CHAPTER-1

INTRODUCTION

1.1 BREAST CANCER

Breast cancer is the most common cancer diagnosed in

women, accounting for more than 1 in 10 new cancer diagnoses each
year. It is the second most common cause of death from cancer among
women in the world. Anatomically, the breast has milk-producing
glands in front of the chest wall. They lie on the pectoralis major
muscle, and there are ligaments support the breast and attach it to the
chest wall. Fifteen to 20 lobes circularly arranged to form the breast.
The fat that covers the lobes determines the breast size and shape. Each
lobe is formed by lobules containing the glands responsible for milk
production in response to hormone stimulation.

Breast cancer always evolves silently. Most of the patients

discover their disease during their routine screening. Others may
present with an accidentally discovered breast lump, change of breast
shape or size, or nipple discharge. However, gastralgia is not
uncommon. Physical examination, imaging, especially mammography,
and tissue biopsy must be done to diagnose breast cancer. The survival
rate improves with early diagnosis.

1.2 STAGES OF BREAST CANCER

TNM staging system

Numbers or letters after T, N, and M provide more details

about each of these factors.

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1.2.1 T categories for breast cancer

T followed by a number from 0 to 4 describes the main

(primary) tumour’s the chest wall under the breast.[4][3] Higher T
numbers mean a larger tumour and/or wider spread to tissues near the
breast.

TX: Primary tumour cannot be assessed.

T0: No evidence of primary tumour.

Tis: Carcinoma in situ (DCIS, or Paget disease of the breast with no

associated tumour mass)

T1 (includes T1a, T1b, and T1c): Tumour is 2 cm (3/4 of an inch) or

less across.

T2: Tumour is more than 2 cm but not more than 5 cm (2 inches) across.

T3: Tumour is more than 5 cm across.

T4 (includes T4a, T4b, T4c, and T4d): Tumour of any size growing into
the chest wall or skin. This includes inflammatory breast cancer.

1.2.2 N categories for breast cancer

N followed by a number from 0 to 3 indicates whether the

cancer has spread to lymph nodes near the breast and, if so, how many
lymph nodes are involved. NX: Nearby lymph nodes cannot be assessed
(for example, if they were removed previously). N0: Cancer has not
spread to nearby lymph nodes. N0(I +): The area of cancer spread
contains fewer than 200 cells and is smaller than 0.2 mm.

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 The abbreviation "I +" means that a small number of cancer
cells (called isolated tumour cells). N0(mol+): Traces of cancer cells
were detected using a technique called RTPCR. N1: Cancer has spread
to 1 to 3 axillary (underarm) lymph node(s), and/or cancer is found in
internal mammary lymph nodes (those near the breast bone) on sentinel
lymph node biopsy. N1mi: The areas of cancer spread in the lymph
nodes are at least 0.2mm across, but not larger than 2mm.

N1a: Cancer has spread to 1 to 3 lymph nodes under the arm with at
least one area of cancer spread greater than 2 mm across.
N1b: Cancer has spread to internal mammary lymph nodes on the same
side as the cancer N1c: Both N1a and N1b apply.
N2: Cancer has spread to 4 to 9 lymph nodes under the arm, or cancer
has enlarged the internal mammary lymph nodes.
N2a: Cancer has spread to 4 to 9 lymph nodes under the arm, with at
least one area of cancer spread larger than 2 mm.
N2b: Cancer has spread to one or more internal mammary lymph
nodes, causing them to become enlarged.
N3: Any of the following:
N3a: either: Cancer has spread to 10 or more axillary lymph nodes,
with at least one area of cancer spread greater than 2 mm,
N3b: either: Cancer is found in at least one axillary lymph node (with
at least one area of cancer spread greater than 2 mm) and has enlarged
the internal mammary lymph nodes,
N3c: Cancer has spread to the lymph nodes above the collarbone
(supraclavicular nodes) on the same side of the cancer with at least one
area of cancer spread greater than 2 mm.

3
1.2.3 M categories for breast cancer
M followed by a 0 or 1 indicates whether the cancer has spread to
distant organs -- for example, the lungs, liver, or bones.
M0: No distant spread is found on x-rays (or other imaging tests) or by
physical exam. [4]
M0(I +): Small numbers of cancer cells are found in blood or bone
marrow (found only by special tests)
M1: Cancer has spread to distant organs (most often to the bones, lungs,
brain, or liver) as imaging tests or by physical exam, and/or a biopsy of
one of these areas proves cancer has larger than 0.2mm.

Fig:1.1 Stages of Breast Cancer

1.3 TREATMENT

The treatment of breast cancer typically depends on various

factors including the stage of cancer, the type of breast cancer, hormone
receptor status, HER2 status, and the patient's overall health and
preferences. Here's an overview of common treatment options:

4
1.3.1 Surgery

The most standard breast surgery approaches are either total

excision of the breast (mastectomy), usually followed by breast
reconstruction, or breast-conserving surgery (lumpectomy).
Lumpectomy entails the excision of the breast tumour with a margin of
surrounding normal tissue. The recommended margins status is defined
as “no ink on tumour”, meaning no remaining tumour cells at the tissue
edge. Studies show that total mastectomy and lumpectomy plus
irradiation are equivalent regarding relapse-free and overall survival
(OS). [2]

Contraindications for breast-conserving surgery include the

presence of diffuse microcalcifications (suspicious or malignant-
appearing), disease that cannot be incorporated by local excision with
satisfactory cosmetic result, and ATM (ataxia-telangiectasia mutated)
mutation (biallelic inactivation).

The surgery to remove axillary lymph nodes is useful to

determine cancerous cell spread and for therapeutic purposes. For
instance, axillary lymph node dissection (ALND) can improve survival
rated by removing remaining tumour cells.

ALND used to be the goal standard for removing positive

lymph nodes. However, clinical trials showed that sentinel lymph node
biopsy (SLNB) had the same effect as ALND regarding disease-free
survival (DFS) and OS. Other clinical trials demonstrated that ALND
was not necessary for all patients with positive lymph nodes.

5
 Moreover, most patients who receive radiation and systemic
treatment after SLNB have negative lymph nodes as these treatments
are sufficient in eliminating residual tumour cells.

1.3.2 Radiotherapy

Radiation therapy has been used to treat cancer since

Rontgen discovered the X-ray in 1895. High-energy radiations are
applied to the whole breast or a portion of the breast (after breast-
conservative surgery), chest wall (after mastectomy), and regional
lymph nodes. A meta-analysis showed that radiation following
conservative surgery offered more benefits to patients with higher-risk
BC while patients with small, low-grade tumours could forego radiation
therapy. Postmastectomy radiation to the chest wall in patients with
positive lymph nodes is associated with decreased recurrence risk and
BC mortality compared to patients with negative lymph nodes.[5][7]

A radiation boost to the regional node radiation treatment

can be incorporated after mastectomy for patients at higher risk for
recurrence. This additional radiation boost to regional nodes following
mastectomy is associated with improved (DFS) but is also associated
with an increase in radiation toxicities such as pneumonitis and
lymphedema. Radiotherapy can be administered concurrently with
personalized therapy (anti-HER2 therapy or endocrine therapy).

