1

PROJECT REPORT

ON

GROUND MOTION SELECTION AND SCALING

USING RESPONSE HISTORY ANALYSIS

Prepared By- Under the supervision of-

Sanjana Jain Prof. Shamsher B.Singh
2016A2PS0587P
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Acknowledgement

This project was possible only because of the support and guidance of
some people, I would like to extend my heartfelt gratitude to all those
people.

I would like to express my special thanks of gratitude to Prof. Shamsher

B.Singh who gave me the golden opportunity to do this wonderful project
on the topic “Ground Motion Selection and Scaling for performing response
History Analysis”,and for providing his invaluable guidance throughout the
course of the project.

I would also like to thanks Mr.Harish Kumar Moolchandani for enlightening

me on how to proceed with the project and constant support. He made sure
that I understand the basics before actually getting on the topic and
constantly motivated me to work harder.

It had been an enriching experience and an experience full of erudition.

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Table of contents

1. Abstract…………………………………………..4

2. Introduction………………………………………5

3. Nanoparticles ……………………….…………..6

4. Preparation of Nanoparticles…………………...8

5. Types of Nanoparticles………………………….9

6. Intracellular delivery……………………………..12

7. Mechanism of uptake…………………………...13

8. Infectious agents………………………………...16

9. Targeted Drug delivery towards infections…….17

10. Conclusion …………………………………….19

11. References …………………………………….20

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INTRACELLULAR DELIVERY OF
NANOPARTICLES IN INFECTIOUS
DISEASES

1.Abstract

To understand the interactions of Drug carriers with cells and how these interactions
influence the cellular uptake is of utmost importance, as targets for most of the
therapeutic agents against several disorders are localized in the subcellular
compartments. Intracellular Drug delivery based on third order targeting is required for
problems related to efficacy of delivery and toxicity.
The Nanoparticles are used for medical purposes for various reasons based on their
unique properties, such as their surface to mass ratio,which as compared to other
particles is much larger; their quantum properties and their ability to adsorb and carry
other compounds. NPs have a relatively large (functional) surface which is able to bind,
adsorb and carry other compounds such as Drugs, probes and proteins, they can be
administered intravenously. Because of the small size they don’t block arteries. They
are also used to reduce toxicity and side effects of Drugs.Nanotechnology is one
approach to overcome challenges of conventional Drug delivery systems based on the
development and fabrication of Nanostructures. Nanoparticles can be recognized by the
host immune system when administered intravenously administered and cleared by
phagocytes so it is coated by polymer, the release is then controlled by diffusion of Drug
from polymeric membrane.

2.Introduction
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Targeted Drug delivery is a method of delivering medication to a patient in a manner

that increases the concentration of the medication in targeted parts of the body relative
to others.

Active Targeting- carrier system bearing Drug reaches to specific site on the basis of
modification made on its surface rather than natural uptake by RES. Active targeting
involves the use of targeting ligands for enhanced delivery of NP systems to a specific
site. Typical targeting ligands include small molecules, peptides, antibodies and their
fragments, and nucleic acids such as aptamers.Conjugating targeting ligands to the NP
surface can facilitate active targeting of NPs to receptors that are present on target
cells, leading to enhanced cell internalization and/or specific uptake through receptor-
mediated endocytosis

● First Order Targeting - Drug delivery system releases Drug in a specific organ,
also called organ targeting.
● Second Order Targeting- Drug delivery system releases Drug to specific cell
types also known as cellular targeting.
● Third Order Targeting- Drug delivery system can enter specific cells and leave
the Drug intracellularly.

Passive Targeting- The accumulation of Drug/Drug carrier systems at the intended site
of action by the action of physicochemical and physiological factors is passive targeting.
Certain tissues under diseases conditions present opportunities in terms of modified
physiologies which can be exploited by passively targeting Nanocarriers.
Presence of leaky vasculature in inflammatory bowel disease and inflammatory
rheumatoid arthritis and in tumor tissues.
The Nanocarriers are cleared by retico endothelial system can be used to
passively target to treat infections that affect the RES.
Passive targeting also benefits from the presence of internal stimuli, such as ph
difference.

Controlled Release Drug delivery- delivery of a Drug at a predetermined rate

according to physiologically / therapeutic needs of the body.

