface.
5.5 IN-VIVO EVALUATION OF BBB PERMEABILITY
The BBB permeability of RT with GQD was studied on Wistar rats. The
concentration of RT in plasma and brain sample was detected using high pressure
liquid chromatography (HPLC).
5.5.1 Standard chromatogram of RT by HPLC

The standard chromatogram 10µg/ml of RT of is shown in Figure 5.20 and the peak
area was found to be 781310.
mAU
254nm4nm (1.00)
4.491
80
70
60
50
40
30
20
10
-10
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.20 Chromatogram of 10µg/ml RT

5.5.2 Standard chromatogram of GQD by HPLC

The standard chromatogram 64µg/ml of GQD of is shown in Figure 5.21 and the peak
area was found to be 123729.
mAU
27.5 254nm,4nm (1.00)
2.683
25.0
22.5
20.0
17.5
15.0
12.5
10.0
7.5
2.863
5.0
2.336
2.5
3.360
0.0
-2.5
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min
Figure 5.21 Chromatogram of 64µg/ml GQD
5.5.3 Analysis of blood samples

Blood samples (0.5ml) were collected from each group at 0.5, 1, 2 and 4 h time
intervals and added anticoagulant and centrifuged. The separated plasma was
subjected to HPLC on Ultra-Fast Liquid Chromatography Shimadzu LC-20AD.
Figure 5.22 shows plasma samples after RT treatment at different time intervals.
Figure 5.22 Plasma collected at different time

intervals after RT injection

Analysis of blood samples: Group 1 (IV administration of RT)

Blood samples were collected from RT treated Wistar rats at 1h and 4 hr, and the
concentrations were calculated from the peak area and are given in Table 5.3.
Sample 1 (1h)
The blood sample after 1 hour intravenous administration of RT was analysed by
HPLC and the chromatogram is shown in Figure 5.23.
mAU
11 254nm4nm (1.00)
10
4
2.813
-1
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.23 HPLC chromatogram of RT in blood sample after 1h

The peak area was found to be 10002 and the concentration of RT in blood after 1h
was found to be 0.128µg/ml.
Sample 2 (4h)
The blood sample after 4 hour intravenous administration of RT was analysed by
mAU
254nm4nm (1.00)
3
2.819
-1
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.24 HPLC chromatogram of RT in blood sample after 4h

The peak area was found to be 4940 and the concentration of RT in blood after 4h was
found to be 0.063µg/ml.
Table 5.3 Concentration of RT in blood of RT

treated rats at different time intervals
Time (h) Concentration of RT in
blood (µg/ml)
1 0.128
4 0.063
The data shows that, the concentration of RT in blood decreases in with time and that
may be due to the distribution of drug into various organs.
Analysis of blood samples: Group 2 (IV administration of GQD)
Blood samples were collected from GQD treated Wistar rats at 0.5, 1, 2 and 4 h, and
the concentrations were calculated from the peak area and are given in Table 5.4.
Sample 1 (0.5h)
The blood sample after 0.5h intravenous administration of GQD was analysed by
mAU
8 254nm4nm (1.00)
3
2.821
-1
-2
-3
-4
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.25 HPLC chromatogram of GQD in blood sample after 0.5h
The peak area was found to be 6563 and the concentration of GQD in blood after 0.5h

Sample 2(1h)
The blood sample after 1 h intravenous administration of GQD was analysed by
mAU
17.5 254nm4nm (1.00)
15.0
12.5
10.0
7.5
5.0
2.799
2.5
0.0
-2.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.26 HPLC chromatogram of GQD in blood sample after 1h
The peak area was found to be 6399 and the concentration of GQD in blood after 1 h
Sample 3 (2h)
HPLC and the chromatogram is shown in Figure 5.27
mAU
254nm4nm (1.00)
3
2.821
-1
-2
-3
-4
-5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min

Sample 4 (4h)
HPLC and the chromatogram is shown in Figure 5.28
mAU
254nm4nm (1.00)
7.5
5.0
2.818
2.5
0.0
-2.5
-5.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Table 5.4 Concentration of GQD in blood of

GQD treated rats at different time intervals
Time (h) Concentration of GQD
in blood (µg/ml)
0.5 3.39
1 3.30
2 3.19
4 2.94
The data shows that, the concentration of GQD in blood decreases in with time and
that may be due to the distribution of GQD into various organs.

