Abstract

Lung cancer remains one of the leading cause of cancer related mortality worldwide,
accounting for approximately 2.2 million new cases and 1.8 million deaths in 2022 according
to GLOBOCAN. In spite of the improvements in diagnostic tools and treatment methods,
the outcomes remain poor, primarily due to late-stage diagnosis, high recurrence rates, and
the limitations of standard therapies such as chemotherapy and radiotherapy which are
accompanied by severe side effects. If left unaddressed, the global burden of lung cancer
may rise significantly by 2030. Nanotechnology-based approaches, particularly titanium
dioxide (TiO2) with its photocatalytic properties and ability to generate reactive oxygen
species under light irradiation induces tumor cell apoptosis. TiO2 nanoparticles will be
synthesized using a reverse microemulsion method, which allows precise control over
particle size, morphology, and homogeneity by confining nanoparticle formation within
nanoscale water droplets stabilized by surfactants. The photocatalytic performance of TiO2
nanoparticles can be significantly improved through doping. Zn-TiO2 shows improved
photocatalytic activity through bandgap narrowing, visible light activation and superior ROS
generation, whereas PEGylated TiO2 offers biocompatibility and stability, making them
potentially suitable for improving anti-cancer efficiency. Titanium (IV) isopropoxide will be
dissolved in ethanol and stirred. Zinc nitrate hexahydrate will be added under continuous
stirring, followed by the addition of distilled water. The resulting sol will be stirred until it
transforms into a gel, which will then be aged, filtered, and dried overnight. The dried
precursor will be calcined at to obtain crystalline Zn-doped TiO₂ nanoparticles. Also, TiO₂
nanoparticles will be added to a PEG 6000 solution and stirred at 750 rpm. To prevent a
thick PEG layer, a low concentration will be used. The PEG-coated nanoparticles will then
be centrifuged at 9000 rpm at room temperature, washed with distilled water, and dried.
After the synthesis of nanoparticles, various characterization techniques will be used such as
UVVis spectrophotometry, Fourier Transform Infra-red spectroscopy (FTIR) and X-ray
diffraction (XRD). After synthesis and characterization, MTT assay will be carried out to
evaluate their cytotoxicity and PDT efficacy. This study aims to evaluate and compare the
effectiveness of Zn-TiO2 and PEGylated TiO2 nanoparticles in lung cancer treatment,
focusing on photocatalytic efficiency, ROS generation, cytotoxicity against lung cancer and
overall biocompatibility.
Keywords: Lung carcinoma, Titanium dioxide nanoparticle, Reverse microemulsion,
Zndoping, PEGylation, Photosensitizer, Photodynamic therapy

i
 Abbreviations
A549: Human Lung Adenocarcinoma
BSA: Bovine Serum Albumin DNA:
Deoxyribonucleic Acid
DMEM-FBS: Dulbecco’s Modified Eagle Medium with Fetal Bovine Serum
FDA: Food and Drug Administration
Fe₂O₃: Iron(III) Oxide (Hematite)
FTIR: Fourier Transform Infrared Spectroscopy
GLOBOCAN: Global Cancer Observatory
IR: Infrared Radiation
MOx: Metal Oxide
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
MW: Molecular Weight
NP: Nanoparticle
NSCLC: Non-Small Cell Lung Carcinoma
OM: Optical Microscopy
PBS: Phosphate Buffered Saline
PDT: Photo Dynamic Therapy
PEG: Poly-Ethyl Glycol
RES: Reticuloendothelial System
ROS: Reactive Oxygen Species
SCLC: Small Cell Lung Carcinoma
SEM: Scanning Electron Microscopy
SHMP: Sodium Hexametaphosphate
TiO₂: Titanium Dioxide
TTIP: Titanium Tetra iso-propoxide
UV: Ultraviolet
Zn: Zinc
ZnO: Zinc Oxide
XRD: X-ray Radiation

ii
 Table of Content
Abstract.............................................................................................................................................. i
Abbreviations ................................................................................................................................... ii
List of Tables .................................................................................................................................... v
List of figures ................................................................................................................................... vi
Chapter 1 Introduction.................................................................................................................... 1
1.1 Background ................................................................................................................. 1
1.2 Statement of Problem .................................................................................................. 3
1.3 Objectives .................................................................................................................... 4
1.4 Hypotheses .................................................................................................................. 4
1.5 Rationale of the study .................................................................................................. 5
Chapter 2 Literature Review .......................................................................................................... 6
2.1 Lung carcinoma ........................................................................................................... 6
2.2 Nanoparticles in Cancer therapy ................................................................................. 6
2.2.1 Types of Nanoparticles......................................................................................... 6
2.2.2 Synthesis of Nanoparticles ................................................................................... 7
2.2.3 History of nanoparticles ..................................................................................... 10
2.3 TiO₂ nanoparticle properties and synthesis ............................................................... 11
2.3.1 TiO2 nanoparticle for cancer treatment ............................................................. 12
2.3.2 TiO2 nanoparticle dispersion ............................................................................. 13
2.4 Zn-doping .................................................................................................................. 13
2.4.1 Previous work on Zn-doping .............................................................................. 14
2.5 PEGylation ................................................................................................................ 14
2.5.1 Previous works on PEGylation .......................................................................... 15
2.6 Photodynamic therapy (PDT).................................................................................... 15
2.6.1 Previous work on Lung cancer treatment using PDT ........................................ 16
2.7 Photosensitizer .......................................................................................................... 16
2.7.1 Role of TiO2 as a Photosensitizer ...................................................................... 16
2.7.2 Previous work of TiO2 NPs as photosensitizer in PDT ..................................... 17
2.8 Characterization of nanoparticles .............................................................................. 17
2.8.1XRD analysis....................................................................................................... 17
2.8.2 Scanning electron microscopy ........................................................................... 18

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 2.8.3 UV-visible spectroscopy .................................................................................... 18
2.8.4 Fourier transform infrared spectroscopy (FTIR) analysis .................................. 18
2.8.5 MTT assay .......................................................................................................... 18
2.8.6 Zeta potential ...................................................................................................... 19
2.9 A549 Cell Culture ..................................................................................................... 19
Chapter 3 Materials and Methodology ........................................................................................ 20
3.1 Materials Required .................................................................................................... 20
3.1.1 Chemicals required ............................................................................................. 20
3.1.2 Instruments required ........................................................................................... 20
3.1.3 Apparatus used ................................................................................................... 20
3.1.4 Software used ..................................................................................................... 20
3.2 Methodology ............................................................................................................. 20
3.2.1 Work completed ................................................................................................. 20
[Link] Zn-doping of NPs ............................................................................................ 22
[Link] PEGylation of NPs .......................................................................................... 22
3.2.2 Work in progress ................................................................................................ 22
3.2.3 Remaining work ................................................................................................. 22
Chapter 4 Results and Discussion................................................................................................. 23
4.1 Results ....................................................................................................................... 23
4.1.1 Synthesis of TiO2 NPs ........................................................................................ 23
4.1.2 Characterization of NPs synthesized from microemulsion method ................... 23
4.2 Discussion ................................................................................................................. 26
Chapter 5 Conclusion and Further Work ................................................................................... 29
5.1 Conclusion ................................................................................................................. 29
Gantt Chart .................................................................................................................................... 30
Cost Estimation .............................................................................................................................. 31
References ....................................................................................................................................... 32

iv
 List of Tables
Table 1 1 Plants from which TiO2 NPs can be extracted [43,52,53,54] ........................... 11
Table 1 2: Bacteria mediated synthesis of TiO2 nanoparticles using various strains........ 12

v
 List of figures
Figure 4 1: Resulted TiO2 NPs .......................................................................................... 23
Figure 4 2: Zeta Potential graph showing stability of TiO2 NPs ...................................... 23
Figure 4.3 XRD of TiO2 nanoparticles…………………………………………………………..24
Figure 4.4: UV-spectroscopy of TiO2 nanoparticles…………………………………............25
Figure 4.5: FTIR of TiO2 nanoparticles…………………………………………………………25

vi
 Chapter 1 Introduction
1.1 Background
Lung cancer remains the leading cause of cancer mortality in men and women worldwide.
About 90% of lung cancer cases are caused by smoking and the use of tobacco products.
However, other factors such as radon gas, air pollution exposures, and chronic infections can
contribute to lung carcinogenesis. In addition, multiple inherited and acquired mechanisms
of susceptibility to lung cancer have been proposed.
A549 is a human lung adenocarcinoma cell line which was isolated from a 58-year-old
Caucasian male in 1972[1]. Lung cancer is divided into two broad histologic classes, which
grow and spread differently: small-cell lung carcinomas (SCLC) and non-small cell lung
carcinomas (NSCLC) [2].
Nanotechnology represents a revolutionary path for technological development that concerns
the management of material at the nanometer scale (one billion times smaller than a meter).
Nanotechnology literally encompasses the fabrication and application of chemical, physical,
and biological systems at scales ranging from individual molecules or atoms to submicron
dimensions, and also the integration of these resulting nanomaterials into larger systems. It
has the potential to change our perspectives and expectations and provide us with the
capability to resolve global issues [3]. In general, the size of nanoparticles spans the range
between 1 and 100 nm, and possess distinctly different physicochemical properties [4].
Depending on the composition, nanoparticles can be classified into carbon-based nps,
organic nps, metal nps, ceramic nps, semiconductor nps, polymeric nps, hybrid nps,
magnetic nps and lipid-based nps.
Microemulsions are clear, thermodynamically stable systems composed of oil, water, and
surfactants, often combined with a co-surfactant. They form spontaneously and contain very
fine droplets in the nanometer size range, which gives them a transparent appearance. Unlike
conventional emulsions, microemulsions do not require high energy for formation and
remain stable for long periods [5]. Because of their unique properties such as low interfacial
tension, high solubilization capacity, and improved stability, microemulsions are widely
used in pharmaceutical, cosmetic, and chemical applications. In drug delivery,
microemulsions are particularly important for enhancing the solubility and bioavailability of
poorly watersoluble drugs [6].
TiO2 is one of the most abundant and widely used metal oxide nanomaterial in the world
[7,8]. As an n-type semiconductor, with a band gap energy of 3.2–3.35 eV, depending on its