Prolonged Release- Drug is made to release in intervals for prolonged or extended

time.

Sustained Release- Drug is released uniformly or constantly. Type of a controlled

release.
While prolonged release have no specifications to time or rate of concentration.
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3.Nanoparticles

Nanoparticles are particles between 1 and 100 Nanometres in size with a surrounding
interfacial layer. Because of their submicroscopic size, they have unique material
characteristics, and manufactured Nanoparticles may find practical applications in a
variety of areas, including medicine,, engineering, catalysis, and environmental
remediation. Nanoparticles have been found useful in development of systemic, oral,
pulmonary, transdermal and other administration routes to study Drug targeting, the
enhancement of Drug bioavailability and protection of Drug bioactivity and stability.
Encapsulation of antimicrobial Drugs in Nanoparticle system has come out as an
inventive alternative that enhances therapeutic effectiveness and minimizes undesirable
side effects of Drugs. There are three major physical properties of Nanoparticles, and all
are interrelated:

● They are highly mobile in the free state.

● They have enormous specific surface areas.
● They may exhibit quantum effects.

Some Other Properties of Nanoparticles-

● Nanoparticles can be administered intravenously.

● Because of the small size, they don’t black arteries.
● They have high surface area to volume ratio. This provides a tremendous driving
force for diffusion, especially at elevated temperatures.
● Surface property of Nanoparticles can be changed for targeted Drug delivery.
● Nanocarriers may overcome solubility or stability issues of the Drug and minimize
Drug induced side effects.
● Suspensions of Nanoparticles are possible because the interaction of the particle
surface with the solvent is strong enough to overcome differences in density.
● To prolong the circulation, they should be small enough or deformable to escape
filtration in spleen.
● Improved bioavailability by enhanced aqueous solubility.
● Targeting Drug to specific location in the body.
● Increasing resistance time in the body (increasing half life for clearance).
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● The large surface area to volume ratio also reduces the incipient melting
temperature of Nanoparticles.
● Decreased patient-to-patient variability.
● More rapid onset of therapeutic action
● Overcoming resistance to common antibiotics
● Can progress therapeutic index and extend Drug circulation.

Some Disadvantages of Nanoparticles-

● Could penetrate skin and cause undesired side effects.
● Easily released into the environment.
● Large surface can make them too reactive and explosive in some situations
● Easily become airborne, breathing in can potentially damage the lungs.
● Changes the properties of a material.

Main advantages of Nanoparticulate systems as compared with free Drugs are

an efficient Drug encapsulation, protection from inactivation, targeting infection
sites and the possibility to deliver Drugs by overcoming cellular barriers. The
Nanoparticles give uniform release in prolonged Drug release while free Drug
doesn’t give. As shown in the figure-
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4.Preparation of Nanoparticles

The selection of appropriate method for the preparation of Nanoparticles

depends on the physicochemical character of the polymer and the Drug to be
loaded. Nanoparticles can be prepared from a variety of materials such as
proteins, polysaccharides and synthetic polymers. The selection of matrix
materials is dependent on many factors including :

• Antigenicity of the final product.

• Biocompatibility and toxicity.
• Degree of biodegradability.
• Drug release profile desired.
• Inherent properties of the Drug (aqueous solubility and stability).
• Size of Nanoparticles required.
• Surface characteristics (charge and permeability).

5.Types of Nanoparticles:-

1. Carbon-Based Nanoparticle- Carbon-based Nanoparticles include two main

materials: carbon Nanotubes (CNTs) and fullerenes.
Applications - Carbon Nanotubes have functionalization enhanced solubility,
penetration to cell cytoplasm and to nucleus, as carrier for gene delivery, peptide
delivery. Conjugation with peptides may be used as vaccine delivery structures
.Tissue-selective targeting and intracellular targeting of mitochondria have been
shown with use of fullerene structures. Fullerenes are also known to exhibit
antioxidant and antimicrobial behavior.