Analysis of blood samples: Group 3 (IV administration of GQD-RT)

Blood samples were collected from GQD-RT treated Wistar rats at 0.5, 1, 2 and 4 hr,
and the concentrations of GQD and RT were calculated from the peak area and are
given in Table 5.5.
Sample 1 (0.5h)
The blood sample after 0.5h of intravenous administration of GQD-RT was analysed
by HPLC and the chromatogram is shown in Figure 5.29.
mAU
254nm4nm (1.00)
4.5
4.0
3.5
3.0
2.5
2.815
2.0
1.5 6.438
1.0
0.5
0.0
-0.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.29 HPLC chromatogram of GQD-RT in blood sample after 0.5h
The concentration of GQD and RT was calculated from standard chromatograms. The
peak area of GQD was found to be 5601and the concentration of GQD in blood after
0.5h was found to be 2.81µg/ml. The peak area of RT was found to be 13014and the
concentration of RT in blood after 0.5h was found to be0.166µg/ml.
Sample 2(1h)
The blood sample after 1 h intravenous administration of GQD-RT was analysed by

mAU
254nm4nm (1.00)
8
2.823
2
6.427
1
-1
-2
-3
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.30 HPLC chromatogram of GQD-RT in blood sample after 1 h
peak area of GQD was found to be 5368 and the concentration of GQD in blood after
1 h was found to be 2.77µg/ml. The peak area of RT was found to be 12315 and the
concentration of RT in blood after 1 h was found to be 0.157µg/ml.
Sample 3 (2h)
The blood sample after 2h intravenous administration of GQD-RT was analysed by
mAU
254nm4nm (1.00)
2.829
1.50
1.25
1.00
6.443
0.75
0.50
0.25
0.00
-0.25
-0.50
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min

Sample 4 (4h)
The blood sample after 4h intravenous administration of GQD-RT was analysed by
mAU
254nm,4nm (1.00)
4.5
4.0
3.5
3.0
2.831
2.5
2.0
1.5
1.0
6.436
0.5
0.0
-0.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min

Table 5.5 Concentration of GQD and RT in blood of GQD-RT

treated rats at different time intervals
Time (h) Concentration of GQD in Concentration of
blood (µg/ml) RT in blood
(µg/ml)
0.5 2.81 0.157
1 2.77 0.166
2 2.36 0.171
4 2.20 0.137
The data shows that, the concentration of GQD and RT in blood decreases in with
time and that may be due to their distribution into various organs

5.5.4 Analysis of brain samples

The animals were euthanized and separated their brain at 0.5, 1, 2 and 4 h. Figure 5.33
shows separated brain of Wistar rat. The brains were weighed and crushed and then
suspended in 10ml of phosphate buffer pH 7.4, and centrifuged at 4000rpm for 15 min
and separated the supernatant solution.. The supernatant solution was subjected to
HPLC on Ultra-Fast Liquid Chromatography Shimadzu LC-20AD.
Figure 5.33 Separated

brain of Wistar rat
Analysis of brain samples: Group 1 (IV administration of RT)

Brain samples were collected after RT treatment at specific time intervals, crushed
and suspended in phosphate buffer, and then centrifuged and collected clear
supernatant solution (Figure 5.34).
Figure 5.34 a) Crushed brain samples of Wistar rats after RT injection at different
time intervals b) Supernatent solution of same brain samples after centrifugation.

The HPLC chromatogram of RT in brain sample is shown in Figure 5.35. No peak

was detected for RT at the selected time intervals. The result indicates that, RT can’t
cross BBB.
mAU
22.5 254nm,4nm (1.00)
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
-2.5
-5.0
-7.5
-10.0
-12.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.35 HPLC chromatogram of RT in brain sample
Analysis of brain samples: Group 2 (IV administration of GQD)

Brain samples were collected from GQD treated Wistar rats at 0.5, 1, 2 and 4 h, and
the concentrations of GQD was calculated from the peak area and are given in Table
5.6.
Sample 1(0.5h)
The brain sample after 0.5h intravenous administration of GQD was analysed by
mAU
8 254nm4nm (1.00)
3
2.821
-1
-2
-3
-4
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.36 HPLC chromatogram of GQD in brain sample after 0.5h

