MARTIN JOSEPH

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KOZHIKODE DISTRIC-673528, KERALA, INDIA

Ph. No: +91 9496189921,

+91 9605189921

Email: mje03111996@gmail.com

OBJECTIVE: THROMBOLYTIC DRUG DELIVERY USING POLY (LACTIC-CO-GLYCOLIC ACID)

NANO MOLECULE AS DRUG CARRIER.

This work replaces the direct injection of thrombolytic drugs without any means of target-oriented action to the
patients with blood vessel block who have possibility of stroke and heart attack. The direct injection to the body
blood vessel causes many side effects and cannot be administered to old people. But when it is guided well so that it
only acts on abnormal clots excluding normal clots, risks come along with the former method can be reduced. Here, the
drug is injected to body along with nano carrier vehicles which come with perfect guide mechanism.
Mode of delivery of drug – nanocomposite: through injection .
Thrombolytic drug treatment is for people who are suffering from aggressive blood clots. They may have to undergo
angioplasty at a later stage, if cannot dissolve the blood clot using thrombolytic drug treatment. Or some patients are
not compatible with this method.

1. PHASE-1:
Finding the chemical for guiding or to direct the drug- nano carrier system to spot of action.

2. PHASE-2:
Method of preparation of polylactic acid based nano molecules which is the carrier of the drugs, and surface
coating it with poly ethylene glycol arms for bridging the drug as well as the protein or antibody, forming an
antibody-PEG-nanocarrier-drug composite.
Mode of action
Protein or antibody or any chemical is identified which flows to the abnormal blood clots. These abnormal clots
cause situations such as deep vein thrombosis (DVT) or pulmonary embolism (PE) and other clots in arteries. Abnormal
blood clots are the clots which goes on forming clots without any regulation or control of anticoagulant mechanism. In
normal clots, once the wound is healed, anticoagulant mechanism start action and dissolve the platelet -fibrin plug.
The special chemical or protein or antibody which is either found in human body or artificially designed has the
ability to attach only to abnormal clots hence they act as perfect guide for drug– nano composite to reach the blood clots.
Till now there is no chemical or biomolecule discovered which has the affinity only to the abnormal clots. So, the
similar attempts to deliver the drug via nano carrier so far done have limitation that some of it miss out of the control
and reach to the site where normal clots are formed. But with the help of the chemical that we are looking for, the drug
delivery is in full control and can be made location specific.
The nano carrier also has the fluorophore linked to it. The fluorophore on a nano carrier show sharp spectral peak in the
visible range and the movement of the drug through the blood can be observed using 2-photon microscopy. The
nanocarrier free of drug shows a different spectral peak. So once the nano carrier drug composite reaches the clots, the
clots will glow more brightly. Hence the drug release at the site of blood clot can be seen with 2- photon microscopy.
The nano carrier that is going to be used in this work is poly lactic acid based nano molecules, which has advantages
such as:
 Biocompatibility (degradation products do not elicit critical responses and are easily metabolized by the body),
Hydrolytic degradation in situ, tailorable properties,
Well-established processing technologies,
Also, it is cytonontoxic.
The drug is released through irradiation by ultrasound (Shan Liu). The drug used for thrombolytic action is tPA(tissue
plasminogenic activator) which lysis the fibrin. The tPA has got one (-NH2 ) arm and a (-OH ) arm which can be
bonded with ( poly ethylene glycol ) arm on suitable preparation. Once the thrombolytic drug is released it readily picks
up the fibrin plug because it has greater affinity towards it.
Major challenge: Finding the perfect guide or artificially designing it.
Possible solution: The best and foremost approach to this challenge is the studying the blood clot sample

Normal blood clots Abnormal clots.

Readily undergo clot lysis automatically, Clot goes on increase in size and no lysis
once wound is healed. happen.

Even though the clot is there, no symptoms of The symptoms of the oxygen deficiency are
oxygen deficiency are shown. So, no much shown. Implies an oxygen concentration
change in oxygen concentration across the gradient. In veins carbon dioxide gradient
blood clot. develops.
No considerable field forms Ionic or charged elements which are big in
size of the blood deposit over on side and
other side has lower concentration of these.
These develops small static electric field.
No change in elastic moduli. Moduli of elasticity may change.

Vasoconstriction by Electrical Stimulation: New Approach to Control of Non-Compressible

Haemorrhage (Mandel): says the blood clotting can be generated using electric field and they
demonstrated the initiation vasoconstriction on the application of small electric field and formation of
blood clot.

