PROJECT OF NANOBIOTECHNOLOGY

Topic
Leukemia and Gene Therapeutics in association with
nanobiotechnology
Submitted to: Dr. Imran
Submitted by: Aasma Zafar (SP19-RMG-001)
Ahmad Raza (SP19-RMG-003)
Syeda Afifa Azhar (SP19-RBS-028)
Ayesha Sarfraz (SP19-RMG-008)
Sanah Ahmad Essa (SP19-RBS-033)
Bibi Maria (SP19-RBS-031)
Group number 6
Date: 07-05-2019
PROBLEM: Prevalence of leukemia around world and Pakistan

Globally: Leukemia is known to be the most common type of cancer among children.
This disease has accounted for one-third of all cancer deaths from 2012 onwards in children <15
years of age. Two major types of leukemia which are common in children: acute myeloid
leukemia (AML) acute lymphoblastic leukemia (ALL). Acute lymphoblastic leukemia is the
most prevalent one in childhood malignancy accounting for almost over 75% of complete forms
of leukemia in children and also adolescents younger than 20 years. Approximately in US every
3 minutes one person is diagnosed with blood cancer. Among new cases of leukemia, lymphoma
and myeloma are expected to be accountable for 10 percent of the total 1,735,350 overall cancer
cases diagnosed in 2018 in the US. For men, highest regional leukemia rate was estimated
around 11.3 per 100,000 population was found in New Zealand and Australia, with United States
and Canada (northern America) next at 10.5 per 100,000. Incidence is also generally much higher
in males, with an overall global male to female ratio of 1.4. The incidence of leukemia in the
world is in total 1 per 100,000 annually. Leukemia contributes to almost 25% of childhood
cancers around globe.

In Pakistan: Approximately 1,48,041 Pakistanis each year are diagnosed with cancer.
According to different surveys over 1,00,000 cancer-related deaths in Pakistan and a prevalence
of around 3,50,000 living cancer patients have been reported mostly suffering from blood
cancer(leukemia) during the past 5 years. leukemia cancer is the fifth most commonly diagnosed
cancer and fourth most prevalent cancer in Pakistan. An estimated 5-year prevalence of 11,917
(3.5%) cases, 5,335 (3.6%) recently diagnosed cases and almost 3,903 (3.9%) deaths occurring
in 2012 and inceasing so far have been due to Leukemia in Pakistan.

INTRODUCTION: Leukemia can simply be defined as the cancer of the leukocytes and bone
marrow. It targets the hematopoiesis carrying out tissues of the body- consisting of the bone
marrow , lymphoid organs and the lymphatic system. Leukemia majorly affects the leukocytes.
Since leukocytes are an integral part of the body’s defence system, patients having leukemia fail
to produce effective leukocytes hence have an immune system which is more impuissant towards
various opportunistic diseases and pathogens. Leukemia can be classified into a number types
and sub types . The main types and sub types can be classified as Acute leukemia and Chronic
leukemia, each of which breaks into further branches.Acute leukemia further branches into Acute
Lymphoblastic Leukemia(ALL) and Acute Myeloid Leukemia (AML). While Chronic
Lymphocytic Leukemia(CLL) and Chronic Myeloid Leukemia(CML) arise from the chronic
lineage. Each type has a different prognosis and treatment. A common ground is that they are all
derived from the bone marrow. Two types of cells are produced in the bone marrow; Myeloid
cells- these are cells where myeloid leukemia arises and Lymphocytes- are cells where
lymphocytic leukemia occurs. Leukemia maybe characterized as either lymphocytic or
lymphoblastic and myelogenous or myeloid, depending on the cells affected by the disease. If the
cell of the bone marrow producing lymphocytes is affected then the case is referred to as
lymphocytic or lymphoblastic and if erythrocytes, platelet and leukocyte forming cells of the
bone marrow are affected the case is referred to as myelogenous or myeloid.