The peak area was found to be 6350and the concentration of GQD in brain after 30
minutes was found to be 3.28µg/ml.
Sample 2 (1h)
The brain sample after 1h intravenous administration of GQD was analysed by HPLC
and the chromatogram is shown in Figure 5.37.
mAU
17.5 254nm4nm (1.00)
15.0
12.5
10.0
7.5
5.0
2.799
2.5
0.0
-2.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.37 HPLC chromatogram of GQD in brain sample after I h

The peak area was found to be 6148 and the concentration of GQD in brain after 1h
Sample 3(2h)
The brain sample after 2 h intravenous administration of GQD was analysed by HPLC
mAU
254nm4nm (1.00)
3
2.821
-1
-2
-3
-4
-5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.38 HPLC chromatogram of GQD in brain sample after 2h

Sample 4 (4h)
The brain sample after 4h intravenous administration of GQD was analysed by HPLC
mAU
254nm4nm (1.00)
7.5
5.0
2.818
2.5
0.0
-2.5
-5.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.39 HPLC chromatogram of GQD in brain sample after 4h
Table 5.6 Concentration of GQD in brain of

GQD treated rats at different time intervals
in brain (µg/ml)
0.5 3.28
1 3.18
2 2.98
4 2.74
The data show that, the maximum concentration of GQD in brain was found to be at
initial time (0.5h) and then the concentration decreases due to it elimination.

Analysis of brain samples: Group 3 (IV administration of GQD-RT)

Brain samples were collected from GQD-RT treated Wistar rats at 0.5, 1, 2 and 4 h,
and HPLC was performed and the chromatograms are given below.
Sample 1 (0.5h)
The brain sample after 0.5h intravenous administration of GQD-RT was analysed by
HPLC and the chromatogram is shown in Figure 5.40. The chromatogram shows only
the peak for G QD. The peak area was found to be 5379 and the concentration of
GQD in brain after 0.5h was found to be 2.78µg/ml.
mAU
8 254nm4nm (1.00)
3
2.821
-1
-2
-3
-4
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
Figure 5.40 HPLC chromatogram of GQD-RT in brain sample after 0.5h
Sample 2 (1h)
The brain sample after 1h intravenous administration of GQD-RT was analysed by
the peak for GQD. The peak area was found to be 5156 and the concentration of GQD
in brain after 1h was found to be 2.66µg/ml.

Figure 5.41 HPLC chromatogram of GQD-RT in brain sample after 1h
Sample 3 (2h)
mAU
254nm4nm (1.00)
7.5
5.0
2.818
2.5
0.0
-2.5
-5.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min

Sample 4 (4h)


mAU
17.5 254nm4nm (1.00)
15.0
12.5
10.0
7.5
5.0
2.799
2.5
0.0
-2.5
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
The concentration of GQD at different time intervals is given in Table 5.7. The data
show that, when GQD-RT is given to the rats, only GQD enters in to the brain and the
rivastigmine couldn’t cross BBB.
Table 5.7 Concentration of GQD in brain of

GQD-RT treated rats at different time intervals
in brain (µg/ml)
0.5 2.78
1 2.66
2 2.29
4 2.12

CHAPTER 6
SUMMARY AND CONCLUSION

CHAPTER 6 S U M M A R Y A N D C O N C L U S I O N | 76
Alzheimer’s is a neurodegenerative disorder characterized by dementia, and its

pathophysiology includes aggregation of amyloid beta peptide in brain and reduced
level of acetylcholine is also observed. The current treatment system for Alzheimer’s
include administration of acetylcholine esterase inhibitors; but the major challenge
exists with this is the inability of these drugs to cross blood-brain barrier (BBB). The
development of nanotechnology could solve this problem by introducing variety of
nanoparticles to carry drugs and to cross BBB. In this study, we used a novel
biocompatible nanocarrier, graphene quantum dots (GQD) that can cross BBB. Many
studies proved that, drugs can be adsorbed on the surface of GQD and they can act as
excellent carriers for drug delivery. Focussing the ability of GQD in preventing
amyloid beta aggregation can promise a new hope in Alzheimer’s treatment.
Here we made an attempt to prepare a conjugate of GQD and rivastigmine tartarate