As one of the major side effects of radiotherapy is

cardiotoxicity, it is critical to minimize exposure to the heart and lungs.
High-energy radiations are applied to the whole breast or a portion of
the breast (after breast-conservative surgery), chest wall (after
mastectomy).
6
Fig:1.2 Stages of breast cancer

7
 Additional techniques can be used to reduce the radiation
exposure to the heart, lungs, and normal tissue such as prone
positioning, respiratory control, or intensity-modulated radiotherapy.
Advanced invasive BC can exhibit radiation therapy resistance.

The hypoxic tumour microenvironment, which lacks

oxygen, leads to increased cell proliferation, apoptosis resistance, and
radiotherapy resistance. The major player of this resistance is the HIF-
1α (hypoxia-inducible factor 1 alpha) protein. Indeed, HIF-1α
overexpression is caused by low oxygen levels within the
microenvironment and promotes the maintenance of hypoxia by
allowing tumoral cells to survive in a hypoxic microenvironment.
Cancer stem cells (CSC) could also have a role in radiation therapy
resistance.

CSC can self-renew and initiate subpopulations of

differential progeny, and a hypoxic microenvironment is ideal for CSC
survival and proliferation. Radiation therapy is used to treat all BC
subtypes, but its implication is more important for TNBC, as there is no
personalized therapy for this subtype. It has been shown that
radiotherapy benefits TNBC patients both after conserving surgery and
mastectomy.

1.3.3 Chemotherapy

BC chemotherapy comprises several families of cytotoxic

drugs, including alkylating agents, antimetabolites and tubulin
inhibitors. Cyclophosphamide is a nitrogen mustard alkylating agent
causing breakage of the DNA strands.[10]

8
 The mechanism of action for anthracyclines (doxorubicin,
daunorubicin, eirenicon, and idarubicin) includes DNA intercalation,
thereby inhibiting macromolecular biosynthesis.

Texans, including docetaxel and paclitaxel, bind to

microtubules and prevent their disassembly, leading to cell cycle arrest
and apoptosis. Chemotherapy can be administered in the neoadjuvant
or adjuvant setting and for metastatic BC treatment.

1.3.4 Neoadjuvant Chemotherapy (NAC)

Neoadjuvant chemotherapy was initially administered for

non-metastatic but inoperable BC, defined as unreachable tumours.
Then, chemotherapy was used before the surgery for operable tumours
to facilitate breast conservation. Studies demonstrated that
chemotherapy administered before surgery is as effective as
administered after surgery. The NSABP-B-18 trial compared the effects
of doxorubicin and cyclophosphamide administered either
postoperatively or preoperatively.

This trial showed that NAC reduces the rate of axillary

metastases in node-negative BC patients. Some patients fail to achieve
pathologic complete response after a full course of NAC. Unfortunately,
there is no consensus regarding the treatment strategy to follow for
patients with residual disease after surgery.

The BC subtype plays an important role in the response to

NAC. Indeed, TNBC and HER2+ BC are more likely to be sensitive to
chemotherapy. Hence, NAC is a good strategy to maximize pathologic
complete response in these BC subtypes.

9
1.3.5 Adjuvant Chemotherapy (AC)

Adjuvant chemotherapy is administered to BC patients with

lymph nodes metastases or a high risk of recurrence. The standard
chemotherapy treatment comprises an anthracycline and a taxeme.

The two most common regimens are cyclophosphamide and

doxorubicin for four cycles followed by paclitaxel for four cycles. Then
patients are given the previous combination of therapies followed by
either weekly paclitaxel for 12 weeks, or docetaxel every 3 weeks for
four cycles. Like neoadjuvant therapy, patients with HR-negative BC
receive more benefits from adjuvant therapy (i.e., reduction of BC
recurrence and mortality) than HR+ BC patients.

However, for patients with HR+, node-negative BC

associated with a high Oncotype recurrence score (≥31), calculated
from the expression of 16 BC-related genes and 5 reference genes,
adjuvant chemotherapy reduces the risk of recurrence. The Tailor
clinical trial showed that HR+ BC patients with a low Oncotype
recurrence score do not benefit from chemotherapy alone.

1.3.6 Endocrine Therapy

Endocrine therapy is the main strategy to treat HR positive

invasive BC. The purpose of this therapy is to target the ER directly
(selective estrogen receptors modulators and degraders) or the estrogen
synthesis (aromatase inhibitors).

The most common types of endocrine therapy are selective

estrogen receptor modulators (SERMs), selective modulators estrogen
receptor degraders (SERDs), and aromatase inhibitors (AIs).

10
 An external file that holds a picture, illustration, etc.
Endocrine therapy mechanisms of action and resistance. The left part of
the figure shows the mechanism of endocrine therapy through
aromatase inhibitors, tamoxifen, and fluvastatin. Endocrine therapy
mechanism of action and resistance are described in Figure.

The right part of the figure describes the mechanisms of

resistance to endocrine therapy through the epigenetic modifications,
the increase of coactivators and cell cycle actors, and the activation of
other signalling pathways

Estrogens can go through the plasma membrane by a.

diffusion as they are small non-polar lipid soluble molecules; b. binding
to membrane ER initiating the activation of Ras/Raf/MAPK and
PI3K/Akt signalling pathways which are blocked by tamoxifen.

1: inhibition of ER dimerization; 2: blockage of nucleus

access; 3: ER degradation. ER: estrogen receptor; AIB1: amplified in
breast cancer 1; IGF-1R: insulin growth factor receptor 1; IGF: insulin
growth factor; HER: human epidermal receptors; EGF: epidermal
growth factor; HB-EGF: heparin-binding EGF-like growth factor;
TGF-α: transforming growth factor alpha; MEK/MAPK: mitogen
activated protein kinase; PI3K: phosphoinositide 3-kinase; mTOR:
mammalian target of rapamycin; Me: methylation; Ac:
acetylation.[13][8]

 Selective Estrogen Receptor Modulators (SERMs)

 Selective Estrogen Receptor Degraders (SERDs)
 Aromatase Inhibitors (AIs).