All blood cells originate from the hematopoietic cells, which gives rise to 2 different lineages i.e
myeloid and lymphoid. The lymphoid group divides into 2 types of cells, the T cells and B cells.
While the myeloid cells produce the cell types; erythrocytes, megakaryocyte, monocytes,
neutrophil, basophil and eosinophil. All of these cells go through a series of development to take
their proper cell form from a hematopoietic cell. The cells differentiate into T lymphoblast and B
lymphoblast for the lymphoid group from a hematopoietic cell. While others may differentiate
into erythroblast, megakaryoblast, monoblast and myeloblast for myeloid group from
hematopoietic cell. These cells differentiate or mature to form the T cells, B cells, erythrocytes,
megakaryocyte, monocytes, neutrophil, basophil and eosinophil respectively.

Leukemia is characterized based on the degree of maturation of the originating cells. If leukemia
originates in the cells of first stage of cell growth i.e in the B lymphoblast, T lymphoblast,
megakaryoblast, myeloblast, erythroblast and monoblast it can be specified as acute leukemia.
While if leukemia emanates in the more mature cells i.e T cells and B cells, erythrocytes,
megakaryocyte, monocytes, neutrophil, basophil and eosinophil it would be characterized as
chronic. Acute leukemia has been seen to grow very rapidly, and the patients start showing
symptoms in a matter of weeks. While chronic leukemia grows slowly, the patients may not
show symptoms for years.
In acute leukemia the blood cell growth gets arrested in the immature stage and the cells divide
rapidly and since they have not differentiated fully into their mature forms they cannot perform
their regular functions. While in chronic leukemia the cells are arrested in the second stage of
development, they have some degree of differentiation in them and if not all, they can perform
some of their regular functions, i.e they are not fully functional. The leukemia cells not only
divide rapidly they also live longer than the normal blood cells. The structure of acute leukemia
structure is very different from their mature specialized cell structure while the chronic leukemia
cells resemble their mature specialized cells both in terms of structure and function.

Leukemia affected cells grow uncontrollably and tend to swarm out unaffected normal cells in
the bone marrow- consequently bringing about a drop in the erythrocyte, platelet and neutrophil
count , making the patients more vulnerable to anemia, excessive bleeding and infections
respectively.

COMMOMN FEATURES AND SYMPTOMS:

Although each type of leukemia has it’s own particular set of signs and symptoms, there are
some common features such as; osteodynia and tenderness, fever and chills, frequent and/or
severe infections, easy and frequent bruising, recurrent nosebleeds, excess blood loos on
incidence of injury, persistent fatigue, undesired and unintentional weight reduction,
lymphadenopathy, petechiae, splenomegaly, hepatomegaly and excessive sweating.

ACUTE LEUKEMIAS

1) ACUTE MYELOID LEUKEMIA:

Abnormal accumulation of abnormal pluripotent proliferative hematopoetic cells. Which are
most of the time abnormally differentiated and sporadically poorly differentiated pluripotent
cells in the circulatory and lymphatic system, bone marrow and other organs results in
development of acute myeloid leukemia, leading to bone marrow failure and death (Dohner et
al., 2005). Since this type of leukemia is in acute form, which means it is quickly proliferating
and so can spread quickly to other parts of the body including spinal cord and central nervous
system.

2) ACUTE LYMPHOBLASTIC LEUKEMIA

Acute lymphoblastic leukemia is one of the most common childhood malignancy (Williams et
al., 2014) and the second most common acute leukemia in adults(Terwilligerl,T & Hay,A.M,
2017). Despite of devising a long term cure for the disease, challenges of relapse still prevail.
Relapse of ALL at extramedullary sites has proven to be one of the top most hurdles in the
pursuance to finding complete cure (Williams et al., 2014).