2. Ceramic Nanoparticles- Ceramic Nanoparticles are inorganic solids made up of

oxides, carbides, carbonates and phosphates. These Nanoparticles have high
heat resistance and chemical inertness. They are superconductive,
dielectric,ferroelectric and ferromagnetic.
Applications - Medical technology used Nanoceramics for bone repair.
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3. Metallic Nanoparticles- They have large surface‐ area‐ to‐ volume ratio as
compared to the bulk equivalents; large surface energies the transition between
molecular and metallic states providing specific electronic structure.
Applications- Drug and gene delivery, highly sensitive diagnostic assays,
thermal ablation and radiotherapy enhancement.

4. Polymeric Nanoparticles- The polymeric Nanoparticles (PNPs) are prepared

from biocompatible and biodegradable polymers in size between 10-1000 nm
where the Drug is dissolved, entrapped, encapsulated or attached to
Nanoparticle matrix.
Applications- Excellent carrier for controlled and sustained delivery of Drugs.
Stealth and surface modified Nanoparticles can be used for active and passive
delivery of bioactives. Polymer based Nanoparticles effectively carry Drugs,
proteins, and DNA to target cells and organs.

5. Lipid based Nanoparticles- They have the least toxicity and hence are used for
in vivo applications, due to this property of lipid based Nanoparticles, they carry a
high potential for research in the area of Drug delivery.

6. Polysaccharide Nanoparticles- because of the amphiphilic nature,

polysaccharide‐ based materials can self‐ assemble into ordered aggregates
within aqueous environment and have various derivable groups on their
molecular chains which can be easily adopted and modified with other chemical
agents. Polysaccharides show a high affinity for mucosal surfaces covering the
nasal, pulmonary and gastrointestinal tract. They are versatility, biodegradability
and biocompatibility.

7. Silica Nanoparticles- mesoporous silica Nanoparticles (MSNs) are widely used

as a delivery reagent because silica possesses favorable chemical properties,
thermal stability, and biocompatibility. The unique mesoporous structure of silica
facilitates effective loading of Drugs and their subsequent controlled release of
the target site. The properties of mesoporous, including pore size, high Drug
loading, and porosity as well as the surface properties, can be altered depending
on additives used to prepare MSNs. Active surface enables functionalization to
change surface properties and link therapeutic molecules.
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8. Liposome - Liposomes are targeted Drug delivery systems consisting of one or

more concentric spheres of lipid bilayers seperated by water or aqueous buffer
components composed of natural or synthetic phospholipids.
Applications- Long circulatory, offer passive and active delivery of gene, protein,
peptide and various other.

9. Polymeric Micelles-Polymeric micelles are core-shell structures synthesized

form amphiphilic block copolymers.They can be used for Drug delivery as the
hydrophobic interior has the capacity to hold Drugs which are poorly soluble in
aqueous solution.They represent an effective delivery system for poorly water-
soluble anticancer Drugs. With small size (10–100 nm) and hydrophilic shell of
PEG, polymeric micelles exhibit prolonged circulation time in the blood and
enhanced tumor accumulation.
Applications- Long circulatory, target specific active and passive Drug delivery,
diagnostic value.

10. Dendrimers-The primary use for DEN's is as a catalyst due to their extremely
high surface area to volume ratio. Advantages that DENs have over other
Nanoparticles is that they are monodisperse and easy to make.
Applications- Long circulatory, controlled delivery of bioactives, targeted delivery
of bioactives to macrophages, liver targeting.

11. Quantum Dots- Quantum dots (QDs) are Nanocrystals of semiconducting

materials measuring around 2–10 nm, consisting of a semiconductor inorganic
core (CdSe), an aqueous organic coated shell (e.g., ZnS) to improve optical
properties.
Applications- Long term multiple color imaging of liver cell; DNA hybridization,
immunoassay; receptor mediated endocytosis.
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6.Intracellular Delivery

Intracellular Delivery is an important technique for treatment of many diseases, since in

many cases the target is located inside the cell, either in the cytoplasm or any organelle
of the cell, such as nuclei, mitochondria, or lysosomes. Based on third order targeting.
Many pharmacologically active substances may not be effective enough in vivo or have
adverse effects due to lack of specific affinity of the drug to the pathological site, so a
large total dose of active molecule is needed to achieve a high local concentration.
Another critical issue consists of the tremendous difficulty of such drugs and active
molecules in crossing biological barriers such as cell membranes and membranes of
intracellular compartments, to reach the site of action. As a result, it is necessary to
administer the drug in large quantities to achieve a therapeutic concentration in a
particular body compartment, leading to non-specific toxicity and other adverse side
effects due to high drug doses.To address these challenges, Drug targeting is aimed to
enhance drug accumulation in the target organ, tissue, or cell, selectively and
quantitatively such that the concentration of the drug at the pathological site is high,
while its concentration on non-target tissues is low, independently of the administration
route.