1
crystal phase, TiO2 acts as an effective photocatalyst during the photocatalytic process for
surface functionalization [9,10,11,12]. It has three crystalline structures: anatase, rutile, and
brookite. Anatase is the most common type and rutile is the most stable form, while brookite
is the rarest [13]. There is also amorphous TiO2, which is a non-crystalline form. TiO2 exists
in various nanostructures, such as nanoparticles, nanotubes, nanorods and nanowires,
nanofilms, nanosheets, and nanocoatings, with remarkable photocatalytic activity, which
attract scientists to develop potential technological applications in multidisciplinary fields
for industrial production [14].
Zinc (Zn) doping is an effective strategy widely used to tailor the physicochemical,
structural, optical, and electronic properties of semiconductor metal oxides. Because Zn²⁺
possesses an ionic radius comparable to many host-lattice cations, it can successfully
substitute into the crystal matrix or occupy interstitial positions without causing severe lattice
distortion. When incorporated into oxide hosts such as TiO₂, ZnO, Fe₂O₃, or other
semiconductor systems, Zn doping modifies defect chemistry, charge carrier concentration,
and band-gap energy. These lattice-level modifications directly influence key material
properties such as charge transport, recombination kinetics, catalytic performance, and
optical absorption [11,12]. Several studies report that Zn-doping introduces shallow donor
states or new localized energy levels within the band gap, enabling enhanced visible-light
absorption and reduced electron–hole recombination [12, 13]. This effect is highly beneficial
for photocatalytic, photo-electrochemical, and optoelectronic applications. In addition, Zn
dopants can stabilize specific crystal phases, promote smaller particle sizes, increase surface
hydroxyl groups, and improve surface reactivity. These changes collectively enhance
photocatalytic degradation of pollutants, antimicrobial activity, and charge separation
efficiency in Zn-doped nanomaterials [11,14].
PEGylation defines the modification of a protein, peptide or non-peptide molecule by the
linking of one or more polyethylene glycol (PEG) chains. This polymer is non-toxic,
nonimmunogenic, non-antigenic and highly soluble in water [15]. PEG copolymer
PEGylating on the surface of TiO2 NPs increases the hydrodipersity and biocompatible [16].
It helps nanoparticles escape the reticuloendothelial system (RES) and increase their half-
life in blood circulation for passive targeting of the anticancer drugs to the tumour cells [17].
Photodynamic therapy (PDT) is an emerging treatment modality that employs the
photochemical interaction of three components: light, photosensitizer, and oxygen. PDT has
been approved by the U.S. Food and Drug Administration for the treatment of microinvasive

2
lung cancer, obstructing lung cancer, and obstructing esophageal cancer, as well as for
premalignant actinic keratosis and age-related macular degeneration. Unlike radiation
therapy, PDT uses nonionizing radiation and can be administered repeatedly without
cumulative long-term complications since it does not appear to target DNA. The wavelength
of light used for PDT is typically in the wavelength range between 600–800 nm, the so called
“therapeutic window. In this wavelength range, the energy of each photon (hv) is high
enough (>1.5eV) to excite the photosensitizer and yet is low enough so that the light has
sufficient penetration into the tissue [18]. Although PDT can induce many cellular and
molecular signaling pathway events in cells, its main purpose is to induce cell death [19].

1.2 Statement of Problem

Lung cancer is the most commonly diagnosed cancer in males and the third most commonly
diagnosed cancer in females worldwide, with approximately 2.2-2.5 million new cases
reported annually. The incidence and mortality rates of lung cancer closely mirror each other,
as the majority of patients ultimately yield to the disease [20]. In 2022 alone, an estimated
2.48 million new lung cancer cases were diagnosed globally, with approximately 1.8 million
deaths reported, accounting for 12.4% of all new cancer cases and 18.7% of cancer-related
deaths [21]. The choice of treatment for lung cancer is primarily determined by the cancer
type and stage at diagnosis. Due to the absence of early clinical symptoms, lung cancer is
frequently detected at an advanced stage, limiting curative treatment options [22]. Surgical
intervention remains the recommended treatment for patients with stage I and II non-small
cell lung cancer (NSCLC) however, complete tumor resection is required, and patients must
be medically fit to tolerate the procedure [23],[24]. Surgical treatment is also associated with
potential complications, including pain, infection, bleeding, and damage to surrounding
tissues. In certain cases, complete tumor removal may not be feasible without risking injury
to vital anatomical structures.
Molecular analysis has revealed that up to 69% of patients with advanced NSCLC may
possess potentially actionable molecular targets [25]. Nevertheless, for patients who do not
qualify for FDA-approved targeted therapies, platinum-based doublet chemotherapy remains
the standard first-line treatment. This treatment plan is associated with significant
gastrointestinal and haematological toxicities. Furthermore, adjuvant chemotherapy is
commonly recommended for patients with resected stage IIA to IIIA NSCLC however, these
patients continue to face a high risk of disease relapse [25]. Chemotherapy-related adverse

3
effects include alopecia, mucositis, gastrointestinal disturbances, fatigue, neuropathy, and
menopausal symptoms and further reduce patient quality of life.
Radiation therapy has been employed in cancer treatment since the discovery of X-rays by
Röntgen in 1895 [26]. In lung cancer management, high-energy radiation is delivered
directly to the tumor site. Despite its therapeutic benefits, radiotherapy is associated with
significant side effects, notably cardiotoxicity, necessitating strict minimization of radiation
exposure to the heart and surrounding normal tissues. Additionally, the hypoxic tumor
microenvironment commonly observed in lung cancer contributes to increased cellular
proliferation, resistance to apoptosis, and reduced sensitivity to radiation therapy,
highlighting critical limitations of this treatment modality.

1.3 Objectives
1.3.1 General objective
To evaluate and compare the photodynamic therapeutic efficiency of zinc doped, PEGylated,
and zinc-doped PEGylated TiO2 nanoparticles in the treatment of lung carcinoma (A549)
cells.
1.3.2 Specific objectives
 To synthesize titanium dioxide (TiO₂) nanoparticles, zinc-doped TiO₂ nanoparticles and
PEGylated TiO₂ nanoparticles.
 To characterize TiO₂, Zn-TiO₂, PEG-TiO₂, nanoparticles using various techniques.
 To evaluate the photodynamic activity of synthesized TiO₂, Zn-TiO₂ and PEG-TiO₂
nanoparticles by assessing their reactive oxygen species (ROS) generation capacity.
 To compare the photodynamic therapeutic effectiveness of Zn-TiO₂ and PEG-TiO₂
nanoparticles in inducing cell death in A549 cells.

1.4 Hypotheses
This study hypothesizes that surface modification of titanium dioxide (TiO₂) nanoparticles
through zinc doping and polyethylene glycol (PEG) functionalization significantly
influences their photodynamic therapeutic efficiency against A549 human lung
adenocarcinoma cells. Zinc doping is expected to alter the electronic structure and charge
separation efficiency of TiO₂, potentially enhancing reactive oxygen species (ROS)
generation upon light irradiation, while PEGylation is anticipated to improve nanoparticle
dispersion, stability, and cellular interaction in biological media. These physicochemical
modifications may result in variations in cellular uptake, ROS production, and cytotoxic

4
response during photodynamic therapy. Therefore, comparative evaluation of zinc-doped
and PEGylated TiO₂ nanoparticles is expected to reveal differences in their photodynamic
therapeutic performance in A549 cells.

1.5 Rationale of the study

Globally, lung cancer is the most common cancer and the leading cause of cancer related
death causing nearly 1.8 million deaths till now. The existing treatments of lung cancer are
not effective enough to provide full cure from this disease [ 27]. PDT is a treatment option
for centrally located early-stage lung cancer and it can preserve lung function, can be
repeated, and can be combined with other therapeutic modalities. [28] TiO2 NPs induce
production of reactive oxygen species (ROS) and is the most effective photosensitizers for
PDT applications. Also, PEGylation and zinc doping of TiO2 NPs increase the efficiency of
ROS. But there is a lack of systematic comparative studies evaluating the photodynamic
therapeutic efficiency of zinc-doped and PEGylated TiO2 nanoparticles under identical
experimental condition. To our best knowledge, there has not been any study relating the
synthesis of zinc doped TiO2 NPs followed by PEGylation.
This study addresses this research gap by conducting a comparative analysis of the
photodynamic therapeutic efficiency of zinc-doped TiO2, PEGylated TiO2 in A549 cells.
By evaluating and comparing their photodynamic effects, this research provides us valuable
insights into how different surface modification strategies influence PDT performance.

5
 Chapter 2 Literature Review
2.1 Lung carcinoma
Lung carcinoma is a leading cause of cancer mortality globally; thus, innovative treatments
are urgently needed. The global geographical patterns in lung cancer deaths closely follow
those in incidence because of poor survival and the high fatality rate of this disease.
Worldwide, lung cancer is the leading cause of cancer death in men and the second-leading
cause in women [14].
In Nepal, cancer accounts for 9% of total annual deaths and is the third leading cause of
noncommunicable disease death. According to the GLOBOCAN 2020 statistics, the number
of new cases of cancer is 20,508 and cancer-related deaths are 13,629 for Nepal. The
agestandardized rates in cancer incidence and mortality per 100,000 were estimated to be
80.9 and 54.8, respectively. Age-standardized incidence and mortality rates of cancer in
Nepal as compared with neighbouring countries, Lung cancer is the most common cancer in
men (18% of new cases diagnosed) and third most common cancer in women (7.7%) in
Nepal [30]. Nevertheless, the true magnitude of the disease in terms of incidence, prevalence,
and mortality is still unknown because Nepal did not have a population-based cancer registry
(PBCR) until recently [31].

2.2 Nanoparticles in Cancer therapy

Cancer continues to be a leading cause of mortality worldwide, with traditional treatment
modalities such as chemotherapy and radiation often limited by their lack of specificity, high
toxicity, and the emergence of multidrug resistance [32]. The advent of nanotechnology has
introduced innovative approaches to cancer diagnosis and treatment through the
development of nanoparticles (NPs). These particles, typically ranging from 1 to 100 nm,
offer unique advantages including enhanced biocompatibility, stability, and the ability to
achieve precise targeting of tumor cells. This literature review explores the current progress
and challenges in utilizing nanoparticles for cancer therapy, particularly focusing on zinc
doped titanium dioxide (Zn-TiO2) nanoparticles and PEGylated titanium dioxide
nanoparticles [33].
2.2.1 Types of Nanoparticles
Nanoparticles can be classified into various categories based on their composition and
structure. Common types include: i. Metallic Nanoparticles

6
Such as gold and silver nanoparticles, known for their optical properties and ability to
generate reactive oxygen species (ROS) upon light activation. ii. Polymeric Nanoparticles:
Biodegradable polymers that can encapsulate drugs, enhancing their solubility and
bioavailability. iii. Lipid-based Nanoparticles:
Including liposomes and solid lipid nanoparticles that facilitate drug delivery through
cellular membranes.
Each type offers distinct advantages for cancer therapy, such as improved pharmacokinetics
and reduced side effects compared to conventional treatments.
2.2.2 Synthesis of Nanoparticles
The NPs are available in a variety of sizes, shapes, and architectures. Many different
synthesis techniques are used for achieving this. Due to their small size, they exhibit unique
physical and chemical properties that differ significantly from their bulk counterparts. These
properties include increased surface area, enhanced reactivity, and distinctive optical
characteristics, making nanoparticles integral to various applications in fields such as
medicine, electronics, catalysis, and environmental science. The synthesis of nanoparticles
is a rapidly evolving field that combines chemistry, physics, and engineering principles [34].
i. General approach
These techniques can be broadly divided into two approaches:
a) Bottom-up Approach
This method involves building material from atoms to clusters to NPs, i.e., building from
simpler substances, hence known as constructive method [35]. Some commonly used
methods are spinning, solgel synthesis, chemical vapor deposition, plasma or flame spraying
synthesis, laser pyrolysis, and biosynthesis.