CHRONIC LEUKEMIAS

1) CHRONIC MYELOID LEUKEMIA:Chronic myeloid leukemia has many firsts

associated with it. It was the first cancer linked to an acquired structural chromosomal anomaly.
It was also the first disease to have a drug synthesized to switch off the elaboration of the gene
product that drives the disease (Digumarti, 2006). This type of cancer occurs because of
translocation of the ABL gene onto BCR gene on chromosome 22. CML has 3 different
phases ;chronic phase-being the least invasive, accelerated phase-here cells divide rapidly and
blast phase-being the most invasive of all three types. Patients diagnosed in chronic phase are
most of the times asymptomatic. Accelerated phase as the name suggests has an accelerated rate
of cell cycle thus cells found in this phase grow at a faster rate and undergo division more
frequently. Patients at this stage experience symptoms like excess blood loss due to petechiae
and bruising and frequent opportunistic infections. The most advance phase of this type is the
blast phase. Here in this stage patients show most of the symptoms.

2) CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia as the name suggests affects the lymphocytic cells of the body,
mostly affecting the B cells. Since this type of leukemia is chronic so it builds up slowly.
Patients don’t show symptoms for up to the first few years. The CLL cell is a cell that had
differentiated up to an extent, where it’s growth gets arrested and thus it has structures that
resemble both the immature cell form and mature B cell structure. The CLL cells are very fragile
compared to a normal B cell, such that the preparation of microscopic slides for observations can
rupture the CLL cell, these cells are then called Smudge cells. Smudge cell appearance in the
microscopic observations is one major indication towards the diagnosis of CLL. The normal
blood cell travels to other organs and attack any potential pathogens, the CLL cells also travel to
lymph node, liver and spleen. The difference being that in the diseased state there are high
numbers of CLL cells, which leads to lymphadenopathy and other conditions.

STAGING AND PROGNOSIS

Staging helps in deciding the general course of treatment for a patient, it helps in predicting the
prognosis of the disease, hence it is crucial to develop an accurate staging plan. Staging for most
cancers is based on the tumor size and extent of metastasis, but since in the case of leukemia a
tumour can not necessarily be observed, which makes staging and visualizing leukemia more
complex. Leukemia affects the different blood cells and is hence is sometimes called fluid
cancer, which can be classified into different stage groups based on the complete blood count
profile and the degree of affected cells in other organs. Staging for leukemia depends on factors
such as;

1) leukocyte count

2) Platelet count

3) Age

4) Bone damage

5) Splenomegaly or liver megaly

6) Previous clinical history

7) Chromosomal mutations and aberrations

For most cancers staging depends on size of tumor, extent of metastasis and age of the patient.
Acute lymphocytic leukemia contrary wise does not form tumors, making it highly difficult to
visualize and by the time it is detected it has already metastasized to other organs . It majorly
affects the bone marrow and blood cell count; thus certain blood markers-observed via lab
reports are used for detection and staging for this type of leukemia

Staging for chronic myeloid leukemia can be divide into three categories;

1) Chronic- is the earliest and least invasive stage. Patients diagnosed in this stage are
usually asymptomatic while others portray mild symptoms.
2) Accelerated- this is a comparatively more advance stage. The cells in this stage divide
more rapidly, making the disease more invasive hence the symptoms get more
aggressive and noticeable
3) Blastic- this is the third and most invasive stage. Symptoms for this stage closely
resemble AML.
 RISK FACTORS:

Risk factors are certain elements that influence the probability of a person catching a
disease. Though it is not necessary that a person having some of the risk factors will
have the disease. The presence of risk factor increase the possibility of having the
disease and themselves do not cause the disease. The risk factors for leukemia like
other cancers include genetic aberrations, family history, previous clinical history,
exposure to radiation, previous chemotherapy and radiotherapy, exposure to certain
chemicals such as benzene and formaldehyde, smoking, obesity and congenital
syndromes.