Nanoparticle Drug delivery platforms including liposomes, polymeric Nanoparticles,

dendrimers, and various inorganic nanoparticles have been increasingly exploited to
enhance the therapeutic effectiveness of existing antibiotics.

Nanoparticles must first be able to traverse the plasma membrane to reach the
subcellular targets inside the cell. Nanoparticles may be incorporated either by directly
interacting with membrane-embedded receptors or indirectly by associating with the
lipid bilayer. In the first method or approach Nanoparticles are made functional with
ligands that bind to receptors on the cell with high affinity and specificity. Internalization
of the resulting receptor–ligand complexes then leads to receptor-mediated endocytosis
of the nanoparticles.

Controlling the route of Nanoparticle uptake is important for biological response.

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7.Mechanism Of Uptake

Nanoparticles can enter cells via phagocytic or non phagocytic pathways. Route of
internalisation determines the site of drug release, which can be in the acidic and
enzyme rich environment of lysosomes, or nanoparticles avoid this compartment and
release drug in cytosol or another organelle. The process can be controlled by a careful
selection of nanoparticle ingredients and precise design of their physicochemical
properties.

Surface conjugation of specific ligands allows nanoparticles to interact with a particular

group of receptors at the cell and tissue surfaces, thereby favourably modifying the
intracellular disposition of nanoparticles. Nanoparticles also offer the potential to protect
the encapsulated molecules from both extracellular and intracellular degradation,
resulting in improved intracellular bioavailability. Following incorporation into the body,
Nanocarriers can translocate from their site of deposition to the elsewhere, such as the
brain and bone marrow, by blood circulation system. The surface of nanoparticles is
commonly coated with a layer of dissolved extracellular molecules in body fluids, such
as proteins, sugars and lipids before their encounter with the cellular membranes.When
reach the site of action, the loaded cargo may be released outside or cells take up
nanoparticles and unload cargo at the desired intracellular compartment. Generally, the
transport of macromolecular carriers such as nanoparticles from the cell surface to the
lysosomal vesicles occurs with a process known as endocytosis. Following the
nanoparticles uptake, they are able to significantly increase the Drug concentration and
act as an intracellular Drug reservoir for long‐ term release.Among natural polymers,
proteins and polysaccharides tend to be associated with cells, rapidly internalized and
degraded, thus enabling the intracellular release of the incorporated drug/gene from the
transporter.

One significant obstacle for the development of effective drug/gene therapies,

particularly anticancer therapies, has been their inability to cross the plasma
membranes of cells. Plasma membrane provides a boundary between the cell and its
environment to maintain the activities that are critical for the normal functioning of
different types of cells.Therefore, nanoparticulate systems need to overcome this barrier
to intrude living cells. Nanoparticulate systems are capable to enter live cells, often
through the several endocytic pathways. Upon endocytosis, nanomaterials are enclosed
within the early endocytic vesicles and are thus not directly carried into the cytosol. In
contrast, the nanomaterials internalized via membrane penetration are directly
transferred into the cytoplasm, which can be the preferred choice particularly for the
targeted Drug delivery
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The term ‘endocytosis’ can be broadly divided into pinocytosis (cell drinking) and
phagocytosis (cell eating). Pinocytosis is commonly involved in the internalization of
fluids and molecules by small vesicles, and phagocytosis is the process by which the
cells such as monocytes/macrophages, neutrophils and dendritic cells engulf large
particulate matter and are able to form intracellular phagosomes. Pinocytosis can be
further subdivided into four different basic categories namely macropinocytosis,
clathrin‐ mediated endocytosis, caveolin‐ mediated endocytosis and clathrin‐ and
caveolin‐ independent endocytosis.

