Modern this research project, a greater understanding of the

Modern science has influenced the way
the diagnosis of diseases and how they are treated. This stems from a greater
understanding of the interactions which occur on a cellular level between the
affected tissues and the applicant atoms used in the treatment, a stellar
example of this being Emil Fischer’s lock and key hypothesis; an early study in
1894 which changed the way proteins are viewed and how they interact with other
molecules in regard to their specific nature, over 100 years on. (1)

With modern techniques now available
such as AFM, FTIR and DLS, it’s now easier than ever to study molecules;
Through the addition of other molecules it’s possible to determine how they can
affect and interact with the body and more importantly how they can help the
study of diseases.

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The research conducted in this project
will observe and ascertain the effectiveness of two different types of
molecules: Peptide and PEG, their
affinity in retrospect to each other but also their affinity with gold
nanoparticles. From this research project, a greater understanding of the use
of nano-particle based drugs and their interaction with cell surfaces will be
gained. This will hope to further advance the study of tailor-made drugs to
achieve both controlled drug release and disease-specific localization by
tuning the polymer characteristics and surface chemistry.

To understand how these particles
interact with each other and how they relate to the improved delivery of drugs,
several areas of research must be conducted.

Preparation of nano-particle based drugs
using gold is the main focus, however the real process begins from the
understanding of the surface interactions from each method such as pegylation
to the AuNP (Gold Nanoparticles) using Polyethylene glycol and Peptides.

Using various solutions, these particles
topographies can be ascertained and further understood through techniques such
as AFM, FTIR, TEM and contact angle measurements. This will allow for a greater
understanding of how these particles change the way that gold mimics or alters
biological processes in relation to cancer treatment and how the particles can
be constructed to possess different properties and release characteristics for the
best delivery or encapsulation of the therapeutic agent. The nanoparticles can
then be adapted and tailor-made to achieve controlled release of the drug and
disease specific localization by fine tuning of the surface chemistry.

Research into these areas improves the
understanding of Immunogenicity; the ability of a particular substance to
create a cell mediated immune response. To further understand the influential
parameters in nonspecific protein adsorption, interacting forces between
proteins and surfaces are discussed, protein attributes that can modify the
surface force profile are explained, and the “gold-standard” surface coating,
polyethylene glycol (PEG), which can greatly reduce the adsorbed amount of
proteins.

Current objectives for the programme of work being undertaken

Working towards understanding how
modifying AuNP can increase the effectiveness of drug delivery, knowledge of
the techniques used to study these particles is required.

Such objectives and deliverables
include:

·        
The use of Atomic
Force Microscopy (AFM) techniques to scan the surface of the specimen and
determine the nano-scale topography of each.

·        
The calculation
of surface tension in solutions to determine the cohesive forces which bind the
molecules together in a substance. This is extremely important to determine how
fluids interact differently at specified cell membranes and how readily they
are absorbed. In conjunction with this and understanding of how contact angle
measurements affect these values will be assessed.

·        
Understanding
the use of FTIR to develop an overall estimation of the particle size and shape
of the modified AuNP and how these interact with specific proteins on cell
surfaces.

·        
Understanding
how differing combinations of modification of nanoparticles through the use of
ligands can affect their biochemistry and physical attributes.

·        
The interaction
of Peptides forwarding the creation of biomaterials used in specific cell
targeting for cancer cells.

·        
Understanding
how Peglygation can improve the active targeting of nano-particle based drugs
and therefore positively influence the therapeutic effect of these.

·        
To develop a
greater understanding of Dynamic light scattering to determine nanoparticle
size and the distinction between a molecule and a particle.

·        
Measure and
quantify the Zeta potential of the modified AuNP and how the differing
modification of these affects the particles stability and its ability to
improve drug delivery.

·        
A critical
assessment of which modification produces the best affinity for the purpose of
drug delivery.

Overview of the published academic and/or technical literature

“Nanotechnology is an idea that most people
simply didn’t believe.” (2)

Nanotechnology
allows for tailor-made particles to be created for complex applications such as
targeting of cancer cells, improved drug delivery and the diagnosis of diseases
through advanced monitoring techniques. All of these can be achieved by fine
tuning the surface properties of these particles to exhibit characteristic for
the suitable application. This review will discuss generation of these
nanoparticles, methods of modification and the analytical techniques used to
image and perform adjustments to each particle.

 

Generation of Nanoparticles

 

Nanoparticles
are the representation of miniscule versions of machines and devices, they work
so well due to their relativistic size compared to the target cells, occupying
the region or area between molecular species and bulk solids. (4)

There
are two main methods for the synthesis of nanoparticles: top-down and bottom up. Top-down
formation involves taking a material in bulk (ex: bismuth) and fragmenting the
material into nano-scale (10-9m) remains, the remains are then made
into uniform particles by shear forces from a mechanism that is quite similar
to emulsion.