Figure 2.1: Scheme of top-down and bottom-up synthesis of nanoparticles (NPs) [18]

7
b) Top-Down Approach
It is also known as the destructive method, which reduces bulk material or substance to
synthesize NPs. A larger molecule is broken down or decomposed into smaller units that are
converted into NPs [36]. It includes techniques such as mechanical milling, nanolithography,
chemical etching, laser ablation, sputtering, electro-explosion, and thermal decomposition.
Remarkably, the morphological parameters such as size, shape and charge of NPs can be
modified by changing the reaction conditions and other synthesis parameters [37]. Besides,
the growth mechanism also determines the chemical properties of NPs. Hence understanding
the growth mechanism is essential to synthesize required NPs.
ii. Green Synthesis

Figure 2.2 : Different types of green synthesis used for the preparation of metal nanoparticle [22]

Green synthesis of nanoparticles (NPs) is an eco-friendly approach to producing nanoparticles,

providing an alternative to conventional physical and chemical methods that often involve toxic
chemicals and high energy consumption. This technique uses biological resources like plant
extracts, bacteria, fungi, and algae as reducing and stabilizing agents to create metallic
nanoparticles. Among its notable advantages are reduced toxicity, lower environmental impact,
cost-efficiency, and scalability for industrial applications[38]. For instance, plant-based synthesis
involves bioactive compounds such as polyphenols and flavonoids that reduce metal ions, forming
nanoparticles like gold (Au NPs) and silver (Ag NPs). These nanoparticles have diverse
applications, ranging from catalysis to antimicrobial uses. Microbial-based synthesis, involving
organisms like yeast and fungi, also plays a crucial role, producing nanoparticles intracellularly or
extracellularly. Specific metal nanoparticles such as copper (Cu NPs) and iron (Fe NPs) are
synthesized using plant extracts, with applications in antimicrobial treatments and environmental
remediation [39].

8
iii. Chemical synthesis
Many different methods are used to synthesize nanoparticles, such as the sol-gel method,
hydrothermal method, solvothermal method, direct oxidation method, chemical vapor
deposition, physical vapor deposition, electrochemical anodization, and biological synthesis.
a. Sol-gel method
The sol-gel method is frequently used to synthesize tin oxide, tungsten oxide, zinc oxide, and
titanium dioxide nanoparticles. This method consists of five main lines; hydrolysis, poly-
condensation, aging, drying, and thermal decomposition. In the hydrolysis step, an initiator
(metal alkoxide) and a solvent (alcohol, water, or solvent with hydroxyl bond) are required.
At this stage, the aim is to separate the metal from the alkoxide and bring it together with the
-OH bond. It is then left to age. Different gel forms are included according to different drying
types, such as supercritical drying, thermal drying, freeze drying, and finally, the product is
obtained by calcination [40].
b. Hydrothermal method
Hydrolysis of metal salt solution can synthesize many nanoparticles, such as a wide variety
of chemical compounds, including oxides, sulfates, carbonates, phosphates, and sulfides.
Hydrothermal synthesis is usually carried out by reaction in aqueous solutions under
controlled temperature and controlled pressure in steel pressure vessels called Teflon lined
or autoclaves. According to the hydrothermal synthesis method, bypassing the gel step of
the sol-gel process, after the hydrolysis reaction of metal alkoxides in an alcohol-water
environment, it is dried to room temperature and directly calcined to obtain solid MOx.
Nanoparticles of desired particle sizes and morphologies are synthesized by controlling
parameters such as temperature, pH, reactant concentrations, and additives [41].
c. Solvothermal method
The main difference between this method from the hydrothermal method is that the solvent
is non- aqueous. Other than that, the steps are almost the same. The advantage of being
anhydrous is that it can operate at high temperatures since high boiling point organic solvents
can be selected. Compared to the hydrothermal method, the size, shape, and crystallinity of
TiO2-NPs can be controlled better [42].
d. Direct oxidation reaction
Oxidants are used for the oxidation of titanium metal. The direct oxidation method is
generally used for the synthesis of TiO2 nanorods. This method takes place by the dissolution
precipitation mechanism. For example, when the anodized titanium plate is heat treated at

9
500 °C for 6 h in an oxygen environment, crystallized TiO2 nanotubes are obtained. It has
also been found that direct oxidation of titanium metal with hydrogen peroxide leads to the
formation of TiO2 nanorods. Acetone and pure water can be used as oxygen sources for the
oxidation of titanium metal. Acetone is a good source of oxygen, and when used at high
temperatures, well-aligned, and highly dense nanorods can be synthesized [43].
e. Emulsion method
Micro-emulsion method is one of the recent and ideal techniques for the preparation of
inorganic nano-particles. Oil and water are immiscible and they separate into two phases
when mixed, each saturated with traces of the other component. An emulsion is formed when
a small amount of an appropriate surfactant is mechanically agitated with the oil and water
resulting in a two-phase dispersion where one phase exists as droplets coated by surfactant
that is dispersed throughout the continuous, other phase. These emulsions are milky or turbid
in appearance due to the fact that the droplet sizes range from 0.1 to 1 micron [44]. The
water-in-oil (W/O) or reversed micelle system, is an effective approach for synthesizing
inorganic nanoparticles with controlled size and morphology. In this technique, nanoscale
water droplets dispersed in an oil phase act as confined microreactors, enabling regulated
nucleation and growth of nanoparticles [45]. Parameters such as surfactant type, counterions,
and the water-to-surfactant molar ratio (ω value) influence the structure of the
microemulsion and thereby affect particle morphology. Unlike conventional chemical
reduction methods, the combination of W/O microemulsions with γ-irradiation introduces
hydrated electrons as strong reducing species, offering enhanced control over nanoparticle
formation. This method has been successfully applied to the synthesis of various metal and
semiconductor nanoparticles and provides valuable mechanistic insights into morphology
control [46].
2.2.3 History of nanoparticles
Nanoparticles in medicine offer a considerable step forward in diagnostic and therapeutic
applications. Nps based drug delivery systems have shown many advantages in cancer
treatment, such as good pharmacokinetics, precise targeting of tumor cells, reduction of side
effects, and drug resistance. The history of nanoparticles in medicine can be traced back to
several key milestones:

• The concept of nanoparticles was first explored in the 1960s, where colloidal gold and
silver nanoparticles were studied. Early studies revolved around using gold NPs for
labelling cells and tissues for electron microscopy [47].

10
• TiO₂ nanoparticles, known for their UV-blocking properties, were used in sunscreens to
protect skin from UV rays while staying transparent. This feature was critical for their
use in dermatology [48].

• TiO2 nanoparticles were first synthesized in the 1990s, gaining attention for their
photocatalytic properties, which allow them to generate reactive oxygen species (ROS)
when exposed to ultraviolet (UV) light. This ability is crucial for PDT, where ROS play
a key role in inducing cell death in cancerous tissues [49].

2.3 TiO₂ nanoparticle properties and synthesis

Titanium dioxide (TiO₂) is a metal oxide widely found in nature and has several notable
properties, including biocompatibility, lightweight characteristics, excellent corrosion
resistance, high thermal stability, minimal ion release, and non-magnetic qualities [50]. TiO₂
occurs in the three crystallographic form rutile, anatase and brookite. The anatase form is
often preferred for photocatalytic applications due to its higher surface area and reactivity
also it is used as a carrier for particles that are biologically active, an example is silver [51].
It has a wide bandgap of about 3.2 eV for anatase and 3.0 eV for rutile, which limits its
absorption to ultraviolet light. However, its optical properties can be modified through
doping or surface modifications [52].
Table 1 1 Plants from which TiO2 NPs can be extracted [43,52,53,54]

11
The preparation of TiO2 nanoparticles can be carried out by various methods such as solgel
method, instantaneous synthesis method, solvothermal method, microwave-assisted
synthesis, simple mixing, and precipitation method. Green synthesis is an eco-friendly
process or rather technique where the extracts of plants (flower, seed, leaves and peels),
fungi, bacteria and also enzymes are used for the synthesis of nanoparticles as a substitute
for chemicals. The synthesis methods and properties of TiO2 nanoparticles play a crucial role
in their diverse applications across environmental science, energy production, and medicine.
Due to its wide synthesis method TiO2 is widely used in cosmetics products like sun scream.
In green synthesis of TiO2 NPs wide numbers of bacteria, fungi and plants can be used.

Table 1 2: Bacteria mediated synthesis of TiO2 nanoparticles using various strains

2.3.1 TiO2 nanoparticle for cancer treatment

Titanium dioxide nanoparticles are widely used in biological applications due to their unique
characteristics and adaptability. The size and morphology of TiO₂ NPs can be tailored
through various synthesis methods, which can influence their biological interactions and
therapeutic efficacy. Research has shown that the size, aggregation tendency, and surface
modifications of TiO₂ NPs play crucial roles in their cellular uptake and subsequent effects
on tumor cells [55].

12
TiO₂ NPs can induce oxidative stress in cancer cells, leading to cell cycle disruption and
apoptosis. The mechanisms of action involve the generation of ROS, which can damage
cellular components such as DNA, proteins, and lipids, ultimately contributing to the
anticancer effects of these nanoparticles [56].
• PDT: TiO₂ NPs can generate reactive oxygen species (ROS) when exposed to ultraviolet
(UV) light. This property is harnessed in photodynamic therapy, where TiO₂ NPs are
used to induce localized oxidative stress in cancer cells, leading to cell death. The ROS
produced can damage cellular components, including DNA, proteins, and lipids, thereby
inhibiting tumor growth [57].