11
 Fig:1.3 Endocrine therapy

Estrogens can go through the plasma membrane by a.

diffusion as they are small non-polar lipid soluble molecules; b. binding
to membrane ER initiating the activation of Ras/Raf/MAPK and
PI3K/Akt signalling pathways which are blocked by tamoxifen. 1:
inhibition of ER dimerization; 2: blockage of nucleus access; 3: ER
degradation. ER: estrogen receptor; AIB1: amplified in breast cancer 1;
IGF-1R: insulin growth factor receptor 1; IGF: insulin growth factor;
HER: human epidermal receptors; EGF: epidermal growth factor; HB-
EGF: heparin-binding EGF-like growth factor; TGF-α: transforming
growth factor alpha; MEK/MAPK: mitogen activated protein kinase;
PI3K: phosphoinositide 3-kinase; mTOR: mammalian target of
rapamycin; Me: methylation; Ac: acetylation.

12
1.3.7 DRAW BACKS

One of the major drawbacks of chemotherapy is its side

effects. The early side effects (0–6 months of treatment) involve fatigue,
alopecia, cytopenia (reduction in the number of normal blood cells),
muscle pain, neurocognitive dysfunction, and chemo-induced
peripheral neuropathy. The chronic or late side effects (after 6 months
of treatment) include cardiomyopathy, second cancers, early
menopause, sterility, and psychosocial impacts. As mentioned
previously in this review, chemotherapy is composed of taxanes,
anthracyclines and cyclophosphamide. Each of these molecules can
lead to resistance.

1.4 STRATEGIES OF DRUG DELIVERY

1.4.1 Organic Drug Delivery

In organic drug delivery, scientists use natural or synthetic

molecules that are carbon-based (organic) to create systems that
transport drugs to specific parts of the body. These systems could be
things like liposomes (tiny fat bubbles), polymers (long chains of
repeating molecules), or even natural proteins. They're designed to
carry drugs safely to their target in the body, like a breast tumour, while
minimizing side effects on healthy tissues. It is mainly used to carry the
drugs accurately in the targeted body like tumor with minimal side
effect to the side healthy tissue.

1.4.2 Inorganic Drug Delivery

In contrast, inorganic drug delivery involves using materials

that aren't based on carbon, such as metals or metal oxides.

13
 These materials can also be engineered to transport drugs to
specific locations in the body, offering unique properties like stability
or responsiveness to external stimuli (like light or magnetic fields) that
can help control drug release.

1.4.3 Receptors

Receptors are like locks on the surface of cells, and they're

specific to certain molecules called ligands, which are like keys. In drug
delivery, scientists can design drug carriers that have ligands on their
surface. These ligands can recognize and bind to receptors that are
overexpressed on cancer cells (like HER2 receptors in breast cancer).
Once bound, the carrier can release its drug payload directly into the
cancer cell, minimizing damage to healthy cells.[1][6]

1.4.4 Localized Drug Delivery

Localized drug delivery means delivering medication

directly to the site where it's needed, rather than letting it circulate
throughout the entire body. For breast cancer, this could involve placing
drug-carrying devices or implants directly into the tumour or nearby
tissue, injecting drugs directly into the tumour, or using targeted drug
carriers that deliver medication specifically to cancer cells while
sparing healthy tissue. This approach helps to maximize the
concentration of the drug at the tumour site while minimizing side
effects on the rest of the body.

1.5 DRUG DELIVERY VEHICLES

These drug delivery vehicles represent a diverse range of

nanotechnologies that hold great promise for improving the efficacy
and safety of breast cancer treatment.
14
 Ongoing research continues to explore novel formulations
and therapeutic strategies to address the challenges associated with
breast cancer therapy.

Drug delivery vehicles for breast cancer are designed to

transport therapeutic agents specifically to breast cancer cells,
maximizing treatment efficacy while minimizing side effects on healthy
tissues. Here are some commonly used drugs delivery vehicles for
breast cancer.

1.5.1 Liposomes:

Liposomes are microscopic lipid vesicles that can

encapsulate a variety of drugs, including chemotherapy agents,
hormones, and targeted therapies. They can be engineered to target
breast cancer cells by conjugating targeting ligands to their surface,
such as antibodies or peptides that recognize receptors overexpressed
on cancer cells. Liposomes protect the drug payload from degradation
and deliver it directly to the tumour site, improving therapeutic
outcomes and reducing systemic toxicity.

1.5.2 Polymeric Nanoparticles:

Polymeric nanoparticles are composed of biocompatible

polymers that can encapsulate drugs and release them in a controlled
manner. These nanoparticles can be surface-modified with targeting
ligands to enhance their specificity for breast cancer cells. Polymeric
nanoparticles offer advantages such as sustained drug release, enhanced
tumour penetration, and the ability to encapsulate both hydrophobic and
hydrophilic drugs.

15
 1.5.3 Dendrimers

Dendrimers are highly branched, tree-like molecules with a

defined structure that allows precise control over drug loading and
release kinetics. They can be functionalized with targeting ligands and
imaging agents to improve specificity and enable real-time monitoring
of treatment response.

Dendrimers have shown promise for delivering a variety of

therapeutic agents, including chemotherapy drugs and nucleic acid-
based therapies, to breast cancer cells.[9]

1.5.4 Nanocrystals

Nanocrystals are solid drug particles with sizes in the

nanometre range that can improve drug solubility and bioavailability.
Nanocrystals offer advantages such as increased drug stability,
improved pharmacokinetics, and the potential for combination therapy
with synergistic drug combinations also it can improve drug solubility.

1.5.5 Gold Nanoparticles

Gold nanoparticles have unique optical and chemical

properties that make them attractive for breast cancer therapy. They can
be functionalized with targeting ligand conjugated to drugs or imaging
cancer therapy. They can be functionalized with targeting ligands. Gold
Nanoparticle is mainly attractive for its optical and chemical properties.
It works as a target ligandin cancer therapy.[11]

16
 Fig:1.4 Drug delivery vehicles

Micelles

Micelles are self-assembling structures formed by

amphiphilic molecules in aqueous solutions. They can encapsulate
hydrophobic drugs within their core and present hydrophilic surfaces for
improved stability and biocompatibility. Micelles can be modified with
targeting ligands to enhance their accumulation in breast tumours.

17
 CHAPTER-2
LITERATURE REVIEW

2.1 CHITOSAN

It uses in many fields. Hence, chitosan derivatives are

synthesized by chemically modifying chitosan. Chemical modification
of chitosan enhances its physicochemical properties and extends the
variety of applications. Chitosan nanoparticles (CSNPs) are safe and
effective nanocarrier systems showing controlled drug release and
targeted drug delivery. CSNPs Chitosan is a naturally occurring amino
polysaccharide obtained by deacetylation of chitin and is second most
commonly used natural polymer. Its non-toxic, biocompatibility and
biodegradability properties have prompted extensive research into
several applications. Additionally, chitosan has important intrinsic
properties like mucoadhesive, permeation enhancer, and antimicrobial
properties. The amino and hydroxyl groups of chitosan [12] are
essential for its properties like mucoadhesive, permeation
enhancement, controlled drug release, in situ gelation, and
antimicrobial. The insolubility of chitosan in water and most organic
solvents has limited have versatile biomedical applications in
therapeutics and drug delivery. Due to diverse physicochemical
properties of chitosan and its derivatives, it has significant potential for
applications in the pharmaceutical and biomedical fields. The present
review highlights a comprehensive information about the properties and
bioactivities of chitosan and its derivative-based nanomaterials for drug
delivery, gene therapy, vaccine delivery, tissue engineering, diagnosis
and other biomedical application.