(RT) to achieve a synergistic effect in Alzheimer’s treatment. Thus, we prepared
GQD from L-glutamic acid and mixed with RT at 3:1 ratio. But the characterization
studies of GQD-RT showed that, RT couldn’t adsorb on the surface of GQD, due to
the presence of tertiary amine groups in RT. However we proceeded with the in-vivo
animal studies with Wistar rats to check the BBB permeability of the GQD-RT
mixture by HPLC analysis of brain samples. The HPLC chromatograms of three
groups were evaluated and the results showed that, RT treated animal’s brain samples
didn’t show peaks and that indicates the inability of RT to cross BBB. The
chromatograms of GQD treated animal’s brain samples indicated that, the presence of
GQD in brain. And finally, the GQD-RT chromatograms were evaluated, and which
showed only the peaks for GQD. That indicates the inability of RT to cross the BBB
when given with GQD.
The study result shows that, RT couldn’t adsorb on GQD surface, and failed to cross
BBB. Thus, detailed study in chemical conjugation reactions are needed for the
successful conjugation of RT on GQD. And this study opens a new horizon to the
researchers and it can be modified in future with new ideas and methods for
conjugation and that can create a new revolution in treatment of Alzheimer’s disease.
CHAPTER 6 S U M M A R Y A N D C O N C L U S I O N | 77
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A N N E X U R E | 95
ANNEXURE
Preparation of pH 7.4 Phosphate buffer
6.8045g of potassium dihydrogen orthophosphate and 1.564g sodium hydroxide

pellets were dissolved in distilled water, and made up to 1000ml.
Preparation of 3.8% sodium citrate
3.8% sodium citrate was weighed and transferred into standard flask, and made up to
100ml with distilled water.

A N N E X U R E | 96

Final Thesis Henna

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Final Thesis Henna

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M.

Thesis submitted to the Kerala University of Health Sciences in

Under the guidance of

I hereby declare that this dissertation entitled “Co-delivery of rivastigmine

This is to certify that the dissertation entitled “Co-delivery of rivastigmine and

This is to certify that the dissertation entitled “Co-delivery of rivastigmine and

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

I am deeply indebted to my guide Dr. Pramod K, Assistant professor,

I express my sincere thanks to Dr. Jose John Mallikasseri, Head of the

I am grateful and fortunate enough to get constant encouragement,

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

I also thank my PG mates Riya Raphey. V and Nivitha.K.P, for their

I also remember my friends Junaid Abdul Majeed. MP, Lubna. T.P,

The warm thanks to my husband Abdussalam. M who supported me in

I can’t conclude my words without thanking my parents and other family

I am also grateful to others, whose name I didn’t mentioned here who

Finally, I understand the word ‘thank you’ is not enough to express my

Thanks for all your encouragement.

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

Alzheimer’s is an age related neurodegenerative disorder characterised by dementia,

Key words: Graphene quantum dot; Rivastigmine tartarate, Alzheimer’s disease;

3 AIM AND PLAN OF WORK 36

4 MATERIALS AND METHODS 38

5 RESULTS AND DISCUSSION 48

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

6 SUMMARY AND CONCLUSION

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

GQD Graphene Quantum dot

EMR Electromagnetic radiation

ROS Reactive oxygen species

PDA Photo diode array

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

1.1 Alzheimer’s disease

Pathologically it is characterized by intracellular neurofibrillary tangles and

1.2 Blood Brain Barrier

Blood-brain barrier (BBB) is the selectively permeable protective layer surrounding

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

Figure 1.1. Representation of formation of amyloid beta fibrils and development of

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

1.2.1 Mechanism of BBB crossing

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

moves from brain back into systemic circulation to prevent accumulation of

Figure 1.2. Representation of different pathways for BBB crossing of some

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

1.2.2 Factors influencing BBB crossing

1.2.3 Approaches for BBB crossing

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

The advancement in nanotechnology could solve these kinds of problems by

1.3 Carbon nanostructures

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

structures. Carbon nanofamily consists of different carbon based structures including

Graphene is an allotrope of carbon, discovered by Geim and Novoselov in 2004, from

1.3.2 Graphene quantum dots

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

GQDs are carbon-based zero-dimensional fluorescent nanomaterials with a graphene

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD

1.3.3 Biomedical applications of graphene quantum dots

Some special characteristics and activities of GQDs are utilized in medical

GQD as an anti-diabetic agent

GQD in hepatitis and wound healing

Immune-mediated fulminant hepatitis is the impairment of hepatic functions due to

COLLEGE OF PHARMACEUTICAL SCIENCES GOVT. MEDICAL COLLEGE, KKD