As reported by Wang et al. these particles are not
ideal for the application due to the differential size of each particle, some
being too small and others being far too varied.

Bottom-up
synthetization involves the use of organometallic compounds, salts or molecular
precursors which are then decomposed by applying a reducing agent or excitation
through thermal or optical effects. These nanoparticles are reported to be far more
useful due to their size distribution, which can be controlled by adjusting the
methods used to decompose the original compounds such as control over
precursor, solvents and organic ligands. 
(5)

 

Controlling the growth of nanoparticles – Ligands

 

The
growth of nanoparticles can be adjusted by compartmentalizing the precursors
using ligands as basic templates to control the nucleation in the inorganic
stage. The ligands are added to solutions with the particles, which form
spherical-like protein structures around their circumference called micelles.
The ligands will then reduce the growth of the nanoparticle in that particular
direction. (6)

The work of W.W. Yu et al. shows
that if the ligands have a particularly high binding strength, they will limit
the amount the particle can grow in this phase and ultimately will develop a
smaller particle. (7)

It can
then be determined that ligands have the ability to kinetically control the
shape of the nanoparticle through their selective adhesion to particular facets
of the particle. X. Peng et al. showed in the case of using a single binding
ligand (in this case, TOPO) for CdSe synthesis, that the ligand will reduce the
growth uniformly, instigating an isotropic growth. (8)

Using a ligand that can bind to
specific facets will produce an anisotropic growth, as the facets lose energy
upon the attachment of the specific ligands, corresponding with W.W.Yu et al’s
findings.  From the figure below it shows
that these techniques can produce highly formed nanorods or cylindrical based
nanotools. The nanoparticle also gathers some useful new bio-applicable
properties from having attached ligands such as protection from oxidation,
prevention of leaching into surrounding solutions and reduce the toxicity of
the nanoparticle as they create a buffer between the particle and the
surrounding environment. They prevent interactions with proteins in the
environment and help avoid cellular uptake. (9)

 

Peptide based ligands

 

           

 

 

Peptides attached to nanoparticles allow
for the NP’s to be readily dispersed into water, making them a prime candidate
to functionalizing NP’s for biological targeting. (10)

These peptide ligands are consisted of a
long chain of amino acids interconnected by amide bonds. From the work of Le
Joncour & Laakkonen, it can be observed that these peptides are rapidly
cleared from non-binding sites and the blood due to their low molecular weight.
They are chemically flexible and easily modified. Supported by the work of Rundra
et al, who showed that the due to the high level of amino acids available for
the creation of peptide chains; chains can become extremely varied, decreasing
the chance of an immune response from T-cells particularly if the chain has
amino acids deleted from their sequence. (11)

E.L. Riché et al. used a mixture of
beta-alanine, gamma-aminobutyric acid and glycine to randomly couple a peptide
sequence with phosphatidylethanolamine before coating gold nanoparticles. The
nanoparticles exhibited a greater life cycle compared to standard NP’s due to
the micelle layer preventing interactions in vivo, acting as a barrier. It can
be noted that peptide based approaches do not produce the same life-span as
Peglygated ligands, from their generation of ABC kinetics (A phenomenon where
components in the blood are cleared at an accelerated rate.)

An application of this can be observed
by the work of Lei Hetian et al. who demonstrated that attachment of these
peptides not only enhance the properties of the particle but also enhance the
targeting ability of an encapsulated drug in its desired delivery. (12)

The study used aldehyde-PEG-PLA block copolymers
synthesized by ring opening polymeriation – where terminal ends of a polymer
chains acts as a reactive centre allowing further cyclic monomers to be
attached by the opening of the ring system to form a longer polymer chain.

From this, nanoparticles were loaded
with paclitaxel – antiangiogenic drug capable to targeting tumours and
preventing them from creating new blood vessels. K237 ligand (which can
specifically target the KDR receptors and attach to them due to its high
affinity to the tumour) was attached to the aldehyde group of the PEG chain.
This ligand inhibits the VEGF-KDR pathway furthering the antigiogenic effect.
The modified nanoparticle displayed a facilitated uptake shown by HUVEC
proliferation, migration and tube formation compared to PTX-NP.

This provides a great deal of confidence
in further applications in this field for targeting as it was successful in vivo
for tumours planted in female mice, offering a new strategy for chemo- &
antigiogenic therapy.