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2.2 TECHNOLOGICAL CHITOSAN PROPERTIES

Solubility

Chitosan is produced by deacetylation of chitin; in this

process, some Acetylglucosamine moieties are converted into
glucosamine units. The presence of large amounts of protonated -NH2
groups on the chitosan structure accounts for its solubility in acid
aqueous media since its pKa value is approximately 6.5. When around
50% of all amino groups are protonated, chitosan becomes soluble
Chitosan solubility depends on different factors such as polymer
molecular weight, degree of acetylation, pH, temperature, and polymer
crystallinity

Viscosity

The viscosity of polymers is a parameter of great interest

from the technological point of view since highly viscous solutions are
difficult to manage. Moreover, viscometry is a powerful tool for
determining chitosan’s molecular weight, as it is a simple and rapid
method even though it is not an absolute method, therefore requiring
the determination of constants that are specific to the solvent. The
average molecular weight is determined by the Mark– Houwink –
Sakurada equation, which relates this parameter with the intrinsic
viscosity:

η = KMvα

Where K and α are constants that must be determined experimentally.

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2.3 BIOLOGICAL PROPERTIES OF CHITOSAN

Chitin, chitosan, oligosaccharides, and derivatives exert

many biological activities including antitumoral, antimicrobial,
antioxidant, and anti-inflammatory activities, which could be used as
therapeutic polymers. It is remarkable that up today chitosan and
chitosan hydrochloride are only accepted as excipients by the regulatory
agencies and not as a drug for the treatment of diseases. [15]

Antimicrobial Activity

Bacterial resistance to antibiotics is a critical public health

concern and, therefore, there is an urgency to find alternatives to
antibiotics. Chitosan, chitosan derivatives and Chito oligosaccharides
exert antimicrobial activity against different microorganisms, including
bacteria, filamentous fungi, and yeast; some examples of the different
growth-inhibitory activity since bacteria is able to grow after the
polymer is removed from the media. This is of importance since
resistant populations might emerge if the cells adapt to chitosan

Antioxidant Activity

Antioxidants are gaining interest due to the relationship

between oxidative stress and several diseases such as Alzheimer’s
disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral
sclerosis, and cancer. Moreover, it is related to complications in other
diseases such as diabetes.

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 Anti-Inflammatory Properties

The inflammatory process is an automatic physiological

response of the body related to tissue damage. The main goal of the
inflammatory response is to bring circulating leukocytes and plasma
proteins to the site of the infection or tissue damage, to eliminate the
causative agent, when possible, and to start the healing process.

Although inflammation is necessary for survival, when it is

very severe, unable to eradicate the causative agent, or is directed against
the host, the inflammatory process may cause damage. The inflammatory
process is strongly related to the generation of free radicals. Again, this
activity seems to be more remarkable when the molecular weight of the
chitosan is reduced and Chito oligosaccharides exhibit higher activity.

Metallic Nanoparticles and Chitosan

Metallic nanoparticles are usually defined as particles of

metal atoms with sizes ranging between 1 nm to a few hundred
nanometres. These particles exhibit optical, chemical, and electronic
properties that differ from individual atoms or bulk materials.

These unique properties are highly appreciated for different

applications such as catalysis, photonics, or biomedicine. Metallic
nanoparticles can be prepared using myriad physical or chemical
methods.

21
Chitosan in Biocatalysis

The use of immobilized enzymes for catalysing chemo-,

regio- and/or stereoselective chemical reactions is a very useful and
well-known technique in this sense, the use of chitosan for
immobilizing enzymes, either as a carrier for covalent linking or as an
encapsulation vehicle, is well reported. Our group described the
production of enantiopure D-p-hydroxyphenyl glycine (D-p-HPG,
Figure 6) using a multi-enzyme system containing D-hydantoins and
Dicarbamoyls encapsulated in chitosan-based materials.[18][20]

2.4 CHITOSAN IN BREAST CANCER

Modification Strategies of CSNPs for BC Therapy Natural

chemotherapeutic medications are more successful and less hazardous
in cancer therapies, according to chemical and pharmacological studies.
Thus, natural chemotherapeutic medication development is a prominent
pharmaceutical issue. The big challenges in the development of
anticancer drugs are: Anticancer drugs are generally hydrophobic.
Small-molecule hydrophobic drugs can be quickly removed from the
tumour site. Poor solubility makes it difficult to dissolve and release the
drug from the tumour. They have a lower half-life and subtherapeutic
tumour concentrations. SiRNA, microRNA, and oligonucleotides for
cancer treatment degrade systemically, lowering t ½. Oral
administration is preferable, easy, and cost-effective. This route must
pass across multiple biological barriers, such as the blood-brain barrier
and tight junction barrier, and be quickly destroyed by digestive fluids
and the liver.

22
 Surface modification of CSNPs enhances their tumour-
targeting ability through different mechanisms such as a receptor or
carrier-mediated transcytosis. CSNPs were more effective than PLGA
NPs because they targeted MCF-7 cells. Modification of chitosan can
use variations in molecular weight and the level of acetylation, which
will provide different properties according to the needs of the drug
delivery system. Chitosan can be used as a solubility-enhancing
polymer backbone.

Advances in polymer chemistry have led to the creation of

smart polymer systems. Polymers used for drug administration can
respond to stimuli such as temperature, light, or pH. Stimulus-
responsive polymers may modify cell adhesion to boost gene
expression or enzyme activity. Chitosan-based gene delivery systems
have attracted considerable attention as a promising alternative to viral-
based gene therapy due to their biocompatibility, low toxicity, and ease
of functionalization.

Scientists have been exploring different ways to deliver

genetic material into cells for gene therapy, which are categorized into
two groups: viral and non-viral methods. Viral vectors have been used
in the majority of gene therapy clinical trials due to their high
transfection efficiency and gene expression levels. However, they also
have significant drawbacks such as immune responses, toxicity,
immunogenicity, low loading capacity, and inflammation. Non-viral
methods, on the other hand, are gaining increased attention because
they can avoid many of these drawbacks.
23
 One promising non-viral method is the use cationic
polymers such as chitosan, which have strong gene complexation and
high transfection efficiency, for gene delivery. However, chitosan-based
gene delivery carriers still face challenges such as poor water solubility,
charge reduction at physiological pH, and poor targeting capability,
which hinder their clinical translation.