GENETIC FACTORS:

Leukemia is the uncontrolled escalation of pluripotent cells. The rate by which a cell
passes through the cell cycle and the rate of it’s division is closely regulated by checkpoints in
the cellular machinery. Any changes in the DNA are readily detected and corrected by DNA
proofreading but in certain cases the proofreading machinery fails to detect alterations, leading to
cases like insertion, deletion, frame shift, missense and nonsense mutations. It is due to these
mutations that the breaks in the cellular machinery become faulty and the cell starts to grow and
replicate uncontrollably. Genetic mutations and aberrations may directly or indirectly impel the
out break of a disease. Directly if the mutation is present within the gene itself and indirectly if
the mutation is present in the genes coding for regulatory elements such as cell regulatory
proteins and factors. The following table shows the occurrence of mutations on different genetic
loci leading to the onset of leukemia
Conventional method for treatment of leukemia:

Conventional method includes chemotherapy, radiation therapies and surgery.

Drawbacks of conventional method:

 Targeting cancer stem cell is difficult.

 Drug resisting property of cancer stem cells makes them immune to anticancer drugs.
 Lack of cancer epigenetic profiling and specificity of existing epi-drug.
 Problems with diagnostic makes it difficult to treat.
 Unavaibility of effective biomarkers for cancer diagnosis and prognosis.
 Limitations of conventional chemotherapeutic agents
 Metastasis posses a huge problem in cancer treatment.
Using Gene therapeutics to overcome these barriers:

Gene therapeutics: gene therapy also called as human gene transfer is the therapeutic
delivery of nucleic acid into a patient’s cell as a drug to treat disease.

Barriers related to gene therapeutics

It is difficult to access some disease sites and local or tropical delivery of genetic
materials usually is not efficient enough to accomplish the desired therapeutic achievement.
Nucleic acids are negatively charged hydrophilic macromolecules, which usually restrain them
to bind to and passively diffuse across lipophilic cell membranes. Even if they are uptake by the
endocytic pathway, the endosomal or lysosomal degradation is also a matter of interest. Tissues
such as tumors, the RES and inflammatory sites have ruptured blood vessels, the capillary vessel
walls of most organs and tissues are not permeable to nucleic acids. In addition, the cytosolic
viscosity and dense organelles may prevent their movement towards target sites.

In case of siRNA-based therapeutics, they must break free from the endosome to reach the
cellular cytoplasm where siRNA acts. In terms of pDNA, the nuclear envelope represents an
extra and formidable barrier and pDNA must translocate to the nucleus for expression.

Nucleic acids are biomolecules of high molecular weight and are subjected to various
environmental factors including pH and nucleases which can degrade them. However, the
translation of therapeutic nucleic acids clinically is largely dependent on the development of an
appropriate delivery system which can overcome all the mentioned biological barriers.

Gene therapeutics using nanobiotechnlogy:

We can overcome above mentioned barriers using nanobiotechnology along with gene
therapeutics. It is important to construct our desired nanosystem for this purpose. But there are
some barriers in making a desired nanosystem for that we have to consider certain elements

Drawbacks in utilization of nano-based drug delivery:

 NPs can be hindered by drawbacks such as size, instability and lack of prolonged
circulation half-lives. These limitations can be overcome by modifying the nanomaterial
composition to improve stability and the surface chemistry to enhance in vivo circulation
rates.
  Primary limitations of these nanocarriers are the lack of control over drug release and
delayed tumor cell uptake. Drug delivery systems may cause disrupt-release effects when
encountered certain body fluids or tissues. Hence, premature drug release from the
rer

carrier in limited time exceeds levels of free drug in the blood.