• Anti-tumor effects: Researches have shown that higher doses of these nanoparticles have
been shown to cause cell death through apoptosis and necrosis. They can also disrupt the
cell cycle, stopping cancer cells from growing and dividing [58].
• SDT: Sonodynamic therapy, is the one of these methods in which the sonosensitizers and
ultrasound (US) are used. Sonosensitizers could be nanoparticles mostly TiO2
conjugated with metals or antibodies. Recent studies show that TiO2 nanoparticles can
be able to kill nanoparticle-impregnated glioma cells after ultrasonic stimulation [59].
2.3.2 TiO2 nanoparticle dispersion
Surfactants help to stabilize titanium dioxide (TiO2) nanoparticles in diverse environments
by lowering surface tension and preventing agglomeration. Proteins like BSA have been
shown to be excellent surfactants in TiO2 nanoparticles. Studies have demonstrated that BSA
considerably increases the stability of TiO2 dispersions in biological media, such as PBS
and DMEM-FBS [60]. Surfactants, such as SHMP, are used to enhance the stability of TiO2
dispersions. SHMP acts as an inorganic surfactant that helps to reduce the surface tension
between the TiO2 particles and the aqueous solution, promoting better dispersion [61].
2.4 Zn-doping
Zinc (Zn) doping is a well-established approach for tuning the physicochemical, structural,
optical, and electronic characteristics of semiconductor metal oxides. Owing to the
comparable ionic radius of Zn²⁺ to many host lattice cations, zinc ions can be effectively
incorporated into the crystal structure either by substituting host ions or by occupying
interstitial sites, without inducing significant lattice deformation. In oxide semiconductors
such as TiO₂, ZnO, Fe₂O₃, and related systems, the introduction of Zn alters defect states,
charge carrier density, and band-gap structure. These atomic-scale modifications have a
direct impact on important material properties, including charge transport behavior,

13
electron–hole recombination dynamics, catalytic activity, and optical absorption efficiency
[11 ,12].
Numerous reports indicate that Zn incorporation leads to the formation of shallow donor
levels or additional localized states within the band gap, which enhances absorption in the
visible region and suppresses the recombination of photogenerated charge carriers [12, 13].
Such effects are particularly advantageous for applications in photocatalysis,
photoelectrochemical systems, and optoelectronic devices. Furthermore, Zn dopants can
contribute to phase stabilization, reduction in particle size, increased density of surface
hydroxyl groups, and improved surface activity. Together, these effects result in superior
photocatalytic pollutant degradation, enhanced antimicrobial performance, and improved
charge separation efficiency in Zn-doped nanomaterials [11, 14].
2.4.1 Previous work on Zn-doping
Research shows that incorporating transition metals and rare earth dopants into ZnO NPs via
methods like sol-gel and hydrothermal synthesis can significantly enhance antimicrobial
activity, biocompatibility, colloidal stability, and potential for bioimaging or drug delivery.
Specifically, doped ZnO NPs exhibit stronger antibacterial effects and greater stability in
biological environments than undoped counterparts, making Zn doping a valuable strategy
to tailor ZnO nanomaterials for nanomedicine and antimicrobial applications [62]. The study
shows, Zn-doped TiO₂ nanoparticles have been shown to exhibit enhanced catalytic and
nonlinear optical properties, where Zn incorporation into the TiO₂ lattice altered the optical
band gap and improved catalytic efficiency for dye degradation, indicating the potential of
Zn-doped TiO₂ for environmental applications and photonic devices [63].
2.5 PEGylation
PEGylation refers to the process of attaching one or more polyethylene glycol (PEG) chains
to proteins, peptides, or other small molecules. PEG is a biocompatible polymer that is
watersoluble and does not trigger toxic, immunogenic, or antigenic responses [15]. When
PEGbased copolymers are coated onto the surface of TiO₂ nanoparticles, they improve
dispersion in aqueous media and enhance overall biocompatibility [16]. In addition,
PEGylation allows nanoparticles to evade rapid clearance by the reticuloendothelial system
(RES), thereby prolonging their circulation time in the bloodstream. This extended half-life
supports passive targeting of anticancer drugs, enabling more effective accumulation at
tumor sites [17].

14
2.5.1 Previous works on PEGylation
In 2019, a study published regarding protein PEGylation for cancer therapy explored the
capability of PEG to alter the physiochemical properties in the parent protein including
electrostatic and hydrophobic properties. This study states that PEGylation allows prolonged
circulation time of conjugated therapeutics in body by reducing proteolysis and opsonisation
[64].
In 2012, current drug research on PEGylation with small molecular agent concluded that
PEGylation effectively increase the solubility of water insoluble compounds, lower the
toxicity, generate a desired pharmacokinetics profile and enhance the accumulation at the
targeted sites [65].

2.6 Photodynamic therapy (PDT)

Photodynamic therapy (PDT) is a treatment that uses a combination of light and a
photosensitizing agent to produce reactive oxygen species (ROS) that can kill targeted cells,
such as cancer cells. The treatment involves three main components: a photosensitizer, light,
and oxygen. When the photosensitizer is exposed to a specific wavelength of light, it
becomes activated and interacts with oxygen to produce ROS, leading to cell damage and
death. PDT is often used for cancer treatment, as it can selectively target tumor cells that
have absorbed the photosensitizer. The therapy has the advantage of being minimally
invasive and has fewer side effects compared to traditional treatments like chemotherapy or
radio therapy. The effectiveness of PDT can be enhanced with the use of nanoparticles, such
as titanium dioxide (TiO₂), which act as photosensitizers and generate ROS when activated
by light [66].
Photodynamic therapy (PDT) offers several advantages, especially in cancer treatment and
other medical applications.
• Minimally invasive: PDT is less invasive than surgery and does not require large
incisions, reducing recovery time and the risk of complications like infection.
• Selective targeting: The photosensitizer accumulates primarily in cancerous or abnormal
cells, and the light activation can be targeted to specific areas, minimizing damage to
healthy tissues.

• Reduced side effects: Compared to chemotherapy and radiotherapy, PDT has fewer
systemic side effects, such as nausea or immune suppression, because it is localized to
the treatment area.

15
• Versatile application: PDT can be used to treat a variety of conditions, including skin
cancer, breast.
2.6.1 Previous work on Lung cancer treatment using PDT
In 2012, a study published in Photodynamic Therapy for treatment of non-small cell lung
cancer pinpointed the use of PDT among 11 patients for central tumor, among which
complete response was achieved within two months and all patients survived with upto 5
years of follow up. This study demonstrated that PDT was well tolerated with fewer side
effects and no secondary cancers and is very effective, especially for small tumor (<1cm)
[67].
In 2025, Research published in Scientific Reports explored the effectiveness of cisplatin
combined with PDT on A549 cells. They mentioned the combination effectively inhibit
tumor growth, and demonstrated a stronger ability to induce apoptosis and impede tumor
regression. This method with the combination, significantly suppressed the tumor volume
growth in the animal model [68].

2.7 Photosensitizer
A photosensitizer is defined as a compound capable of absorbing light energy and
transferring that energy to other molecules, leading to chemical changes. In the context of
PDT, when a photosensitizer is exposed to specific wavelengths of light, it becomes excited
and generates reactive oxygen species (ROS), which can damage or kill nearby cancer cells
[69].
2.7.1 Role of TiO2 as a Photosensitizer
Because TiO2 nanoparticles can produce reactive oxygen species (ROS) when activated by
light, they have shown great promise as photosensitizers in photodynamic treatment (PDT).
Mechanism of TiO2 as a photosensitizer
• TiO₂, when exposed to UV or visible light, undergoes excitation leading to the generation
of electron-hole pairs. These charge carriers can interact with molecular oxygen to
produce ROS, such as singlet oxygen (1O2), superoxide (O2 -), and hydroxyl radicals
(OH). These ROS can induce apoptosis in tumor cells and are essential for the therapeutic
efficacy of PDT [70].

• TiO2 exhibits unique photocatalytic properties, allowing it to absorb light, particularly

in the ultraviolet (UV) range. Upon irradiation, electrons from the valence band are
excited to the conduction band, creating electron-hole pairs that facilitate the generation

16
 of various ROS such as hydroxyl radicals, hydrogen peroxide, and superoxide anions
[71].

• The generated ROS can induce oxidative stress within cancer cells, leading to cellular
damage and apoptosis. The effectiveness of TiO2 as a photosensitizer is significantly
influenced by the wavelength of light used for activation; UV light is commonly
employed, although there is ongoing research to enhance its efficacy under visible light
through doping or hybridization with other materials [72].
2.7.2 Previous work of TiO2 NPs as photosensitizer in PDT
In 2016, a study published in Scientific Reports demonstrated that nitrogen-doped TiO₂ NPs
could induce ROS-mediated autophagy and terminal differentiation in leukemia cells when
activated by visible light. This research indicated that well dispersed N-TiO₂ NPs had no
growth inhibitory effect without light but significantly reduced cell viability when irradiated,
suggesting a controlled approach to inducing cell death through PDT [73].
In 2016, a Research on graphene oxide/TiO₂ (GOT) hybrid nanoparticles showed their
potential as new photosensitizers for melanoma cells. The study found that irradiation with
visible light led to growth arrest and cell death in a dose-dependent manner, indicating the
effectiveness of GOT NPs in PDT while minimizing toxicity compared to traditional
methods [74].

2.8 Characterization of nanoparticles

Nano particles having different shape and size exhibit different properties from their bulk
parts. Mainly two of the main parameters are studied in the characterization of NPs, they are
size and shape [75]. Different analytical techniques are used to confirm the synthesis and
characterize the morphological and structural features of biological NPs like:
• XRD analysis
• Scanning electron microscopy (SEM)
• UV-visible spectroscopy
• Fourier transform infrared spectroscopy (FTIR) analysis
• Zeta potential
2.8.1XRD analysis
XRD is the analytic technique that determines the crystallographic structure of materials. In
synchrotrons, X-ray radiation is produced by accelerating electrons to nearly the speed of
light. These high-energy electrons are generated by an electron gun, then boosted and

17
introduced into a storage ring. As they travel through the ring, they emit radiation across a
wide range of wavelengths, from infrared to gamma rays. This method allows scientists to
choose specific energy levels of X-rays, making it ideal for characterizing materials like
silver-doped TiO2 nanoparticles [76].
2.8.2 Scanning electron microscopy
In general, two types of microscopy are available, OM and SEM. OM and SEM are two
important methods for analyzing materials. OM uses light and gives lower magnification
with true color images, while SEM uses electrons to achieve much higher magnification
and detailed gray-scale images, making it better for studying silver-doped TiO2
nanoparticles. SEM is more advanced and often works with Energy Dispersive X-ray
Spectroscopy to analyze the elements in the samples [77].
2.8.3 UV-visible spectroscopy
Spectroscopy looks at how light interacts with materials, helping us learn more about them.
UV spectroscopy is an important method that measures how much UV and visible light
(200800 nm) is absorbed by a sample, which can reveal its composition and concentration.
This technique is useful for studying nanoparticles by analyzing their light absorption
patterns. This technique is useful for studying silver-doped TiO2 nanoparticles by analyzing
their light absorption patterns [78].
2.8.4 Fourier transform infrared spectroscopy (FTIR) analysis
The FTIR spectrophotometer uses a black-body source to emit IR radiation, which passes
through an interferometer for spectral encoding, creating an interferogram. The beam then
enters the sample compartment, where the sample absorbs specific IR frequencies. The
detector measures the interferogram, and Fourier transformation software generates the IR
spectrum by subtracting the background. The resulting spectrum, typically in the mid-IR
region (4000–400 cm⁻¹), reveals information about functional groups, with specific bond
types (single, double, triple) detectable in different wavenumber ranges. This process aids
in identifying molecular vibrations and functional groups in the material [79].
2.8.5 MTT assay
The MTT assay is a colorimetric assay used to measure cell viability and proliferation by
assessing the metabolic activity of cells. It is based on the reduction of the yellow MTT
reagent (a tetrazolium salt) to purple formazan crystals by mitochondrial dehydrogenase
enzymes in metabolically active cells, with the intensity of the color being proportional to
the number of viable cells [80].