Low-molecular-weight chitosan and a low N/P ratio (the

ratio of the amine groups of chitosan to the phosphate groups of DNAS)
are more suitable for designing chitosan-based nonviral vectors for gene
therapy. This is because the physicochemical and biological properties,
as well as the stability of nanoparticles formulated with low-molecular-
weight chitosan, are better than those formulated with higher-
molecular-weight chitosan.

2.5 CHITOSAN IN BIOMEDICAL APPLICATION

In the biomedical field, chitosan finds several applications

due to its unique properties.

Drug Carrier

Drug discovery and development involve highly

challenging, laborious, and expensive processes. Most of the drugs in
the clinical phase, however, fail to achieve favourable clinical outcomes
because they do not have the ability to reach the target site of action. A
significant amount of the administrated drug is distributed over the
normal tissues or organs that are not involved in the pathological
process, often leading to severe side effects.[12]

24
 An effective approach to overcome this critical issue is the
development of targeted drug delivery systems that release the drugs or
bioactive agents at the desired site of action. This could increase patient
compliance and therapeutic efficacy of pharmaceutical agents through
improved pharmacokinetic- is and biodistribution.

The targeted drug delivery system is comprised of three

components: a therapeutic agent, a targeting moiety, and a carrier
system. The drug can be either incorporated by passive absorption or
chemical conjugation into the carrier. The chemical modification of
chitosan imparts amphiphilicity, which is an important characteristic for
the formation of self-assembled nanoparticles, potentially suited for
drug delivery applications.

The hydrophobic cores of the nanoparticles could act as

reservoirs or microcontainers efficiency of the drug. Chitosan has been
widely utilized as drug delivery systems for low molecular drugs,
peptides and genes. The liver is a critical target tissue for drug delivery
because many fatal conditions including chronic hepatitis, enzyme
deficiency, and hepatoma occur in hepatocytes.

In general, liver-targeting systems employ passive trapping

of microparticles by reticuloendothelial or active targeting based on
recognition between hepatic receptor and ligand-bearing particulates.
the preparation of chitosan particles, several techniques are available
such as emulsion, ionotropic gelation, reverse micellar, solvent
evaporation, spray drying, coacervation, and sieving methods.

25
Wound Accelerator

Wound dressings before the 1960s were considered to be

only the so-called passive products having a minimal role in the healing
process.50 The pioneering research of Winter initiated the concept of
an active involvement of a wound dressing in establishing and
maintaining an optimal environment for wound repair.

This awareness resulted in the by development of wound

dressings from traditional passive materials to functional active
dressings which, through the 125 interactions with the wounds they
cover, create and maintain a moist and healing environment.

An ideal wound dressing should protect the wound from

bacterial infection Slow healing and non-healing wounds, such as
ulcers, as well as wounds caused by major or minor injuries, surgery, or
burns, represents the most widespread treat- able conditions
encountered by humans and animals. Wound repair is a well highly
coordinated process that involves a series of overlapping phases:
inflammation, cell proliferation, matrix deposition and tissue
remodelling.

Alara stated in his study that catalysis of chitosan

degradation is known to be caused by lysozyme, an enzyme transported
to the wound sites by the inflammatory cells (polymorphonuclear
leucocytes).

The abundant lysozyme at the wound site would break

chitosan 2 to the active N-acetyl-d-glucosamine dimer and provide for
its sustained release.

26
Haemostatic Agent

Haemostatic materials have different surface properties

from blood contacting materials that are nonchromogenic. The
development of artificial biomaterials that contact blood (e.g.,
prosthetic vascular grafts) have a primary consideration for being anti-
thrombogenic.

Thrombosis on the material surface remains a serious

bioengineering problem despite demonstrated good blood
compatibility. Thrombosis is assumed to be caused by plasma protein
adhesion, platelet activation and clot formation.

For example, polyurethane (PU) is a well-known

biomaterial with good biocompatibility and mechanical properties for
biomedical applications. However, the usage of PU is still limited
because of the poor cell attachment/growth or the induction of
thrombogenesis on the surface.

Many attempts have been made, such as chemically or

photochemically modifying the PU surface to overcome these
problems. When chitosan is applied as a blood contacting material, such
anticoagulant modifications are usually applied.

The effect of chemical structure modifications and physical

form of the chitosan upon haemostasis was recently reviewed.66 In
addition, several commercially available haemostatic agents including
a chitosan derivative approved by Food and Drug. Administration have
been used in recent combat operations and their effectiveness has been
reported.

27
Antimicrobial Agent

Chitosan is a biomaterial widely used for effective delivery

of many pharmaceuticals, 100 Accordingly, chitosan may be suitable
for incorporating other antipyretic for the preparation of long-acting
antibacterial wound dressing.

The antimicrobial activity of chitosan also increases with

increasing degree of deacetylation, due to the increasing number of
ionisable amino groups. Antibacterial activity also increases with
increasing molecular weight of chitosan, though too high a molecular
weight or concentration is counterproductive. The optimum molecular
weight of chitosan for anti-microbial activity is about M_{v} = 9.16 *
10 ^ 4 The antimicrobial activity of chitosan was observed against a
wide variety of microorganisms including fungi, algae, and some
bacteria.

2.6 HYALURONIC ACID

Hyaluronic acid, as a natural linear polysaccharide, has

attracted researchers’ attention from its initial detection and isolation
from tissues in 1934 until the present day. Due to biocompatibility and
a high biodegradation of hyaluronic acid, it finds wide application in
bioengineering and biomedicine: from bio revitalizing skin cosmetics
and endoprostheses of joint fluid to polymeric scaffolds and wound
dressings.

However, the main properties of aqueous polysaccharide

solutions with different molecular weights are different. Moreover, the
therapeutic effect of hyaluronic acid-based preparations directly
depends on the molecular weight of the biopolymer. [13][16]

28
 The present review collects the information about relations
between the molecular weight of hyaluronic acid and its original
properties. Particular emphasis is placed on the structural, physical and
physio-chemical properties of hyaluronic acid in water solutions, as
well as their degradability.

2.7 DOXORUBICIN

Doxorubicin is an antibiotic derived from the Streptomyces

paucities bacterium. It has widespread use as a chemotherapeutic agent
since the 1960s. Doxorubicin is part of the anthracycline group of
chemotherapeutic agents.

Doxorubicin may be used to treat soft tissue and bone

sarcomas and cancers of the breast, ovary, bladder, and thyroid. It is also
used to treat acute lymphoblastic leukaemia, acute myeloblastic
leukaemia, Hodgkin lymphoma, and small cell lung cancer.

This activity will highlight the mechanism of action,

adverse event profile, pharmacology, monitoring, and relevant
interactions of doxorubicin, pertinent for interprofessional team
members in the treatment of patients with cancers for which it is
indicated.[20]

DDB1 and CUL4 associated factor 13 (DCAF13) is a

substrate receptor in the CUL4‐DDB1 E3 ligase, and its expression is
associated with the prognosis of certain cancers. In the present study,
we report evidence that DCAF13 is aberrantly overexpressed in human
breast cancer and its expression is positively associated with cancer
progression.