 However, half-life is also a major issue. Drug-encapsulated NPs with shorter half-lives
can be instantly eliminated from systemic circulation, reducing the efficacy to produce a
desired result. Longer half-lives of nano-based drug delivery systems are beneficial for
tumor treatment. NPs must cross multiple barriers to reach the tumor site. These barriers
include the endothelial barrier, interstitial pressure gradient at the tumor site, abnormal
tumor vascularization and tumor cell heterogeneity.
 Intravenous treatment of hematological malignancies emphasizes on rapid contact of
nanocarriers with tumor cells in the blood and delivers drugs directly to the target site.
Instead, leukemic cells are also located in the bone-marrow and in cases of relapsed acute
lymphocytic leukemia and CNS. This requires drug-encapsulated NPs to overcome
barriers such as the Marrow-Blood Barrier (MBB), the BBB, and the Blood-Testes
Barrier (BTB). Therefore, nanocarriers with short half-lives may be suitable to target
leukemic blasts in circulation; those with longer half-lives may accumulate sufficiently in
the bone-marrow and CNS. Thus, NPs with differential half-lives could be beneficial to
treat Leukemia.
 Not to mention the fact that that long-term retention capacity of NPs in the bone-marrow
may hinder the self-renewal, proliferation and maturation of normal stem and progenitor
cells.
 The drawbacks associated with these novel nanoparticles, such as poor uptake and
inability to cross the blood–brain barrier (BBB), can be achieved by surface modification
of the polymers. Surface modified nanoparticles can penetrate the BBB. The process is
influenced by the endocytosis of the nanoparticles via the low-density lipoprotein
receptor of the endothelial cells of the brain followed by the release of the drug inside the
brain.
 One of the biggest hurdles encountered in cancer therapy includes the problem of dose-
dependent toxicity toward the non-cancerous cells because high doses of drug treatment
were generally required owing to poor drug accessibility to the tumor site. These
drawbacks led to the development of new modified NPs with increased cellular and sub-
cellular targeting capabilities.

Construction of Nanoliposome:

As we need to deliver the active gene to the target cell, so we will use second generation
non-viral gene delivery carriers known as lipolpolyplexes. Lipopolyplexes are a combination of
lipids (lipids that can be neutral, cationic or anionic) and polyplexes (cationic polymers). To
make our lipopolyplex, we will use 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) as
cationic lipid and Polyethylenimine (PEI) as cationic polymer. Combination of these two is
reported to have less cytotoxicity and higher transfection efficiency. We need to assure that our
lipopolyplex crosses the barriers in bone marrow and reaches to the desired white blood cell. For
this purpose, we need to engineer the lipopolyplex surface with a ligand for receptors that are
overexpressed on the defected cell. EGFR, epidermal growth factor receptors, are overexpressed
on most of the cancer cells and we aim to target them. The desired size preferred for making a
nanoliposome in vivo is less than 100nm as nanoliposome greater than 100nm can undergo
phagocytosis. So, we will make our lipopolyplex of 40-50nm in size.

Materials and Method:

We need to construct 40-50nm unilamellar lipopolyplex. To achieve this, we will use

different techniques for instance reverse phase method, continuous cell disruption system, high
pressure extrusion etc.

Preparation of Cationic Polymer:

First, we will prepare the polymer by mixing the polymer in oraganic solvent. We will
use methanol as an organic solvent which will be followed by mixing of Polyethylenimine (PEI)
in methanol and heating the mixture at 45-50 °C. After heating, evaporation will be done to
assure the removal of organic solvent. For evaporation, we will use rotary evaporator. As a
result, dried cationic polymer will be formed.

Preparation of Lipopolypexes:

After making the cationic polymer, we will mix it with our cationic lipid DOTAP at 1:1 molar
ratio by using an organic solvent. Chloroform: methanol will be used as an organic solvent at
(2:1 v/v).

Reverse Phase Method:

We will use reverse phase method to get the desired lipopolyplex. DOTAP: PEI (1:1) will
be mixed in organic solvent and poured in a round bottom flask. After pouring the mixture, we
will evaporate the solvent by rotary evaporator. Now, we will add water to the thin liposome
layer present inside round bottom flask. These layers will swell and detach themselves and form
Multi-Lamellar-Vesicles (MLV).

High Pressure Extrusion:

As we need unilamellar vesicles, so we will pass these MLV through small pore to
achieve ULV.
Continuous Cell Disruption System (CCDS):

We will use CCDS to achieve our desired size that is 40-50nm. By applying 200Mpa
pressure in 5-6 passes.