Conversely, this supports the thought -blood clot produce small electric field

1st Approach to discover the perfect guide

• The refractive index, dielectric constant, capacitance, permittivity, density of clot, hole size of the
clots, number of holes, chemical potential are measured for both normal and abnormal clots and
see the difference in them.
• The corresponding bio molecules which are selective to each of the difference are attached to nano
carrier and analysed their guiding capacity towards the abnormal clots.
• We combine all the chemical guides involved with above action on the nano carrier.
• This composite is first placed in to normal clots and upon varying the chemicals adsorbed on the
nanocarrier, we can identify the least reactive phase (Mandel).
 • Then it is added to sample of abnormal clotting and changed the chemicals number adsorbed for
maximum guiding and attachment of nanocarrier on the clot.
• Now it is added to a mixture containing both normal as well as the abnormal clot and changed the
number of molecules on the surface of the nanocarrier for the maximum attachment on the
abnormal clots and less affinity to normal clots.

2nd Approach to discover the perfect guide

The second approach is artificially introducing the guiding chemical.
• I suppose to study the biological activity which responds differently to different concentration of same
entity.
• Adopt useful things out of it and apply in the current problem.
• Initially, normal clot are introduced to an animal body, then study and isolate the antibody produced.
(Animals, like Monkey or mice)
• Then the abnormal clots, introduce into another animal of same species and study the antibody
produced. If two are different, the antigen part of the second antibody is cut down and can be used as
guide for abnormal clots and these don’t affect normal clots.

3rd Approach
Autoimmune thrombocytopenia (AITP) is a disorder of low blood platelet counts in which platelets
are destroyed by antibodies produced by the immune system.(National Heart)
Antibody from the serum of the patients are isolated and designed for our purpose with antibody engineering
which intent to remove the destroying capacity of the antibody but keep the selective attachment of it on
platelets. Also, modification is introduced to selectively attach on abnormal clots.

This part of finding the guide for target specific action is the difficult part of the work. I have got the motivation for this
work after reading the therapeutic application of quantum dots for cancer treatment.

INTRODUCTION
Nano Particles are tiny materials having size ranges from 1 to 100 nm. They can be classified into different classes based
on their properties, shapes or sizes. The different groups include fullerenes, metal Nano Particles, and polymeric NPs.
NPs possess unique physical and chemical properties due to their high surface area and nanoscale size. Their optical
properties are reported to be dependent on the size, which imparts different colors due to absorption in the visible region.
Their reactivity, toughness and other properties are also dependent on their unique size, shape and structure. Due to
these characteristics, they are suitable candidates for various commercial and domestic applications, which include
catalysis, imaging, medical applications, energy-based research, and environmental applications.
Controlled drug delivery systems (DDS) have several advantages compared to the traditional forms of drugs. A drug is
transported to the place of action, hence, its influence on vital tissues and undesirable side effects can be minimized.
Accumulation of therapeutic compounds in the target site increases and, consequently, the required doses of the drugs
are lower. This modern form of the therapy is especially important when there is a discrepancy between the doser or the
concentration of a drug and its therapeutic effects. Cell-specific targeting can be accomplished by attaching drugs to
specially designed carriers. Various nanostructures, including liposomes, polymers, dendrimers, silicon or carbon
materials, and magnetic nanoparticles, have been tested as carriers in drug delivery systems.
When a blood clot forms where it should not have been developed, it is called a thrombus. A blood clot is also called a
thrombus. The clot may stay in one spot (called thrombosis) or move through the body (called embolism or
thromboembolism). The clots that move are especially dangerous. Blood clots can form in arteries (arterial clots) or
veins (venous clots).
Any blood clots that form in arteries (arterial clots) or veins (venous clots) can be serious. A clot that forms in one of
your bodies larger veins is called a deep vein thrombosis (DVT). A stationary blood clot, or one that stays in plays, may
not hurt you. A blood clot can block the arteries to the heart. This can cause a heart attack, when part of the heart muscle
dies due to the lack of oxygen being delivered by the blood.
Thrombolytic work by dissolving a major clot quickly. This helps restart blood flow to the heart and helps prevent the
damage to the heart muscle. Thrombolytics can stop a heart attack that would otherwise be larger or potentially deadly.
Outcomes are better if the person receives a thrombolytic drug within 12 hours safter the heart attack starts. But the
sooner treatment begins, the better the results. Adverse effects of any fibrinolytic agents are almost similar, which
include bleeding, hypotension, allergic reactions, angioedema, and reperfusion arrythmias (when used in the acute MI).
Among all of the fibrinolytic agents, streptokinase is most antigenic, thus most frequently complicated by allergic
reaction and hypotension. (MedlinePlus)
Bleeding is the most frequent complication of thrombolytic therapy and can occur in puncture site or spontaneously
anywhere inside the body. Intracranial hemorrhage or hemorrhagic stroke is the greatest concern. Risk factors associated
with hemorrhagic complications include elderly patient, uncontrolled hypertension, recent stroke or surgery, presence
of bleeding diathesis, and concurrent use of anti-coagulants. Overdose most often occurs when administered in a non -
body – weight adjusted manner and can cause severe hemorrhagic complications.
This work attempt to identify the chemical or the biomolecule that only responds to abnormal clots, there by using it in
the thrombolytic drug delivery by nanoparticle – fluorophores conjugated system. The particular biomolecule which
only has the affinity towards abnormal clots ensures the target specific precise drug delivery. PLGA nanoparticles
associated with PEG arms and fluorophores is the drug carrier. The biomolecules act as perfect guide for the transport
of the drug loaded nanocarrier system. Ultrasound irradiation is the mechanism by which drug is released. Thrombolytic
drug used is tPA protein.