29
 Although the cellular substrates recognized by the DCAF13
have not been fully uncovered, a series of recent studies indicate that
DCAF13 regulates a few biological processes by targeting some key
players for polyubiquitination and proteasomal degradation. In human
embryos, DCAF13 is expressed from eight‐cell stage to morula stage
and the knockout of DCAF13 leads to preimplantation lethality.
Interestingly, the H3K9 trimethylation levels are dramatically elevated
in DCAF13‐null embryos. H3K9 trimethylation is a key barrier to gene
expression reprogramming in early embryo development.
Mechanistically, DCAF13 recognizes SUV39H1, a H3K9 methyl
transferase, and targets it for polyubiquitination and proteasomal
degradation, thus promoting H3K9 trimethylation removal. In oocytes,
DCAF13 was identified as a nucleolar protein and an important
component of the rRNA‐processing complex. [22]

Conditional knockout of DCAF13 results in defects in

oocyte development and female fertility. Further analysis revealed that
DCAF13 is implicated in 18S rRNA processing in growing oocytes. A
few studies also suggest that DCAF13 is an important player in cancers.
In osteosarcoma cells, CRL4BDCAF13 E3 ligase has been shown to
specifically target the tumour suppressor PTEN (phosphatase and
tensing homolog deleted on chromosome 10) for polyubiquitination and
proteasomal degradation. The DCAF13 gene was also found to be
amplified in 14.7% of cases of hepatocellular carcinoma and its
expression was upregulated in hepatocellular carcinoma. DCAF13
expression is positively correlated with advanced hepatocellular
carcinoma grade and closely associated with poorer survival.

30
 Besides hepatocellular carcinoma, DCAF13 was also
shown to be of prognostic value in lung adenocarcinoma. In the present
study, we show that DCAF13 is aberrantly overexpressed in human
breast cancer. Further analysis shows that DCAF13 promotes
epithelial–mesenchymal transition (EMT) in breast cells, whereas it
does not have significant impact on breast cell proliferation, cell cycle
progression and apoptosis.

2.9 NOVEL BANANA PEEL SYNTHESIS

The two main parthenocarpy species of banana are Musa

acuminate Colla and Musa balbisiana Colla. There are several health-
promoting and disease-preventing effects of Musa acuminate Colla,
which are attributed to its important bioactive compounds, including
phenolics, carotenoids, biogenic amines, phytosterols, and volatile oils,
found in the stem, fruit, pseudo stem, leaf, flower, sap, inner trunk, root,
and inner core.[5]

 Hydroxyapatite (HAP) nanoparticles synthesis using banana peel pectin

as template
 Biological synthesis is inexpensive, ecofriendly and uses non-
hazardous chemicals.
 Nano-HAP is pure, low crystalline, reduced size at high concentration
of pectin.
 HAP nanoparticles showed antimicrobial activity used for biomedical
applications.

31
 CHAPTER-3

PROPOSED SYSTEM

3.1 Objectives

 Pretreatment of banana peel for banana fiber (BP) isolation

 Preparation and characterization of chitosan nanoparticle (CS NP)
 Conjugation of hyaluronic acid with chitosan nanoparticle (HA-CS)
 Preparation of banana fiber conjugated HA-CS (BF-HA-CS) scaffold
 Characterization of BF-HA-CS scaffold by FTIR, XRD, TGA and SEM
analysis

3.2 Materials and methods

Materials

Chitosan, Hydrochloric acid (HCl), Sodium Tri Phosphate

Penta basin (TPP), Hyaluronic Acid, Acetic acid, Doxorubicin, Banana
peel, Sodium chlorite solution, Glutaraldehyde, Hydrogen
Sulphate(H2SO4) Phosphate Buffered Saline (PBS), Fluorescein
isothiocyanate (FITC), Sodium Chloride (Nacl), Potassium.

Fig 3.1 Smashed Banana Peel Fig 3.2 Hypochlorite solution

32
3.3 Preparation of chitosan nanoparticles

 Take 500 mg of chitosan was dissolved in 20 ml water & add with 5 ml

acetic acid.
 Then 500 mg of sodium was added in TPP solution.
 After that sodium TPP was added dropwise into mixture and was kept
for 12 hours.
 After completely dissolved, the mixture was centrifuged at 4000 rpm
kept for 20 minutes Supernant was discarded. Pellet was dried and used
for characterization.

Fig 3.3 Chitosan Nanoparticle

3.4 Banana Fiber isolation

 Take 30ml ml of hypochlorite solution was prepared and added in 5

ml H2SO4 solution.
 12 g of banana peel were smashed and added into the solution
followed by stirrer for overnight.
 The mixture was centrifuged at 4000 rpm for 15 minutes. Supernatant
was discarded and pellet was kept it for dried.
33
 Fig 3.4 Dried banana peel

Fig 3.5 Banana Peel powder

3.5 Preparation of final sample

 Dried banana peel was made into powder and 20 ml of water was added
in 0.2 g of banana peel powder & was kept in stirrer for 1 1/2 hour.
 After that 0.2 g of CS NP was added and kept in stirrer for 1 hour.
 Then it was sonicated for 30 minutes and stored in -200C.

34
Fig 3.6 12g Measured peel powder Fig 3.7 H2SO4 Mixed

Fig 3.8 stirrer for overnight Fig 3.9 Final sample

35
3.6 FITC loaded sample preparation

 0.5 g of chitosan nanoparticle was dissolved in 20 ml of water and 5 ml

acetic acid was added.
 After fully mixing 0.5 g of sodium TPP was added in 20 ml water
dropwise and kept for 12 hours.
 After that 500 𝜇l of FITC was added into the mixture and keep it in
stirrer for 1 hour. Then after dried it was smashed to get powder form.
10 ml of water was added in banana fiber powder and keep it in stirrer
for 30 minutes.
 0.2 g of chitosan nanoparticle conjugated hyaluronic acid was added in
FITC and kept in stirrer for 1 hour.