Encapsulating DNA:

After this, we will encapsulate the DNA with desired gene in liposome
which would be our desired lipopolyplex.

Targeted Delivery:

As we need to deliver our gene to the target cell, so we will engineer the surface of
lipopolyplex with ligand for epidermal growth factor (EGFR). Some commonly used EGFR
ligands include GE11, D4 and AE, so we will use one of them.

Repairing Gene Defect by Integration of regulator using Nanovehicles:

The underlying concept of integration of regulators in genome is to modify the expression of

genes so as to eliminate the root cause of Leukemia. AML (Acute Myeloid Leukemia) is divided
into 9 subtypes that differ with respect to particular myeloid lineage involved & degree of
leukemic cell differentiation.

In subtype M4Eo FISH analyses identified an insertion of 16q22 material (CBFB) in band 16p13
(MYH11), generating a CBFB-MYH11. The CBFB gene transcribe protein core binding factor
beta (CBFβ) which helps the RUNX protein to bind to DNA & protect it from being degraded.
RUNX1 protein is responsible for controlling the activity of genes involved in the development
of blood cells (hematopoiesis). The MYH11 gene transcribe for a protein called smooth muscle
myosin heavy chain 11 which is a part of smooth muscle myosin. All cases of AML subtype
M4Eo express CBFB-MYH11 fusion gene encoding CBFβ Smooth Muscle Myosin Heavy
Chain SMMHC which blocks the definitive hematopoiesis & adult hematopoietic stem cell.

Observation:

In M4Eo AML insertion of 16q22 (CBFB) gene near 16p13 (MYH11) formed a fusion
gene CBFB-MYH11 transcribing SMMHC is observed in FISH analysis.

Hypothesis:

If CBFB-MYH11 is interrupted by promoter to have separate expression of each gene,

then problem (Leukemia) can be eliminated.

Experiment:
Three groups of mice are taken & organism of group 1, 2 & 3 gene present at 16p13 was
silenced. In group 1 mice hematopoietic stem cells are mutated by insertion of gene 16p13
without promoter in band of 16q22. In group 2 mice hematopoietic stem cells, gene 16p13 with
promoter is inserted in band of 16q22. In group 3 gene for MYC11 was silenced. In group 4
organisms are not mutated instead given condition similar to experimental groups (control).
Organisms are grown & blood samples of organisms are checked after regular interval.

Expected Results:

In group 1 organisms, blood test would be positive for leukemia. In organisms of group 2
& 4 no disease symptoms would be observed while in organisms of group 3 there would be some
pathological symptoms observed due to knock down of MYC11 but no symptoms of Leukemia
would be observed.

Inference:

Problem can be eliminated by inserting a piece of DNA (either promoter to have separate
expression of CBFB) using lipopolyplex. If inserted successfully, it will help to eliminate the
negative aspects of chromosomal aberration by modifying the expression of CBFB-MYH11
complex. Stem cell would behave normally as thus the progeny (new stem cell & differentiated
one).

Why gene therapeutics using nanosystem:

1.Adult cancer treatments are quite well-developed but pediatric malignancy is somehow under
developed so first objective of this study was to aim at proposing a better solution for it.

2.This Nano system was aimed to overcome the side effects of conventional treatments and anti-
cancer drugs in every possible aspect.

3.Conventional cancer drugs are low weighted but this property results in there excessive
wasting and low therapeutic efficacy to overcome this limitation this Nano system was made in
organic solvents which increases its availability in body for more time and decrease cytotoxicity
(Bharali DJ et.al, 2010).

4.It was aimed to design such Nano system which is within specified size range (10-100nm)
which is required for its effective function as it need to cross barriers like endothelium to reach
tumor and perform

5.To make this Nano system target specific it was aimed to conjugate its surface with a
biomarker so this lipopolyplex is conjugated with ligand for epidermal growth factor (EGFR)
(Godin B et.al, 2010).
6.Vigilant design of the physicochemical specificities of nanoparticles and their binding affinity
to tumor cells and the targeted delivery of the drug can advance diagnostic and treatment results.