18
2.8.6 Zeta potential
Nanoparticles with a zeta potential between −10 and +10 mV are considered approximately
neutral, while nanoparticles with zeta potentials of greater than +30 mV or less than −30 mV
are considered strongly cationic and strongly anionic, respectively. Since most cellular
membranes are negatively charged, zeta potential can affect a nanoparticle’s tendency to
permeate membranes, with cationic particles generally displaying more toxicity associated
with cell wall disruption.
2.9 A549 Cell Culture
The A549 cell line was first developed in 1972 by Giard et al., through the removal and
culturing of cancerous lung tissue in the explanted tumor of a 58-year-old Caucasian male
[81]. They are human alveolar basal epithelial cells, are squamous in nature and responsible
for the diffusion of substances, such as water and electrolytes, across the alveoli of lungs.
They grow adherently, as a monolayer, in vivo. A549 cells have characteristic features of
Type II cells of the pulmonary epithelium, including lamellar bodies [82].

19
 Chapter 3 Materials and Methodology
3.1 Materials Required
3.1.1 Chemicals required
1. Cyclohexane (MW- 84.16)
2. Tween-80
3. Titanium tetraisopropoxide [TTIP] (MW-284.22)
4. Ethanol
5. Deionized Water
6. Polyethylene glycol (MW-6000)
7. Zinc nitrate hexahydrate [Zn(NO3)2.6H2O] (MW-297.48)
3.1.2 Instruments required
1. Micropipette
2. Magnetic stirrer
3. Hot air oven
4. Centrifuge machine
5. Muffle furnace
3.1.3 Apparatus used
1. Beaker
2. Falcon tube
3. Petridish
4. Measuring cylinder
5. Crucible
6. Test tubes
7. Spatula
3.1.4 Software used
1. Origin

3.2 Methodology
3.2.1 Work completed

[Link] Synthesis of TiO2 NPs

For the synthesis of TiO2 nanoparticle, we used reverse microemulsion method. Two
separate solutions A and B were created. For solution A, 60 ml cyclohexane was taken in a

20
beaker in magnetic stirrer. Then, 8ml tween-80, 4ml ethanol followed by 3ml TTIP was
added dropwise. It was left to stir for an hour. Simultaneously for solution B, 19ml
cyclohexane was taken in another beaker in magnetic stirrer. Then, 3ml tween-80, 4ml
ethanol followed by 3ml TTIP was added dropwise. After that, solution A was added
dropwise to beaker containing solution B. The resultant mixture was let to stirred for 12
hours in magnetic stirrer. The stirred solution was centrifuged and washed twice with ethanol
and once with deionized water for 7 minutes at 5000 rpm. The precipitate was dried in oven
for 12 hours at 60-70 C followed by calcination at 400 C for 3 hours.

Step 6: TiO2 NPs after calcinate

Figure 3.1: Synthesis of TiO2 NPs through microemulsion method

21
[Link] Zn-doping of NPs
For the synthesis of 1% zinc doping of TiO2 nanoparticle, 1 gram of TiO2 nanoparticle was
taken in a beaker containing 20ml deionized water in magnetic stirrer at 1200 rpm. 15ml of
deionized water was slowly added to it making a total of 35ml. In another beaker, 0.01 gram
of zinc nitrate hexahydrate was taken in a beaker containing 10ml of deionized water in
magnetic stirrer at 500 rpm. 5ml of deionized water was slowly added to it and stirred for 10
minutes. The zinc solution was added dropwise to Tio2 solution and stirred for 1 hour at
1200rpm. The resulting mixture was then let to age for 4 hours. The mixture was then filtered
and washed with ethanol and deionized water. The precipitate was dried in oven for 12 hours
at 60-70 ℃ followed by calcination at 450 ℃ for 2 hours [83].
[Link] PEGylation of NPs
For coating TiO2 with PEG, 20mg of TiO2 nanoparticle was taken in a beaker containing
25ml of deionized water. 250mg of PEG6000 was dissolved to the mixture. The obtained
mixture was ultrasonicated for 15 minutes to break any big clumps of nanoparticles and help
PEG molecules attach to surface of [Link] was then stirred for 12 hours at room
temperature. After stirring, the suspension was centrifuged at 4000rpm for 15 minutes [84].
3.2.2 Work in progress
For the characterization of Zn-doping and PEGylation of TiO2 NPs, samples of Zn-doped
and PEGylated TiO2 are taken to NAST for XRD and Zeta potential and to RECAST for UV
and FTIR. The results are yet to be obtained.
3.2.3 Remaining work
We will focus on the characterization of zinc-doped and PEGylated TiO₂ nanoparticles to
determine their optical properties, surface functional groups, surface electrical charge, and
crystalline size. Efforts will also be directed toward the development of a controlled and
suitable light source for photodynamic therapy applications. Furthermore, A549 cells will be
cultured under standard laboratory conditions for experimental evaluation. Finally, a
comparative analysis of the photodynamic efficiency of TiO₂, zinc-doped TiO₂, and
PEGylated TiO₂ nanoparticles on A549 cells will be carried out to assess the influence of
zinc doping and surface modification on therapeutic performance.

22
 Chapter 4 Results and Discussion
4.1 Results
4.1.1 Synthesis of TiO2 NPs
TiO2 nanoparticles was synthesized using microemulsion method which resulted in 0.550g
of white TiO2.

Figure 4 1: Resulted TiO2 NPs

4.1.2 Characterization of NPs synthesized from microemulsion method

A. Zeta potential

Figure 4 2: Zeta Potential graph showing stability of TiO2 NPs

The zeta potential distribution of TiO₂ nanoparticles shows a single sharp and dominant peak
centered at approximately –59.36 mV, indicating a strongly negative surface charge on the
nanoparticles. This high magnitude of zeta potential reflects strong electrostatic repulsion
among particles, which effectively prevents aggregation and confirms excellent colloidal
stability of the dispersion [85]. The narrow peak profile suggests a uniform surface charge
distribution and good monodispersity, while the absence of significant secondary peaks

23
indicates minimal presence of aggregates or particles with different surface properties.
Overall, the zeta potential result demonstrates that the TiO₂ nanoparticles are highly stable
in suspension, with low tendency toward agglomeration, making them suitable for stable
colloidal applications.
B. X-Ray Diffraction

Figure 4.3 XRD graph showing diffraction pattern of TiO2 NP

The above graph illustrates X-ray diffraction pattern of TiO2 nanoparticles. The diffraction
peaks were obtained at approximately 25°,37°,48°, 54°, and 52° corresponding to (101),
(104), (200), (105), and (204) planes of anatase phase of TiO2 respectively. The prominent
peak is observed at around 25° corresponding to the (101) plane which confirms that anatase
is the dominant crystalline phase [86]. No significant diffraction peak corresponding to rutile
phase were observed, suggesting that the synthesized TiO2 nanoparticles are predominantly
anatase in nature. To estimate the crystallite size of the synthesized nanoparticles, the
Scherrer equation was applied which is given as:
𝐾𝜆
D=
𝛽𝑐𝑜𝑠𝜃

Where,
D = crystallite size (nm)
K = shape factor, taken as 0.9
λ = X-ray wavelength of Cu-Kα radiation = 0.15406 nm

24
β = Full Width at Half Maximum of the peak (in radians)
θ = Bragg angle (in radians), θ=2θ/2
Using the most intense peak observed at 2θ = 25.22514°, corresponding to the (101) plane
of anatase TiO2, the crystalline size was found to be 8.14nm.
C. UV-Vis Spectrophotometry

Figure 4.4 UV-Spectroscopy of TiO2 nanoparticles

In the above figure, the absorbance spectrum of TiO2 NPs shows a large range of absorbance
value also with a strong peak of absorption of 346 nm indicating that there are uniformly
distributed particles. The curve starts low at shorter wavelengths (~200 nm), rises to a peak,
and then gradually decreases as the wavelength increases. For λ=346 nm, the equivalent band
gap energy is found to be 3.58ev as calculated according to Planck’s equation. This value
suggests that the material is anatase-phase TiO2. This value suggests that the material is
anatase-phase TiO2, which typically has a band gap of ~3.2 eV [87].
D. FTIR
The FT-IR spectrum was recorded in the range of 4000–500 cm⁻¹ to identify the functional
groups present and to confirm the formation of TiO₂.
A broad absorption band observed around 3200–3600 cm⁻¹ corresponds to the O–H
stretching vibration, which indicates the presence of surface hydroxyl groups and adsorbed
water molecules on the TiO₂ surface. These hydroxyl groups are commonly formed due to
moisture adsorption from the atmosphere. The absorption peak observed in the region of
2800–3000 cm⁻¹ is attributed to the C–H stretching vibration, suggesting the presence of

25
residual organic compounds originating from the precursor or surfactant used during the
synthesis of TiO₂.

Figure 4.5 FTIR of TiO2 nanoparticles

A band near 1600–1650 cm⁻¹ corresponds to the O–H bending vibration (δ O–H) of
physically adsorbed water molecules on the TiO₂ surface. The sharp peak around 1700–1750
cm⁻¹ is assigned to the C=O stretching vibration, further confirming the presence of residual
organic species in the sample. In the low wavenumber region below 1000 cm⁻¹, strong
absorption bands are observed, which are characteristic of Ti–O and Ti–O–Ti lattice
vibrations. This region confirms the successful formation of the TiO₂ metal oxide framework
[88].
4.2 Discussion
The XRD patterns confirm that the reverse microemulsion route successfully produced
crystalline TiO₂ nanoparticles predominantly in the anatase phase, as evidenced by sharp,
well-defined diffraction peaks indicative of good crystallinity and controlled particle growth.
The dominance of the anatase phase is important because anatase TiO₂ is widely reported to
exhibit higher photocatalytic activity and electron mobility than rutile or brookite, suggesting
that the synthesized nanoparticles are structurally suitable for photocatalytic and related
surface-driven applications [89]. The UV–Visible spectra, showing strong and broad
absorption in the ultraviolet region with maxima near 260nm, further confirm the formation
of nanoscale TiO₂ with a typical semiconductor-like absorption edge and minimal molecular
impurity features [90].