Characterization

1. Scanning Electron Microscopy (SEM)

Scanning electron microscopy images were collected with a
Quanta 200 scanning electron microscope (FEI). The accelerating
voltage was 20 kV. SEM characterization usually occurs in vacuum,
and sample preparation is achieved in solid state.
2. Fourier Transform Infrared (FTIR) spectroscopy:
Spectra were collected using a Thermo Nicolet, Nexus,
870spectrometer. This instrument is equipped with a MTEC model 300
photoacoustic accessory. Continuous mode FTIR spectra were
collected in the energy range of 400-4000cm−1 at a mirror velocity of
0.158cm s−1, and 1000 scans were collected for each sample at a
resolution of 4cm−1.
The sample preparation method used most often for the
analysis of solid samples involved a KBr matrix; specifically, the solid
samples were dispersed and supported on a KBr matrix tablet.
36
3. X-ray Diffraction (XRD):
Sample was prepared for XRD analysis. A wide-angle
XRD pattern of all the samples was recorded using an x-ray
diffractometer, D8 Discover, Bruker AXS Instruments. Cu Kα radiation
with a wavelength range of 1.54 °A was used in this experiment. A
quartz single-crystal zero-background sample holder was used to
minimize the background signals.
The dried sample were protected from oxygen by applying
a few drops of 10% glycerol in 95% ethanol and allowed to dry for 1-2
more days. The glycerol-coated specimens were then analyzed in
ambient air with using a Philips X’Pert MPD diffractometer
(PW3040/00) operated at 40 KVP and 50 mA. Successive scans, as well
as scans taken after 24 h of exposure to ambient air, showed no change
in the structural properties of the glycerol coated sample, verifying the
protective effect of the glycerol film with respect to oxidation. The
mean crystallite dimension was estimated using the Scherrer equation,
after correction for instrumental broadening.
4.Thermogravimetric Analysis (TGA):
It has two types of TGAs, a top-loading TGA 4000(it
supports the sample pan above the balance via a “stem” support rod,
and a bottom loading or hang down, TGA 8000(supports the sample
pan via a “hang down” below the balance). The sample size should be
between 2 and 50 mg. If minimum amount of sample, run at least 1 mg.
After that cover the bottom of the pan with the same
material. Temperatures, perform a survey scan. A survey scan is run at
200C per minute and it begins and ends 1000C below and above the
transition of interest.

37
Porosity and density analysis

The Densities of all aerogels were calculated by dividing the mass of an

aerogel by its apparent volume. The volume of each aerogel was
calculated by taking diameter and height measurements of each aerogel
with a digital calliper.

The volume was calculated assuming the aerogel was a

perfect cylinder. The mass of each aerogel was measured with an
analytical balance (Mettler Toledo Inc., Mississauga, Canada,
readability of 0.0001 g). The density values reported are an average of
three separate samples foreach type of CNC aerogel.

Swelling studies
In order to study the swelling studies, MA-GG-SS of
0.05 g was immersed in different pH buffer medium (4, 7.4 and 9) at
temperature 25 °C until a swelling equilibrium was attained. Samples
were placed in swelling solution and the weight of swollen samples was
measured against time after the excess surface water was removed by
filter paper.
The degree of swelling was defined as volume of hybrid
polymer before and after swelling at time t and calculated using
following formula.
Swelling ratio % = Wt. – Wo × 100 ÷ Wo
Where,
Wt. - Mass of swollen sample,
Wo - Mass of initial sample, respectively.

38
DOX release from BF-HA-CS scaffold
DOX@BF-HA-CS scaffolds were dialyzed against 30 mL
phosphate buffer solutions of different pH and in presence and absence
of GSH and incubated at 37 °C with stirring.
At specific time interval, 5 mL of solution was taken at
every time interval for investigation. Simultaneously, 5 mL of fresh
buffer solution was replaced to maintain the solution quantity and
identical pH value.
The amount of DOX release was analysed using a UV–vis
spectroscopy at 360 nm. FITC@ BF-HA-CS scaffolds were done by the
same method to check the release the fluorescence by
photoluminescence study.

39
 CHAPTER-4
RESULTS AND DISCUSSION

Preparation and characterization of BF-HA-CS scaffold:

A B

Chitosan NP HA-CS
C D

Banana Peel Fiber Final Scaffold

Fig 4.1: Chitosan nanoparticle (A), Chitosan conjugated hyaluronic

acid (B), Isolated banana fiber from banana peel (C), Final BF-HA-
CS scaffold (D)
40
 HA and CS nanoparticle was formed first and the final BF-
HA-HAP-FIB scaffold was formed by surface modification method.
The BF-HA-CS forms porous scaffold structure and improve the
solubility.

A B

C F F
B 1 2
F

Fig 4.2: (A) hight of BF-HA-CS scaffold, (B) Width of BF-HA-CS

scaffold, (C) Density and porosity calculation for banana fiber
scaffold (BF) and final scaffolds with 2% HA-CS (F1) and 5% HA-
CS(F2).

41
 Density calculations indicated that density was decreased
with increased concentration of HA-CS. Banana fiber had the highest
density (1.45g/cm3). But with increased concentration of HA-CS (2%
and 5%) in the final sample (F1, F2 respectively) the density was
decreased respectively.
Calculated porosity from density values indicates F2 sample
with 5% HA-CS have the highest porosity of 99.819%, while only
banana Fiber have a very less porosity of 78.221%.
This indicates that the porosity of our scaffold depends on
the amount of HA-CS. HA-CS makes the scaffold more porous which
will be helpful for breast cancer drug delivery because pores will
facilitate cell distribution, integration with the host tissue and capillary
in growth.

Fig 4.3: FTIR analysis of CS NP, HA, HA-CS, BF, 2%

HA-CS conjugated BF (F1) and 5% HA-CS conjugated
BF (F2)

42
 FTIR spectrophotometer was used to obtain IR spectra to
analysis the change in functional group. Fig. shows IR spectra of CS,
HA, HA-CS, BF, F1, F2 scaffolds. A peak at 3500 to 3700 cm−1 was
observed for the main functional group of chitosan and is due to the O-
H group of stretching vibrations.
The presence of peaks at 1600 cm−1 is due to the N-H
bending vibration of amino (−NH2) group and C-H bending vibration
of the alkyl group. The peaks at 1200 to 800 cm−1 are recognized due
to the stretching vibration of C-O-C bridges and assigned to
glucopyranose ring in chitosan matrix. (Saharan et al.,2006). HA
showed an intense band at 2000 cm-1 corresponding to C-O stretching.
3000 cm−1, 2500 cm−1 for amide secondary N-H bonding.
Similarly in case of the FTIR spectrum of HA-CS, the peak
at 3000-2800 cm−1 confirmed the presence of HA and peak at 1200-800
cm−1 confirmed the presence of CS. The Banana fiber (BF) showed a
typical cellulose fingerprint at the region 1000-1500 cm−1 associated
with complex vibration associated with C–O, C–C stretching.
In case of BF O–H stretching is evident at 3400 cm−1 and
C–H stretching in methyl and methylene group is seen around 2900
cm−1 In case of F1, F2, all represents final BF-HA-HAP scaffold with
2% and 5% HA-CS respectively.
These scaffolds showed peaks at 1000-1500 cm−1 region,
2800-3000 cm−1 region due to vibrations of C-OH side groups, -OH
stretching and 1500 cm−1 and 1400 cm−1 for C–O, C–C stretching which
represents that HA-CS is successfully conjugated with BF (T. Schön et
al,2011)

43
 Fig 4.4: XRD analysis of CS NP, HA, HA-CS, BF, 2% HA-CS
conjugated BF (F1) and 5% HA-CS conjugated BF (F2)

XRD micrographs of HA, CS, HA-CS, BF and Final (F1,

F2) scaffolds were shown in Fig. For HA alone it has not given any
specific peak due to its amorphous structure. For CS alone, there was
no significant peak.