METHOD
Blood clot analysis:
Hall effect is used to measure the electric field on the abnormal as well as the normal clots
The classical HE describes the charge separation phenomenon in a conductive object moving in a magnetic field . This
charge separation is the result of the opposing Lorentz forces on the positive and negative charges, and leads to an
externally detectable voltage, the Hall voltage. The Hall voltage amplitude is determined by the strength of the Lorentz
force and the charge density and mobility. The Lorentz force is proportional to the magnetic field B0 and the velocity of
motion ν, while the charge density and mobility are characterized by the overall conductivity σ of the object, including
the dielectric contribution. Thus, the Hall voltage Vh α σνB0. (Han Wen). To find the conductivity, a method called hall
effect imaging where in a hall effect coupled with ultrasound imaging is used. The capacitance, permittivity dielectric
constant of the samples are measured. Otaki et al. suggested that dielectric blood coagulometry may be a useful method
for measuring blood clotting and could provide the detailed assessment for the status of anticoagulant therapy (Li Gun).
To measure the refractive index, total reflection ellipsometry is used. (Jian-Cheng Lai)
Hole size as well as the hole number found out using light diffraction through the clot sample. A low energy laser beam
is allowed to fall on the tissue with the help of a optic fibre. The photo of the diffraction pattern is analysed for find the
hole sizes as well as the number of holes.
The chemical potential μ, which is simply the free energy per molecule, is probably the most useful thermodynamic
quantity for describing and thinking about chemical systems. Because μ represents an energy for one molecule, it is easy
to think about concretely. In fact, for a system consisting of molecule types A, B, C, etc., each occurring with NA, NB,
... copies, we can write the total free energy at constant pressure exactly as

(zuckerman)
Density of the blood clot can be find out. The mass of the clot cane be measured. For the measurements of clot density,
the volume of the clots can be determined using a fluid displacement method. A standard laboratory scale is used to
assess the mass of each clot in physiologic saline at room temperature. Thus, the influence of physiologic variability
and experimental uncertainty were captured in the standard deviation of the density and calorimetric measurements.
Attempts are done for finding the difference between normal clots and abnormal clots. Number of normal clots as well
as abnormal clots are collected which are studied by following methods.
1. 2d gel electrophoresis
 2. Mass spectrometry analysis
3. Fibrinogen purification from plasma
4. Western Blot analysis
Formation of nanoparticles: A solution of the polymer and the dye in a water-miscible solvent (here acetonitrile) was
quickly added to a large excess of an aqueous phase, resulting in supersaturation and formation of nanoparticles. chosen
dye systems with the bulky perylene dye lumogen red, which combines very high brightness and photostability . Based
on data available, chose 1 wt% of the dyes with respect to the polymer as optimum loading. The most straightforward
approach to increase NP brightness is to increase their size, since the number of encapsulated dyes increases with the
third power of the radius. A tenfold increase in brightness can thus be achieved by a 2.2-fold increase in size. In the case
of nanoprecipitation, the particle size depends on the concentration of the polymer (View ORCID ProfileIgor Khalin).
Coating dye loaded PLGA nano molecules with PEG: PLGA and PEG were mixed in 1:1 (wt. ratio) in chloroform.
The solvent was evaporated under a gentle stream of air and the obtained film was redispersed in 250 μl of
dichloromethane followed by precipitation in ice-cold diethyl ether thrice. The precipitated copolymer was filtered out
and dried under vacuum at 40°C for 24h (Margaret Lashof-Sullivan).
Linking tpa with PEG-PLGA-FLUROPHORE:The nanoparticles were generated by covering PEG-PLGA with a
mixture of human tissue plasminogen activator and bovine serum albumin.100 µL PEG-PLGA (0.15 mg in toluene) was
mixed with 600 µL BSA (5 mg mL-1 in water) and 100 µg tPA (human tissue plasminogen activator,1 mg mL-1 in
water). 4 mL of 0.1 M sodium bicarbonate buffer was added to this mixture and probe sonicated for 1–2 min on an ice
bath until a homogeneous milky solution was formed. The precipitated copolymer was filtered out and dried under
vacuum at 40°C for 24h (Eszter Voros)
Molecular weight of the product is determined by gel permeation chromatography. Particle size can be determined by
dynamic light scattering and scanning electron microscopy.