26
The preservation of the anatase phase after zinc doping is consistent with literature reports
showing that low-level Zn incorporation can occur within the TiO₂ lattice without disrupting
its crystal structure while subtly altering its electronic properties. Similarly, PEGylation as a
surface modification strategy is widely used to enhance nanoparticle stability, dispersibility,
and biocompatibility without significantly affecting the core crystallinity of TiO₂, which
aligns with the retention of sharp anatase peaks in this study [84].
Reported XRD patterns of anatase TiO₂ characteristically show an intense (101) reflection
near 25°, matching the dominant peak position observed here and reinforcing the
identification of the phase as anatase. Previous studies on thermally treated TiO₂
nanoparticles also describe strong UV absorption associated with high surface area and
nanoscale dimensions, which is in good agreement with the broad ultraviolet absorption band
obtained for the synthesized samples. The successful synthesis of phase-pure, crystalline
anatase TiO₂ via the reverse microemulsion method demonstrates that this route provides
reliable control over nucleation and growth, supporting its suitability for producing uniform
nanomaterials [91].
The fact that crystallinity is preserved after zinc doping and PEG surface modification
indicates that these post-synthesis treatments can be applied without compromising the
structural integrity of the TiO₂ core. These structural and optical characteristics suggest that
the obtained nanoparticles are promising candidates for applications where surface reactivity
and stability are essential, such as photocatalysis, UV blocking coatings, and biomedical or
drug-delivery interfaces. Moreover, the established microemulsion protocol can serve as a
platform for further compositional tuning and surface functionalization aimed at tailoring
band-gap, dispersion stability, or biological interactions [92]. During microemulsion
synthesis, excessive addition of Tween surfactant occasionally led to gel like phase
formation, indicating disruption of the microemulsion region and highlighting the narrow
processing window required for stable droplet formation. Repeated washing steps resulted
in partial loss of TiO₂ material, necessitating multiple rounds of optimization of oil and
surfactant composition, which may have limited overall yield and reproducibility. Attempts
to introduce zinc via a sol gel route did not yield clear evidence of successful doping,
suggesting possible incompatibilities in precursor chemistry or reaction kinetics under the
conditions employed; similar sensitivity of sol gel TiO₂ to synthesis parameters has been
emphasized in earlier reports [89,91]. In addition, the current work focused mainly on
structural (XRD) and optical (UV-Vis) characterization, without complementary analyses

27
such as band-gap determination, direct verification of Zn incorporation, quantification of
PEGylation, or evaluation of photocatalytic and biological performance, which limits the
ability to fully correlate structure with function.
Future investigations should therefore integrate advanced structural and surface‑sensitive
techniques unambiguously verify zinc distribution and PEGylation efficiency on the TiO₂
surface and analyses that would provide quantitative insights into surface area and
hydrodynamic size, enabling direct correlation between microemulsion parameters, particle
morphology, and dispersion behaviour. Additional work is also needed to systematically
map the microemulsion composition space varying water/oil/surfactant ratios and surfactant
types to mitigate gel phase formation and improve process robustness and yield [90].

28
 Chapter 5 Conclusion and Further Work

5.1 Conclusion
TiO2 nanoparticles has been successfully synthesized through reverse microemulsion
method. The characterization of synthesized NPs confirms successful formation of anatase
phase TiO2 with nanocrystalline size of 8.14nm indicating small coherent diffraction
domains. The nanosize significantly increases the surface area. FTIR analysis validate the
presence of hydroxyl (-OH) group on the surface indicating active hydrophilic surface which
promotes better interaction with cellular membranes. Zeta potential result reveals a strong
negative surface charge suggesting strong electrostatic repulsion among Ti02 nanoparticles
and confirming excellent colloidal stability.
Since it has been stated in the previous research that the cytoxicity of nanoparticles depend
on size i.e., the smaller the size better the cytotoxicity. Thus, the size of synthesized TiO2
Nps being 8.14nm will have more effective results than bigger-size nanoparticles. Also,
TiO2 NPs are photo catalytically active in the UV range so, ROS production will
significantly increase when it is exposed to UV light. The enhanced ROS production can
induce oxidative stress, damage cellular components and ulimately lead to increase cytotoxic
effects.

29
Gantt Chart

30
 Cost Estimation
S.N Materials Rate(Rs) Quantity Amount

2. Cyclohexane 1000 per 500ml 2ltr 4000

3. Ethanol 325 per 500ml 5ltr 3250

4. Tween-80 8500 per 500ml 500ml 8500

5. De-ionized water 250 per jar 2 jar 500

6. TTIP 30,000 per 500 500ml 30,000

ml
7. A549 cell line 25,000 1 25,000

8. Culture media 25,000 25,000

9. Annapurna neuro lab 30,000 30,000

10. NAST lab (XRD test, Light 15,000 15,000

intensity test)
11. Blue Light source, Resistor, 1000 1000
Capacitor
12. 96 well plate 400 per plate 3 1200

13. Matrix Board 400 2 800

14. Miscellaneous 10,000 10,000

Grand Total Rs.1,54,250

31
 References
[1] Koparal, A. Tansu, and Melih Zeytinoglu. "Effects of carvacrol on a human non-small cell lung
cancer (NSCLC) cell line, A549." Cytotechnology 43, no. 1 (2003): 149-154.
[2] H. Lemjabbar-Alaoui, O. Hassan, Y.-W. Yang, and P. Buchanan, “Lung cancer: Biology and
treatment options,” Biochimica et Biophysica Acta (BBA) – Reviews on Cancer, vol. 1856, no. 2,
pp. 189–210, 2015.
[3] F. M. P. Tonelli, F. C. P. Tonelli, and H. G. Cordeiro, “TiO₂ nanoparticles in cancer therapy as
nanocarriers in paclitaxel’s delivery and nanosensitizers in phototherapies and/or sonodynamic
therapy,” Curr. Pharm. Biotechnol., vol. 25, no. 2, 2024, doi:
10.2174/1389201024666230518124829.
[4] K. A. Altammar, “A review on nanoparticles: characteristics, synthesis, applications, and
challenges,” Frontiers in Microbiology, vol. 14, p. 1155622, 2023.
[5] D. O. Shah and J. H. Schulman, “Microemulsions—A new class of transparent, stable
dispersions,” J. Pharm. Sci., vol. 57, no. 10, pp. 1793–1797, 1968.
[6] M. J. Lawrence and G. D. Rees, “Microemulsion-based media as novel drug delivery systems,”
Adv. Drug Deliv. Rev., vol. 45, no. 1, pp. 89–121, 2000.
[7] X. Zhang, W. Li, and Z. Yang, “Toxicology of nanosized titanium dioxide: An update,” Archives
of Toxicology, vol. 89, pp. 2207–2217, 2015, doi: 10.1007/s00204-015-1594-6.
[8] S. Shabbir, M. F.-E.-A. Kulyar, Z. A. Bhutta, P. Boruah, and M. Asif, “Toxicological
consequences of titanium dioxide nanoparticles (TiO₂NPs) and their jeopardy to human
population,” BioNanoScience, vol. 11, no. 2, pp. 621–632, 2021.
[9] A. R. D. A. Scharnberg, A. C. de Loreto, T. B. Wermuth, A. K. Alves, S. Arcaro, P. A. M. dos
Santos, and A. D. A. L. Rodriguez, “Porous ceramic supported TiO₂ nanoparticles: Enhanced
photocatalytic activity for Rhodamine B degradation,” Boletín de la Sociedad Española de
Cerámica y Vidrio, vol. 59, no. 6, pp. 230–238, Nov.–Dec. 2020.
[10] H. H. Do, D. L. T. Nguyen, X. C. Nguyen, T.-H. Le, T. P. Nguyen, Q. T. Trinh, S. H. Ahn, D.-
V. N. Vo, S. Y. Kim, and Q. Van Le, “Recent progress in TiO₂-based photocatalysts for hydrogen
evolution reaction: A review,” Arabian Journal of Chemistry, vol. 13, no. 2, pp. 3653–3671, 2020.
[11] V. Hiremath, V. G. Deonikar, H. Kim, and J. G. Seo, “Hierarchically assembled porous TiO₂
nanoparticles with enhanced photocatalytic activity towards Rhodamine-B degradation,” Colloids
and Surfaces A: Physicochemical and Engineering Aspects, vol. 586, Art. no. 124199, 2020.

32
[12] X. Bai, J. Jia, Y. Du, X. Hu, J. Li, E. Liu, and J. Fan, “Multi-level trapped-electrons system in
enhancing photocatalytic activity of TiO₂ nanosheets for simultaneous reduction of Cr(VI) and RhB
degradation,” Applied Surface Science, vol. 503, Art. no. 144298, 2020.
[13] A. M. Fresegna, C. L. Ursini, A. Ciervo, R. Maiello, S. Casciardi, S. Iavicoli, and D. Cavallo,
“Assessment of the influence of crystalline form on cyto-genotoxic and inflammatory effects
induced by TiO₂ nanoparticles on human bronchial and alveolar cells,” Nanomaterials, vol. 11, no.
1, Art. no. 253, 2021.
[14] A. M. Alotaibi, B. A. D. Williamson, S. Sathasivam, A. Kafizas, M. Alqahtani, C.
Sotelo-Vazquez, J. Buckeridge, J. Wu, S. P. Nair, D. O. Scanlon, and I. P. Parkin, “Enhanced
Photocatalytic and Antibacterial Ability of Cu-Doped Anatase TiO₂ Thin Films: Theory and
Experiment,” ACS Applied Materials & Interfaces, vol. 12, no. 13, pp. 15348–15361, 2020.
[15] F. M. Veronese and G. Pasut, “PEGylation, successful approach to drug delivery,” Drug
Discovery Today, vol. 10, no. 21, pp. 1451–1458, 2005.
[16] Q. Sun, K. Kanehira, and A. Taniguchi, “PEGylated TiO₂ nanoparticles mediated inhibition of
cell migration via integrin β1,” Science and Technology of Advanced Materials, vol. 19, no. 1, pp.
271–281, 2018.
[17] D. Venkatasubbu G., R. S., V. Ramakrishnan, and J. Kumar, “Folate-targeted PEGylated
titanium dioxide nanoparticles as a nano-carrier for targeted paclitaxel drug delivery,” Advanced
Powder Technology, vol. 24, no. 6, pp. 947–954, Nov. 2013.
[18] T. C. Zhu and J. C. Finlay, “The role of photodynamic therapy (PDT) physics,” Medical
Physics, vol. 35, no. 7, pp. 3127–3136, Jun. 2008.
[19] C. A. Robertson, D. Hawkins Evans, and H. Abrahamse, “Photodynamic therapy (PDT): A
short review on cellular mechanisms and cancer research applications for PDT,” Journal of
Photochemistry and Photobiology B: Biology, vol. 96, no. 1–2, pp. 1–8, 2009.
[20] C. S. Dela Cruz, L. T. Tanoue, R. A. Matthay, et al., "Lung cancer: epidemiology, etiology,
and prevention," Clinical Chest Medicine, vol. 32, no. 4, pp. 605–644, 2011.
[21] Y. Jin et al., “Global burden of lung cancer in 2022 and projections to 2050: incidence and
mortality estimates from GLOBOCAN,” Cancer Epidemiology, vol, Art. no. 102693, 2024.
[22] S. Hammerschmidt and H. Wirtz, "Lung cancer: current diagnosis and treatment," Deutsches
Ärzteblatt International, vol. 106, no. 49, pp. 809–818, 2009.
[23] J. Vansteenkiste, L. Crinò, C. Dooms, et al., "2nd ESMO Consensus Conference on Lung
Cancer: early-stage non-small-cell lung cancer consensus on diagnosis, treatment and follow-up,"
Annals of Oncology, vol. 25, pp. 1462–1474, 2014.