● Macropinocytosis is a form of actin‐ driven endocytic mechanism by which

extracellular fluid and its debris are internalized in a nonspecific manner
within large, heterogeneous vesicles, called macropinosomes.
● Each clathrin‐ mediated internalization pathway is started by the specific
interactions between ligands and extracellular receptors. Following entry
into the cell, the internalized nanoparticles are typically trapped inside the
endosomal/lysosomal vesicles, resulting in the degradation of the
sequestered cargo material by the lysosomal enzymes.
● Caveolae/raft‐ dependent endocytosis is involved in clustering of the lipid
raft domains at the plasma membrane into the flask‐ shaped invaginated
structures called ‘caveolae’ and formed through the interaction of cellular
membranes with the different types of proteins, especially caveolin
Endocytosis by clathrin‐ coated pits or uncoated pits traffics the material to the
lysosomal degradative compartment, while caveolae‐ mediated endocytosis mediates
the translocation to the Golgi apparatus, to endoplasmic reticulum or through the cell .
Therefore, it is useful to develop other strategies such as caveolae‐ mediated pathway
and macropinocytosis which are somewhat non‐ specific, and neither acidic nor
digestive and can preferentially internalize nanoparticles through an alternative
pathway, preventing lysosomal degradation after internalization.

7.1 Factors Affecting the Efficacy of Nanoparticle Uptake-

The efficiency of endocytosis depends on not only the size of nanoparticles, but also the
charge, and surface coating. The smaller the particle size, the easier they can be
internalized by cells as smaller particles have a larger surface area than same mass of
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larger particles, allowing them more contact with the biological membranes. Elongated
Nanoparticles have been reported to yield a higher efficiency in adhering to the cells as
compared to the spherical nanoparticles, especially after surface modification as
elongated nanoparticles have a higher surface area and the ability to interact more
efficiently with the cell surface membranes with respect to their shape. Generally,
positively charged nanoparticles exhibit better internalization level as a result of
attractive electrostatic interaction with the negatively charged cell membrane that has a
profound effect supporting the uptake of nanoparticles.

8.Infectious agents
Infectious agents can be extracellular and intracellular.

Extracellular Infectious Agents- To produce disease, these pathogens use any portal
of entry provided a satisfactory fluid medium be recognized at site of injury or wound.
They utilize virulence mechanism to avoid antimicrobial capabilities of humoral immunity
and phagocytosis thus advancing extracellular reproduction
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Intracellular Infective Agents- They promote entry into host cells containing
macrophages and non professional phagocytes such as epithelial cells. They are able
to invade and survive in the cells of the organisms in active or latent forms over
prolonged period of time.

Bacteria gains antibiotic resistance due to these reasons-

● Modification of active site of the target resulting in reduction in efficacy of binding

of Drug.
● Direct destruction or modification of antibiotic by enzymes produced by the
organism.
● Efflux of antibiotic from the cell.
Nanoparticles can target antimicrobial agents to the site of infection so that higher
doses of drug can be given at the infected site, thereby overcoming existing resistance
mechanism with fewer harmful effects upon the patient. Nanoparticles targeting the site
of infection can release high concentration of antimicrobial drugs at the site of infection
while keeping total dose of drug administered low.

9.Targeted Drug delivery towards infections

9.1 Tuberculosis -
Tuberculosis bacteria are taken up by the macrophages/monocytes in the
lungs.Delivery of antituberculosis drugs by nanoparticles offers potential
advantages over free Drug, including the potential to target specifically the
tissues and cells that are infected by Mycobacterium tuberculosis, thereby
simultaneously increasing therapeutic efficacy and decreasing systemic toxicity,
and the capacity for prolonged release of drug, thereby allowing less-frequent
dosing. We have employed mesoporous silica nanoparticle (MSNP) drug delivery
systems either equipped with a polyethyleneimine (PEI) coating to release
rifampin or equipped with cyclodextrin-based pH-operated valves that open only
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at acidic pH to release isoniazid (INH) into M. tuberculosis-infected

macrophages.To take advantage of the fact that macrophages are particularly
efficient in internalizing particles and delivering the particles to acidified
endosomes, we have utilized MSNP as a delivery vehicle for two first-line
antituberculosis drugs, INH and RIF. MSNP are internalized efficiently by M.
tuberculosis-infected macrophages, selectively release drugs intracellularly in
macrophages, and kill M. tuberculosis more effectively than an equivalent
amount of free drug.