EXPECTED OUTCOME
The proposed study involves finding the differences between normal and abnormal blood clots, thereby identifying the
chemical which reach out only to the abnormal clots, synthesis of tPA-PEG-PLGA -fluorophore nano composite. This
method of drug treatment is experimented on animals. The outcomes of the proposed study can be used to develop
nanocarrier drug delivery for dissolving of the abnormal blood clots.

IMPACT ON INDIA
Cardiovascular diseases have assumed epidemic proportions in India as well. The Global Burden of Diseases (GBD)
study reported the estimated mortality from coronary heart disease (CHD) in India to be 1.6 million in the year 2000
which is estimated to increase to ∼64 million by the year 2015. The projected rise in disease burden due to CVD is
expected to make it the prime contributor of total mortality and morbidity.
Reports on CAD in Indians have shown that Asian Indians are at 3–4 times higher risk of CAD than white Americans,
6 times higher than Chinese, and 20 times higher than Japanese. Various independent epidemiological studies conducted
in North India suggest that the prevalence of CAD has increased from 1% in 1960 to 10.5% in 1998 in the urban
population. A higher prevalence of CAD, ranging from 11.0% to 14.2%, has been reported from South India. Taking
into account the size of the Indian population, these prevalence rates indicate that a large number of deaths can be
attributed to CAD.
In smaller cities of India, thrombolysis continues to be a vogue as the facilities for primary angioplasty in myocardial
infarction (PAMI) are not available and transport facilities are limited. Also, in India the ambulance facilities are poor
and those available are often not up to the mark. Most of the times, patients with STEMI condition present late after the
onset of symptoms and the desired goals like, door-to-needle time and door-to-balloon time, as specified in guidelines,
are rarely met. In CREATE registry, only 41.6% of patients with STEMI presented within 4 h of the onset of chest pain
and 31% of patients presented after 12 h.
Presently, there are over 500 centres with facilities for coronary angiography in India; however, the number is quite
disproportionate to the size of CAD patient burden in the country. Extrapolation of the Global Burden of Diseases
(GBD) study showed that current burden of CAD in India is as high as >32 million patients with a sizeable proportion
in rural (3–5%) and urban (7–10%) population. Hence, in Indian conditions with delayed access to the limited number
of catheter laboratories and insurance benefits being a rarity, pre-hospital or in-hospital thrombolysis should be the
treatment of choice for patients with Acute Myocardial infarction
Approval of third generation thrombolytics by the Drug Controller of India (DCGI) was a major step towards developing
viable STEMI systems of care in the country. These new generation fibrinolytic drugs improve reperfusion rates and
relative clinical outcomes in ST Elevation Myocardial Infarction patients. The ability of third generation thrombolytics
to be administered as a bolus has made it possible to consider pre-hospital treatment in certain settings. New data on
adjunctive therapy with Clopidogrel may also expand the benefits of fibrinolytic therapy. Finally, an improved
understanding of the role of pharmacoinvasive approach, i.e., the combination of immediate pharmacological
reperfusion with invasive cardiac procedures, suggests the possibility of targeting PCI in high-risk patients (Jamshed
Dalal).
The statistics tells that Indian population which is second in the world is much vulnerable to stroke and other medical
conditions regarding abnormal clots. As of now, nano particle guided drug delivery is not practised anywhere in India
for the treatment of above-mentioned disease even though they come with more sophisticated treatment and better
results. It is mainly because those treatment are not cost effective in developing countries like India, where the
thrombolytics are bought off from foreign countries. In some countries the Government produce the thrombolytic drugs.
Also, the nanoparticle drug delivery is not as precise because it lacks a perfect guide which binds to abnormal clots
alone. Considering the situations, it is understandable that India sides with oral administering as well as IV-line transfer
of drugs. With the success of this work, our Country might take a turn over. As I am sure that this work on its success
will inspire medicine manufactures to produce nano particle guided drugs in large scale which cause a cheaper drug
availability in the market. Following are some limitations of the current thrombolytic therapy.
1. Thrombolytic drugs are administered orally or through IV line which enables the thrombolytic to acts on normal
clots too.
2. This cannot be prescribed to pregnant women, or aged people since they may suffer from significant bleeding.
3. In addition, other clotting factors such as factor V and factor VIII may be consumed which is undesirable.
4. It may be catastrophic in those who have had a recent surgery, trauma, or undetected peptic ulceration. The
incidence of serious hemorrhage varies between 4 and 11 % of patients. Significant bleeding has also been
reported in patients with cerebrovascular accidents and central retinal vein occlusion.

The above limitations arises because the thrombolytics act all over the body rather than target-oriented action. But
with employment of proper guide chemical with precise target specific delivery, the proposed drug nano carrier
composite in the work, can overcome above listed limitations.

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