33
[24] Y. Alduais, et al., "Non-small cell lung cancer (NSCLC): A review of risk factors, diagnosis,
and treatment," Medicine, vol. 102, no. 8, p. e32899, 2023.
[25] F. R. Hirsch, et al., "Lung cancer: current therapies and new targeted treatments," The Lancet,
vol. 389, no. 10066, pp. 299–311, 2017.
[26] E. H. Grubbé, “Priority in the therapeutic use of X-rays,” Radiology, vol. 21, no. 2, pp. 156–
162, 1933.
[27] S. Chakraborty and T. Rahman, “The difficulties in cancer treatment,” ecancermedicalscience,
vol. 6, p. ed16, 2012.
[28] J. Usuda et al., “Photodynamic therapy (PDT) for lung cancers,” Journal of Thoracic Oncology,
vol. 1, no. 5, pp. 489–493, 2006.
[29] M. B. Schabath and M. L. Cote, “Cancer progress and priorities: lung cancer,” Cancer
Epidemiol. Biomarkers Prev., vol. 28, no. 10, pp. 1563–1579, 2019.
[30] S. Karki and A. Yadav, “Status of the rising number of patients with lung cancer in Nepal,”
Cancer Research, Statistics, and Treatment, vol. 8, no. 1, pp. 85–86, Jan.–Mar. 2025, doi:
10.4103/crst.crst_37_25.
[31] R. Shilpakar et al., "Lung Cancer in Nepal," J. Thorac. Oncol., vol. 17, no. 1, pp. 2229, Jan.
2022.
[32] S. Gavas, S. Quazi, and T. M. Karpiński, “Nanoparticles for Cancer Therapy: Current Progress
and Challenges,” Nanoscale Res Lett, vol. 16, no. 1, p. 173, 2021.
[33] A. A. et al., “Advances in nanoparticles-based approaches in cancer theranostics,” Adv.
Drug Deliv. Rev., vol. 197, pp. 114843, 2023. doi: 10.1016/[Link].2023.114843.
[34] H. F. Mahbubul, M. R. Islam, and F. H. A. Afsar, “Nanoparticles: properties, applications and
toxicities,” Arab. J. Chem., vol. 12, no. 7, pp. 908–931, Nov. 2019, doi:
10.1016/[Link].2017.05.011.
[35] Q. Yang, S. W. Jones, C. L. Parker, W. C. Zamboni, J. E. Bear, and S. K. Lai, “Evading Immune
Cell Uptake and Clearance Requires PEG Grafting at Densities Substantially Exceeding the
Minimum for Brush Conformation,” Mol Pharm, vol. 11, no. 4, pp. 1250– 1258, Apr. 2014.
[36] A. Marukurti et al., “Evaluation of anti-vibriocidal, antioxidant properties and cytotoxicity of
bio fabricated/green synthesized silver nanoparticles using Euphorbia hitra L. leaf extract,” Next
Materials, vol. 7, p. 100355, 2025.
[37] Y. Omidi and J. Barar, “Targeting tumor microenvironment: crossing tumor interstitial fluid
by multifunctional nanomedicines,” Bioimpacts, vol. 4, no. 2, p. 55, 2014.

34
[38] A. Zafar, M. Kanwal, N. Mumtaz, A. Rameez, F. Khan, and M. Bilal, “Green synthesis of
nanoparticles: a sustainable approach for environmental remediation,” Policy Res. J., vol. 3, no. 3,
pp. 279-292, 2025, doi: 10.15680/IJMRSET.2025.0805248.
[39] S. Ying et al., “Green synthesis of nanoparticles: Current developments and limitations,”
Environ Technol Innov, vol. 26, p. 102336, 2022.
[40] B. V.-C. Catalysis and undefined 2018, “Photocatalytic degradation of dyes: an overview,”
[Link] ViswanathanCurrent Catalysis, 34 2018•[Link], vol. 7, no. 2,
pp. 0–000, Dec. 2018, doi: 10.2174/2211544707666171219161846
[41] S. M. Hunagund, V. R. Desai, J. S. Kadadevarmath, D. A. Barretto, S. Vootla, and A. H. Sidarai,
“Biogenic and chemogenic synthesis of TiO 2 NPs via hydrothermal route and their antibacterial
activities,” [Link] Hunagund, VR Desai, JS Kadadevarmath, DA Barretto, S Vootla, AH
SidaraiRSC advances, 2016•[Link], vol. 6, no. 99, pp. 97438– 97444, 2016.
[42] P. Nyamukamba, O. Okoh, H. Mungondori, R. Taziwa, and S. Zinya, “Synthetic methods for
titanium dioxide nanoparticles: a review,” Titanium dioxide-material for a sustainable environment,
vol. 8, pp. 151–175, 2018
[43] D. F. Emerich and C. G. Thanos, “The pinpoint promise of nanoparticle-based drug delivery
and molecular diagnosis,” Biomol Eng, vol. 23, no. 4, pp. 171–184, Sep. 2006.
[44] P. Kale and S. Agashe, "Microemulsion based synthesis of nanoparticles," Advances in Colloid
and Interface Science, vol. 133, no. 1, pp. 129–138, Jan. 2007.
[45] C. Stubenrauch, T. Wielpütz, T. Sottmann, C. Roychowdhury, and F. J. DiSalvo,
“Microemulsions as templates for the synthesis of metallic nanoparticles,” Colloids and Surfaces
A: Physicochemical and Engineering Aspects, vol. 317, pp. 328–338, 2008, doi:
10.1016/[Link].2007.10.031.
[46] Q. Chen, X. Shen, and H. Gao, “Formation of nanoparticles in water-in-oil microemulsions
controlled by the yield of hydrated electron: The controlled reduction of Cu²⁺,” J. Colloid Interface
Sci., vol. 308, no. 2, pp. 491–499, 2007, doi: 10.1016/[Link].2006.12.021.
[47] X. Chen and S. S. Mao, “Titanium dioxide nanomaterials: Synthesis, properties, modifications
and applications,” Chem Rev, vol. 107, no. 7, pp. 2891–2959, Jul. 2007.
[48] G. Raja, S. Cao, D. H. Kim, and T. J. Kim, “Mechanoregulation of titanium dioxide
nanoparticles in cancer therapy,” Feb. 01, 2020, Elsevier Ltd.
[49] A. Weir, P. Westerhoff, L. Fabricius, K. Hristovski, and N. Von Goetz, “Titanium dioxide
nanoparticles in food and personal care products,” Environ Sci Technol, vol. 46, no. 4, pp. 2242–
2250, 2012.

35
[50] M. Coronell, G. Toscano-Lucas, R. Solano, and A. Herrera, “Green Synthesis of SilverDoped
Titanium Dioxide Nanostructures for Acetaminophen Degradation Under Solar Radiation,”
Ingeniería y Universidad, vol. 26, 2022. “Synthesis titanium dioxide nanoparticles doped with silver
and Novel antibacterial activity”, doi: 10.1088/1742-6596/1999/1/012033.
[51] M. N. Aravind and O. D. Naidu, “Parameter Estimation of Power Transformer in Presence of
Bad Measurement Data,” ICPEE 2021 - 2021 1st International Conference on Power Electronics
and Energy, Jan. 2021.
[52] D. Masekela, N. C. Hintsho-Mbita, B. Ntsendwana, and N. Mabuba, “Thin Films
(FTO/BaTiO3/AgNPs) for Enhanced Piezo-Photocatalytic Degradation of Methylene Blue and
Ciprofloxacin in Wastewater,” ACS Omega, vol. 7, no. 28, pp. 35 24329– 24343,Jul.2022.
[53] P. Balaraman, B. Balasubramanian, … W. L.-E., and undefined 2022, “Sargassum
myriocystum-mediated TiO2-nanoparticles and their antimicrobial, larvicidal activities and
enhanced photocatalytic degradation of various dyes,” ElsevierP Balaraman, B Balasubramanian,
WC Liu, D Kaliannan, M Durai, H Kamyab, M AlwetaishiEnvironmental research, 2022•Elsevier,
Accessed: Sep. 24, 2024.
[54] M. Ahamed, M. A. M. Khan, M. J. Akhtar, H. A. Alhadlaq, and A. Alshamsan, “Agdoping
regulates the cytotoxicity of TiO2 nanoparticles via oxidative stress in human cancer cells,”
Scientific Reports 2017 7:1, vol. 7, no. 1, pp. 1–14, Dec. 2017, doi: 10.1038/s41598-017-17559-9.
[55] S. Y. Chae, M. K. Park, S. K. Lee, T. Y. Kim, S. K. Kim, and W. I. Lee, “Preparation of size-
controlled TiO2 nanoparticles and derivation of optically transparent photocatalytic films,”
Chemistry of Materials, vol. 15, no. 17, pp. 3326–3331, Aug. 2003, doi: 10.1021/CM030171D.
[56] H. G. Park and M. K. Yeo, “Comparison of gene expression changes induced by exposure to
Ag, Cu-TiO2, and TiO2 nanoparticles in zebrafish embryos,” Mol Cell Toxicol, vol. 9, no. 2, pp.
129–139, Jun. 2013, doi: 10.1007/S13273-013-0017-0.
[57] P. V. AshaRani, M. P. Hande, and S. Valiyaveettil, “Anti-proliferative activity of silver
nanoparticles,” BMC Cell Biol, vol. 10, no. 1, p. 65, Sep. 2009.
[58] S. Yamaguchi et al., “Sonodynamic therapy using water-dispersed TiO2- polyethylene glycol
compound on glioma cells: Comparison of cytotoxic mechanism 36 with photodynamic therapy,”
Ultrason Sonochem, vol. 18, no. 5, pp. 1197–1204, Sep. 2011, doi:
10.1016/[Link].2010.12.017.
[59] R. I. MacCuspie, A. J. Allen, and V. A. Hackley, “Dispersion stabilization of silver
nanoparticles in synthetic lung fluid studied under in situ conditions,” Nanotoxicology, vol. 5, no.
2, pp. 140–156, Jun. 2011, doi: 10.3109/17435390.2010.504311.