 

PEG based ligands

 

These
are ligands based upon biologically inert polyethylene glycol (PEG) which helps
the nanoparticle from being cleared in vivo as quickly, allowing for a greater
exposure time to complete their tasks.

Modifying
the nanoparticles with PEG creates a steric barrier/palisade for the which
helps reduce the onset of hydrophobic and electrostatic interactions with parts
of the cellular environment as well as reducing any effect the particles will
have on each other.

These
interactions would include exposure to proteins and monocytic cells which would
look to destroy the particle registering it as a foreign substance due to
attachment of proteins. (13)

Leading
to an improved circulation time in the designated target zone, similar to
studies on peptide based modification by the previously noted authors. The PEG
process is not perfect and raises the same issue as peptide; accelerated blood
clearance, worsened by continuous exposure to the same PEG chain causing a reaction
to Immunoglobin M – the largest and first antibody in initial response to
antigens. (14)

This
issue is thought to be caused by the nature of the PEG barrier, serving as a
“net” picking up proteins which subsequently form a corona around the particle.
(15)

This issue is worsened by a lower density of PEG
molecules on the surface as this increases the volume of the entrapment and
increasing the amount of proteins which are picked up, evoking a faster immune
response. (16)

 

Keeping
the density of PEG as high as possible will reduce this from happening, but not
stop its affects entirely; Phagocytosis will occur regardless due to its
non-specificity.

Other studies such as the work undertaken by Spadavecchia
et al have shown in practice the applications for nanomedicine and in
particular: Cancer targeting. The experiment in question used a peglygated gold
nanocarrier loaded with doxorubin (a chemotherapy agent) which was intended to
be intravenously administered to a patient for the treatment of pancreatic
cancer.

The
premise of this was to find the balance between removing stabilizers involved
in preventing aggregation of nanoparticles in vivo without affecting the
stability of the nanoparticles. The main issue with stabilizers is they are
highly toxic, causing issues translating nanoparticle approaches from in vitro
to in vivo.

Spadavecchia
et al. used a one-step method for synthesis of dicarboxylic acid terminated PEG
gold nanoparticles, loaded with doxorubin and an antibody targeting component
added (anti-kv11.1 polyclonal antibody pAb). (17)

The
study evaluated the effect of active targeting functionality from modified gold
nanoparticles (AuNPs) and their efficiency in anti-tumour administration.  

Findings
showed that peglygation of the AuNP’s provided a greater drug delivery and
active targeting at the intended site with 30x decrease in the half maximal
effective concentration in cell nuclei and cell cycle blockage; a lower
concentration was required to acquire the same potency, with the drug being
internalized in the membrane of the pancreatic cells.

Active
targeting of the AuNPs through the attachment of Kv11.1 positively influenced
cell uptake by targeting the antibodies surrounding the pancreatic cells and
the increased lysosomal function functionalised by the translocation of the
nanoparticles into the lysosomes of the cells. It can be suggested that
controllable pegylgaton will be a key factor into the success of nanomedicine
and combination of these techniques, along with antibody targeting, will
increase surface affinity.

Other
studies presented findings which supported the evidence of Spadavecchia such as
the work of Park et al. Park used a unique method to create the encapsulated
delivery; an avidin-biotin coupling system. The encapsulation of these
particles did not reduce the effectiveness of the chemotherapy agent as the
free molecules where found to be at least as potent as A20 murine B-cell
lymphoma cells studied in vitro compared to tumours in vivo. The cardiotoxicity
was also reduced, measured with echocardiology and histopathology.

Below is a figure showing the % encapsulation of
doxorubin and the effective concentration against a function of the square root
of time.

 

Below
is a figure showing Cytotoxicity of DOX in PEGylated nanoparticles (?), Doxil
(?) or free DOX (?) vs. no treatment (?) against A20 lymphoma cells after 24h treatment.
(18)

 

Further
supporting to the work of both Park et al. and Spadavecchia et al. Kirpotin et
al. showed that tumour internalization is increased form the use of antibody
targeting chains attached to Peglygated PLGA nanoparticles with encapsulated
doxorubin. These gold nanoparticles showed a 6x greater uptake into the
cellular structure whereas the non-coated particles where mostly found in the
extracellular stroma or within macrophages. The attachment of anti-HER2 Mab
fragments targeted the HER2 antigens within cancer cells directed the drug to
the correct location, but in contrast to studies performed by Park et al. they
did not increase the tumour localization as both non-targeted and targeted
nanoparticles showed the same concentration in tumour tissue (7/8%). (19)

The use of Gold Nanoparticles

 