7.This Nano system was aimed to use in both diagnostics and therapeutics because it offers more
profound imaging approaches that effect in earlier detection that offer noninvasive and effective
treatment (Galvin P et.al, 2012).

8.These dynamic Nano molecules have optical, structural and magnetic properties that make
them a potent candidate for treating leukemia.

Drawbacks in using gene therapeutics using nanosystem:

The major problems are potential chronic and acute toxic effects; the potential toxicity of
NMs cannot be ignored in antitumor therapy. NMs may be attached to the surface of biological
membranes by adsorption or electrostatic interactions, and they can cause damage to cells by
producing reactive oxygen species, leading to protein denaturation, lipid peroxidation, DNA
damage, and ultimately cell death.

Alternative Approaches :

 The identification and detection of leukemia are difficult. A recent approach for the
diagnosis of leukemic cells in whole blood is the development of fluorescence-based
anisotropy. This technique involves the combined action of near-infrared fluorescent
nanoparticles with target cell-specific aptamers. The fluorescence anisotropy method
involves a rapid homogeneous recognition technique for detection of leukemic cells in
whole blood without the need of complicated separation time.
 Another alternative approach to provide solution to cancer problems is the establishment
of the combination therapy for better long-term prognosis. Combination therapy can
modulate different signaling pathways, enhancing the therapeutic effect by overcoming
toxicity and, can overcome the mechanisms of drug resistance associated with cancer
treatment.
 Another electrochemical approach to detect the cancer cells is the development of gold
NP modified glassy carbon electrodes (GCE). These NPs were utilized to displace
difference between drug sensitive leukemic cells and drug resistant leukemic cells.
 Magneto-PCR-enzyme linked gene technique allowed separation of PCR products and
were highly specific for leukemic cells. Nanoparticle based cancer diagnostics could be
significantly maximized by selective tissue localization. The selective targeting ligand
can be conjugated on nanoparticles by developing multiple nanoparticle systems for
different diseases.
Expected result:

An innovative idea created to examine if our hypothesis could be successful and if our
gene therapeutic in association with nanosystem could actually target the abnormal cells. The
abnormal cells which are responsible for leukemia.

Conclusion:

Nanotherapeutics have shown promising outcomes for many diseases especially for
different types of malignancies so we designed a Nano system for acute lymphoblastic leukemia
(ALL) to overcome the drawbacks of conventionally used drugs and therapies. Pediatric cancers
are less researched as compared to adult cancers so much work is needed on this field so in
present study Nano system is designed for it. This Nano based solution is beneficial for both
diagnostics and therapy as it poses imaging properties as well as it has EGFR conjugated
lipopolyplexes which specifically target it to tumor cells of ALL reducing the cytotoxic effects
on healthy cells. This solution has shown considerable results as it increased circulation,
retention time, degree of target specificity and minimal side effects. This Nano system has shown
additional benefits such as targeted delivery, lesser dosage, increased gap time between
subsequent doses, lesser immunogenicity and minimal toxicity. Accessibility of Nano carriers
that marks leukemia in the CNS, blood, testes and the bone-marrow concurrently as a sole
formulation ought to aid treatment of ALL in with negligible side-effects. Accomplishing this
goal needs systematic methods joint with long-term struggles and obligations by interdisciplinary
biomedical scientists and clinicians. Determining the worth of blood retention time for short-
term and long-term consequences in treating leukemia require in vivo studies. It is significant to
note that long-term retaining of Nano systems in the bone-marrow may obstruct the self-renewal,
propagation and growth of normal stem and progenitor cells. Another aspect to be taken in
account is the burst release of drug which may result in toxicity. Eliminating leukemia requires
the personalized treatment so as to have complete information of chromosomal aberration(site of
insertion, deletion, inversion) so as to plan the appropriate strategy to eliminate the problem.
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