36
[60] U. Kreibig and L. Genzel, “Optical absorption of small metallic particles,” Surf Sci, vol. 156,
no. PART 2, pp. 678–700, Jun. 1985.
[61] M. Carofiglio, S. Barui, V. Cauda, and M. Laurenti, “Doped zinc oxide nanoparticles:
Synthesis, characterization and potential use in nanomedicine,” Applied Sciences, vol. 10, no. 15,
Art. no. 5194, 2020, doi: 10.3390/app10155194.
[62] S. Rao, M. P. Shilpa, S. J. Shetty, and S. S. Bhat, Zn-doped TiO₂ Nanoparticles:
Enhanced Catalytic and Nonlinear Optical Properties, J. Mater. Sci.: Mater. Electron., vol. 36, art.
1144, 2025.
[63] V. G. Gupta, S. Bhavanasi, M. Quadir, K. Singh, G. Ghosh, K. Vasamreddy, A. Ghosh, T. J.
Siahaan, S. Banerjee and S. K. Banerjee, “Protein PEGylation for cancer therapy: bench to bedside,”
Journal of Cell Communication and Signaling, vol. 13, no. 3, pp. 319–330, 2019, doi:
10.1007/s12079-018-0492-0.
[64] W. Li, P. Zhan, E. De Clercq, H. Lou, and X. Liu, “Current drug research on PEGylation with
small molecular agents,” Prog. Polym. Sci., vol. 38, no. 3–4, pp. 421–444, 2013, doi:
10.1016/[Link].2012.07.006.
[65] P. Agostinis et al., “PHOTODYNAMIC THERAPY OF CANCER: AN UPDATE,” CA
Cancer J Clin, vol. 61, no. 4, p. 250, Jul. 2011.
[66] C. B. Simone, et al., "Photodynamic therapy for the treatment of non-small cell lung cancer,"
Journal of Thoracic Disease, vol. 4, no. 1, pp. 63–75, 2012.
[67] C. B. Simone, et al., "Photodynamic therapy for the treatment of non-small cell lung cancer,"
Journal of Thoracic Disease, vol. 4, no. 1, pp. 63–75, 2012. “Photosensitization | Photodynamic
Therapy, Light Therapy & Photobiology | Britannica.” Accessed: Sep. 26, 2024.
[68] S. Sargazi et al., “Application of titanium dioxide nanoparticles in photothermal and
photodynamic therapy of cancer: An updated and comprehensive review,” J Drug Deliv Sci
Technol, vol. 75, p. 103605, Sep. 2022.
[69] R. Asahi, T. Morikawa, T. Ohwaki, K. Aoki, and Y. Taga, “Visible-Light Photocatalysis in
Nitrogen-Doped Titanium Oxides,” Science (1979), vol. 293, no. 5528, pp. 269–271, Jul. 2001.
[70] A. Karbalaei, Z. Mohammadalipour, M. Rahmati, A. Khataee, M. Amin Moosavi, and
A. Professor, “GO/TiO2 Hybrid Nanoparticles as New Photosensitizers in Photodynamic Therapy
of A375 Melanoma Cancer Cells,” Journal of Skin and Stem Cell 2017 4:1, vol. 4, no. 1, p. 63984,
Jun. 2017.
[71] M. A. Moosavi et al., “Photodynamic N-TiO2 Nanoparticle Treatment Induces
Controlled ROS-mediated Autophagy and Terminal Differentiation of Leukemia Cells,” Scientific
Reports 2016 6:1, vol. 6, no. 1, pp. 1–16, Oct. 2016.
37
[72] A. Karbalaei, Z. Mohammadalipour, M. Rahmati, A. Khataee, M. Amin Moosavi, and A.
Professor, “GO/TiO2 Hybrid Nanoparticles as New Photosensitizers in Photodynamic Therapy of
A375 Melanoma Cancer Cells,” Journal of Skin and Stem Cell 2017 4:1, vol. 4, no. 1, p. 63984,
Jun. 2017.
[73] A. Sohail Shah et al., “IMPACT OF SILVER DOPANT ON STRUCTURAL AND OPTICAL
PROPERTIES OF TiO2 NANOPARTICLE IMPACT OF SILVER DOPANT ON STRUCTURAL
AND OPTICAL PROPERTIES OF TiO 2 NANOPARTICLES.”
[74] J. Epp, “X-Ray Diffraction (XRD) Techniques for Materials Characterization,” in Materials
Characterization Using Nondestructive Evaluation (NDE) Methods, Elsevier Inc., 2016, pp. 81–
124.
[75] A. Mohammed and A. Abdullah, “SCANNING ELECTRON MICROSCOPY (SEM):
A REVIEW.” A. Mandru, J. Mane, and R. Mandapati, “A Review on UV-visible spectroscopy,”
2023.
[76] M. A. Mohamed, J. Jaafar, A. F. Ismail, M. H. D. Othman, and M. A. Rahman, “Fourier
Transform Infrared (FTIR) Spectroscopy,” in Membrane Characterization, Elsevier Inc., 2017, pp.
3–29.
[77] G. Y. Nigussie et al., “Antibacterial activity of Ag-Doped TiO2 and Ag-Doped ZnO
nanoparticles,” International Journal of Photoenergy, vol. 2018, 2018.
[78] R. Sundararajan, R. Rajendran, S. Shahid, D. K. Santosh, K. Ramakrishnan, et al., “Effect of
irreversible electroporation on cancer cells,” in Proc. IEEE Conf. Electr. Insul. Dielectr. Phenomena
(CEIDP), 2011, pp. 164–167.
[79] M. Lieber, B. Smith, A. Szakal, W. Nelson-Rees, and G. Todaro, “A continuous tumorcell line
from a human lung carcinoma with properties of type II alveolar epithelial cells,” Int. J. Cancer, vol.
17, pp. 62–70, 1976.
[80] M. Ahamed, M. A. M. Khan, M. J. Akhtar, H. A. Alhadlaq, and A. Alshamsan, “Role of Zn
doping in oxidative stress mediated cytotoxicity of TiO₂ nanoparticles in human breast cancer MCF-
7 cells,” Scientific Reports, vol. 6, no. 1, p. 30196, Jul. 2016.
[81] S. Bhullar, N. Goyal, and S. Gupta, “FericipXT-coated PEGylated rutile TiO₂ nanoparticles in
drug delivery: in vitro assessment of imatinib release,” RSC Advances, vol. 14, no. 33, pp. 23886–
23901, Jul. 2024.
[82] B. A. Miu, M. S. Stan, M. Mernea, A. Dinischiotu, and I. C. Voinea, “Pure epigallocatechin-3-
gallate-assisted green synthesis of highly stable titanium dioxide nanoparticles,” Materials, vol. 16,
no. 2, Art. no. 679, Jan. 2023, doi: 10.3390/ma16020679.

38
[83] Lee, Man Sig, Gun-Dae Lee, Chang-Sik Ju, and Seong-Soo Hong. "Preparations of nanosized
TiO2 in reverse microemulsion and their photocatalytic activity." Solar energy materials and solar
cells 88, no. 4 (2005): 389-401.
[84] D. Ziental et al., “Titanium Dioxide Nanoparticles: Prospects and Applications in
Medicine,” Nanomaterials, vol. 10, no. 2, p. 387, Feb. 2020, doi: 10.3390/NANO10020387. [88] S.
A. Khan, S. B. Khan, L. U. Khan, A. Farooq, K. Akhtar, and A. M. Asiri, “Fourier Transform
Infrared Spectroscopy: Fundamentals and Application in Functional Groups and Nanomaterials
Characterization,” pp. 317–344, 2018.
[85] M. H. Nateq and R. Ceccato, “Sol–gel synthesis of TiO₂ nanocrystalline particles with
enhanced surface area through the reverse micelle approach,” Advances in Materials Science and
Engineering, vol. 2019, Art. no. 1567824, 2019, doi: 10.1155/2019/1567824.
[86] O. U. Akakuru, Z. M. Iqbal, and A. Wu, “TiO₂ nanoparticles: Properties and applications,” in
TiO₂ Nanoparticles: Applications in Nanobiotechnology and Nanomedicine, A. Wu and W. Ren,
Eds. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2020, pp. 1–66, doi:
10.1002/9783527825431.ch1.
[87] I. Chandraprabha, R. Kottayi, V. Ilangovan, and R. Sittaramane, “Structural and optical studies
of zinc-doped TiO₂ nanoparticles,” IOSR Journal of Applied Physics, vol. 13, no. 1, pp. 49–55, Jan.–
Feb. 2021, doi: 10.9790/4861-1301024955.
[88] B. D. Napruszewska et al., “TiO₂ nanoparticles with adjustable phase composition prepared by
an inverse microemulsion method: Physicochemical characterization and photocatalytic
properties,” Nanomaterials, vol. 14, no. 13, Art. no. 1130, Jun. 2024, doi: 10.3390/nano14131130.
[89] M. H. Nateq and R. Ceccato, “Sol-Gel Synthesis of TiO₂ Nanocrystalline Particles with
Enhanced Surface Area through the Reverse Micelle Approach,” Adv. Mater. Sci. Eng., vol. 2019,
Art. no. 1567824, 2019, doi: 10.1155/2019/1567824.

[90] O. U. Akakuru, Z. M. Iqbal, and A. Wu, “TiO₂ Nanoparticles: Properties and Applications,”
in TiO₂ Nanoparticles: Applications in Nanobiotechnology and Nanomedicine, A. Wu and W.
Ren, Eds. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2020, pp. 1–66, doi:
10.1002/9783527825431.ch1

[91] I. Chandraprabha, R. Kottayi, V. Ilangovan, and R. Sittaramane, “Structural and optical

studies of Zinc doped TiO₂ nanoparticles,” IOSR Journal of Applied Physics, vol. 13, no. 1, pp.
49–55, Jan.–Feb. 2021, doi: 10.9790/4861-1301024955.

[92] B. D. Napruszewska, A. Walczyk, D. Duraczyńska, J. Kryściak-Czerwenka, R. Karcz, A.

Gaweł, P. Nowak, and E. M. Serwicka, “TiO₂ Nanoparticles with Adjustable Phase Composition

39
Prepared by an Inverse Microemulsion Method: Physicochemical Characterization and
Photocatalytic Properties,” Nanomaterials, vol. 14, no. 13, Art. 1130, Jun. 2024, doi:
10.3390/nano14131130.

40