In case of HA-CS characteristic peaks were observed at

2θ = 20.0°, 30.0°, 35.0°, 550 and the well-defined multiple peaks
denotes the crystalline structure of HA-CS. HA-CS crystalline structure
contributed on amorphous BF to the final sample.

In case of F1, F2 they showed a crystalline nature but

compared to F1, F2 has little more intensity in peak due to higher
concentration of HA-HAP. (Jozef S. et al,2014)

44
 Figure 4.5: TGA analysis of BF, 2% HA-CS
conjugated BF (F1) and 5% HA-CS
conjugated BF (F2)

TG/DTA curves of BF and final scaffolds (F1, F2) are

displayed in Fig. Thermal decomposition of all the polymers was
carried out at a heating rate of 10 °C/min under nitrogen atmosphere
over the temperature range 50–700 °C.

The structural transformation is observed by TG curves

which are supported by DTA studies. This analysis confirms the water
content in the nanocomposites and the thermal stability under controlled
heating rate is at around 180 °C and is simple and is a one-step reaction.
It has been seen that F2 (BF with 5% HA-CS)

45
 A B

5µ 5µ

E F

10µ 10µ
m m

C D

30µ 10µ
m m
Fig 4.6: (A, B) Chitosan nanoparticle SEM image, (C,D)
Banana Fiber SEM image, (E,F) HA-CS coated banana fiber SEM
image
Fig A and B described the morphology of chitosan
nanoparticle. The spherical shape confirmed the nanoparticle
confirmation, The SEM image of isolated fiber from banana sticks has
shown here in this figure (C, D) to analyse the morphological nature of
banana fiber.

46
 The fibrous structure at 30µm confirmed the banana fiber
morphology. They existed in an aggregated structure. The SEM image
of HA-CS conjugated banana fiber (BF-HA-CS) was observed for
morphology analysis.

The fig (E, F) detects the attached HA-CS on banana fiber.

At higher magnification spherical conjugated HA-CS was seen on the
top of the fiber which resembles the successful attachment and
distribution of HA-CS on fiber scaffold. Attached HA-CS will help in
the bone growth regeneration. The distribution of HA-CS on the fiber
scaffold can also be observed in the SEM image.

A B

40µ 30µ
m m
C D

10µ 3µ
m m
Fig 4.7: HR-SEM analysis of HA-CS coating on banana fiber
scaffold

47
 HR-SEM analysis of final scaffold gave the confirmation of
scaffold morphology. At higher magnification attached HA-CS was
observed on fiber. HR-SEM has detected the distribution of HA-CS
particle on scaffold.

Fig 4.8: Degree of swelling of HA-CS conjugated BF scaffold

The swelling ability is related to the pore size, the

interconnection conditions and an important index for the scaffolds for
the absorption of body fluid and transfer of cell nutrients and
metabolites. Swelling ability of BF-HA-CS scaffold seems to be
increasing with change in time with different pH (pH 5, pH 7 and pH
9). In case of BF-HA-CS scaffold initial stage nanocomposites showed
18% of swelling at 10 h which increased to 75% at 50h. The main
factors for high degree of swelling at pH 5 as compared to pH 9 is due
to the strong interaction of BF-HA-CS scaffold.

48
 This also caused by increase in ionic osmotic pressure. At
particular time it reaches equilibrium, it reaches steady state at 100 h
shows 70% at 9.0 and 90% at pH 5. Mainly, swelling is directly
proportional to more drug capacity and sustained release of drug. So,
effective swelling at acidic pH denotes the higher release of drug with
time in acidic environment. This higher degree of swelling will have a
larger surface area/volume ratio thus allowing the samples for cell
infusion as well as maximum drug release in acidic tumour
environment.

Fig 4.9: pH and Redox (GSH) responsive drug release from

BF-HA-CS scaffold

49
 As stated in the introduction, the extracellular fluids and
normal cells have a 7.4 pH, in which the GSH concentrations are 2-20
μM and 2-10 mM, respectively; while the tumour cells contain slightly
acid environment (pH 5.0) and have a higher GSH level (at least 4-fold
related to normal cells).

Therefore, we chose the following three kinds of media for

drug release studies: pH 5,7,9 medium in presence and absence of 5 μM
and 10μM GSH. Fig. shows the drug release behaviour from the DOX-
loaded BF-HA-CS scaffold in the above three media. In pH 7 and 9
medium the release ratio was only about 60%.

When the pH was 5, the release of the drug increased. The

concentration of GSH in the medium further increased the release, the
DOX release rate increased to 70 %. Whereas in pH 5.0 medium with
10 mM GSH, the DOX release significantly increased to highest value
93%, suggesting that the DOX-loaded scaffold have an excellent
controlled release of DOX.

Fig 4.10: Photoluminescence Study from

BF-HA-CS scaffold

50
 Figure shows absorption spectra of dye loaded BF-HA-CS
scaffold (pH 5, pH 7, pH 9). Pure FITC shows broad absorption
spectrum whereas the dye loaded scaffold showed narrow absorption at
pH 5.0 at around 520 nm.

A slight red shift was noted in case of pH5.0 similar to

previous reports when compared to pH 7, and pH 9. Figure shows the
PL spectra for FITC doped nano scaffold. The excitation wavelength
was 365 nm and a clear emission peak was noted around 490–550 nm
for all particles.

These emission peaks stand typical for green fluorescein

FITC. Thus, the absorption and PL spectrum complement each other
very well depicting successful loading of FITC. The typical green
fluorescence was maximum in case of pH 5.0 indicating maximum
release of FITC at pH 5.0.

51
 CHAPTER-5

CONCLUSION

Natural Scaffold is a class of new therapeutic material that

meet the necessities such as biocompatibility, bio functionality, and
tenable properties for therapeutic agent delivery. We prepared
hyaluronic acid functionalized chitosan nanoparticle conjugated banana
Fiber scaffold. BF-HA-CS scaffold and their controlled release in
response to redox and ph. Hyaluronic acid (HA) is a natural polymer
act as a ligand was used to conjugate with CS in order to increase the
surface characteristics of the prepared composite material. This natural
scaffold is the right prime for targeting cancer cells. As all components
are biodegradable, biocompatible and natural polymers so a strong
cellular internalization was exhibited without any toxicity. The prepared
conjugated material (BF-HA-CS) formed fibral shaped scaffold which
have high potential for drug loading and can be developed for
anticancer applications.

52
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55
CERTIFICATES

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