Gold
is a one of the most widely used metals for the formation of nanoparticles due
to its unique properties both biochemically and electronically and it’s
biologically inert/nontoxic. Because of their oxide-free nature and the fact
that when gold is coated with organic molecules it exhibits a highly catalytic
nature, they can be used for many applications such as molecular electronics
and quantisation of tags in biological arrays. (20) (21)

Gold nanoparticles have a large surface to
volume ratio allowing for a great deal of surface modification, making them
readily modified for various uses including drug delivery. (22)

AFM imaging and
modification

 

For
the creation of complex nano-objects, a deep understanding of the surface
properties is required and without adequate imaging methods this is
unattainable. One of the most used methods for imaging is atomic force
microscopy which not only produces 3D topographies of the substance but allows
surface modification of the particles. Being able to control nanoscale
interactions is paramount in understanding the behaviour of the particles for
assembly.

Many
authors have shown differing methods of modification using AFM. The most
relevant for the tapping mode of AFM was Paollicelli et al. who manipulated
AuNPs of differing sizes on a pyrolite graphite surface. These particles were
selectively moved, and their energy detachment thresholds were estimated from
the force required to move each. (23)

Mougin
et al. manipulated functionalised AuNPs on silicon substrates with dynamic AFM
however on many attempts, the probing tip was damaged from collisions with
particles due to the high electrostatic interactions and friction between
particles. (24)

Progress on the
core deliverables of the work plan

 

So far in the project, multiple meetings have been
undertaken with the Project supervisor Prof. Patrick Lemoine in which various
trainings procedures have been discussed such as AFM training and methods to
calculate surface tension of liquids.

Health and safety training was the first discussion;
providing knowledgeable information on key factors for working in a laboratory
environment such as locations of fire exits, exposure to infectious specimens,
unfixed tissues or body fluids, poor work practices e.g. eating or drinking in
the laboratory, poor hygiene, inappropriate or inadequate PPE.

AFM and surface tension training involved a key
breakdown of how the machine works, the functions of the methods used and how
to analyse the data provided. An understanding of adjustments to make to
improve sample accuracy and results has also been attained.

An understanding of the generation of nanoparticles
and their functions has been gained through independent research. Understanding
the interactions between peptides and PEG and their respective affinity to
nanoparticles has been advanced along with theoretical methods to modify them
with AFM.

Preliminary results & findings to date

 

Currently
the level of results is not at a high level with much of the work performed in
the starting weeks based around background knowledge on the project and the
provided training to set up laboratory work. Training provided has currently
covered the use of AFM and surface tension calculations from pendant drop
procedures.

AFM

 

AFM or atomic force
microscopy is a type of high resolution scanning probe microscope with a
resolution measured in fractions of a nanometer. It’s an extremely important
tool for imaging of nanoparticles and plays a key part of the project and the
future data analysis of solutions and the modifications of each. AFM works on
the basis of a probe tip attached to a moveable cantilever which is then used
to scan the surface of the material in question. As the probe moves across the
surface of the specimen it will generate a force between its tip and the sample
according to Hooke’s law. This force will deflect the cantilever and in turn
this will cause changes in the laser on the to surface of the cantilever. The
Laser is targeted at an array of photodiodes which will generate a measurement
of the original force created.

AFM has two primary modes:
contact and non-contact. In contact mode the probe is used to drag across the
surface of the specimen and create a 3D topography of he given surface from the
contours on its surface. In non-contact mode the cantilever will vibrate above
the specimen at a frequency which is slightly above the resonance frequency. A
feedback loop will prevent the probe from crashing into the surface and a
recorded distance between the sample will allow the software to generate an
image of the surface. It can be noted however, that any moisture on the surface
of the sample will make imaging hard through this mode.

Tapping mode is the primary
mode of use for the measurement of nanoparticles and categorisation of their
properties. In this mode a piezoelectric element is attached t the top of the
cantilever causing it to oscillate near its resonance frequency with an adjustable
amplitude. As the probe becomes closer to the tip, the forces will cause the
amplitude to be reduced. The cantilever will then adjust in height to regulate
the amplitude at a constant level. This method is more accurate than
non-contact when moisture interference is present and much less likely to
damage the probe tip. This technology is incredibly useful in the measurement
of very small samples as it provides a high degree of accuracy on an unmodified
sample without the requirement of a vacuum. However, a major disadvantage of
AFM is its small scanning size for imaging which is 150×150 microns, a fraction
of the size of a scanning electron microscope, this coupled with a slow scan
speed can lead to drift and noise in the sample, therefore reducing accuracy.

From AFM samples taken from
bare glass slides, unmodified gold nanoparticles, peglygated and hybrid
nanoparticles, particle analysis has been performed on each and histograms have
been produced.

The threshold values and histograms
for each can be found below.