9.2 Malaria-
Modification of different liposome prototypes that had been developed in our
group for the targeted delivery of antimalarial drugs to Plasmodium-infected red
blood cells (pRBCs). Infection starts when a parasitized female Anopheles
mosquito inoculates sporozoites of the malaria parasite, the protist Plasmodium
spp., into a person while taking a blood meal. Within a few minutes, sporozoites
have migrated through the skin and bloodstream to the liver, where they invade
hepatocytes. Sporozoites develop into merozoites,which enter the circulation,
invade red blood cells (RBCs). Nanoparticle based targeted delivery approaches
can play an important role for the treatment of malaria because they might allow
(i) low overall doses that limit the toxicity of the drug for the patient
(ii) administration of sufficiently high local amounts to minimize the evolution of
resistant parasite strains
(iii) improvement of the efficacy of currently used hydrophilic (low membrane
trespassing capacity) and lipophilic antimalarials (poor aqueous solubility), and
(iv) use of orphan drugs never assayed as malaria therapy. One of the limitations
of liposomes as carriers for Drug delivery to Plasmodium-infected RBCs (pRBCs)
is that because of the lack of endocytic processes in these cells, a relatively fluid
liposome lipid bilayer is required to favor fusion events with the pRBC plasma
membrane.
As a result, these liposomes are leaky for small drugs encapsulated in their
lumen, and when membrane fusion occurs, only a relatively small fraction of the
originally contained drug is delivered into the cell. On the other hand, liposomes
made of saturated lipids have less fluid bilayers that retain drugs with high
efficacy, although fusion events with pRBC membranes are greatly diminished,
which might also reduce the amount of luminal cargo delivered to the target cell.

9.3 HIV infections-

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HIV is known for its long period of latency. This is achieved to a certain extent by
the formation of reservoirs.The intracellular reservoirs are the resting CD4+ T-
lymphocytes and the macrophages. Like the CD4+ T-cells, monocyte-lineage
cells like tissue macrophages act as reservoirs of HIV infection.. Drug delivery to
HIV-infected tissues/ cells with selectivity can be achieved by targeting surface
markers on CD4+ T-cells, macrophages, dendritic cells and towards the CNS.
Macrophages can be actively targeted with the help of targeting ligands: against
mannose receptor, formyl peptide receptor 1 and other similar receptors on the
macrophages. Antiviral Drug azidothymidine(AZT) was encapsulated with
PEGylated Nanoparticles with surface functionalization by transferrin which
enhanced the brain localization of AZT.
Actively target the lymphatic system where the HIV is known to colonize and from
reservoirs. Liposomes loaded with zidovudine were surface modified with a
lymphatic site specific ligand- mannose: effective in uptake and localization into
the lymph nodes and the spleen.

10.Conclusion

This report deals with targeted drug delivery, active and passive targeting and its types.
It also explains different types of drug releases for better understanding of the difference
between the use of free drugs and nanoparticles. The preparation of nanoparticles is
also quite a challenge as they should fulfil certain criterias and specifications. Here we
also discussed about the preparation of nanoparticles. The report throws light on the
properties, advantages and disadvantages of using nanoparticles for Drug delivery. It
also gives a brief description of intracellular delivery. Nanoparticulate systems are
capable to enter live cells, often through the several endocytic pathways. We also
discuss the mechanism of uptake of nanoparticles and the factors affecting the efficacy
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of uptake. Toward the end of the report I have elaborated the targeted Drug delivery
towards certain infections, like Malaria, HIV infections and Tuberculosis.

11.References:

1. https://onlinelibrary.wiley.com/doi/full/10.1111/jcm
m.13110\
2. https://pubs.rsc.org/en/content/articlehtml/2015/nr
/c5nr01285f
3. https://www.beilstein-
journals.org/bjnano/articles/5/174
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC55
71529/
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5. https://www.researchgate.net/publication/3000646
21_Nanoparticle_based_Drug_Delivery_Systems_
for_Treatment_of_Infectious_Diseases