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Synthesis, Preparation and Characterization of Nanoparticles for Biomedical

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Research Proposal · December 2018

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 Research Proposal

Synthesis, Preparation and Characterization of Nanoparticles for Biomedical Applications

Dr. Abolfazl Akbarzadeh1 & Dr. Siamak Saghfi2

1
Associate professor, Department of Medical Nanotechnology, Faculty of Advanced Medical
Science, Tabriz University of Medical Sciences, Tabriz, Iran
Email: a.akbarzadeh@northeastern.edu

2
Postdoctoral Researcher, Department of Medical Nanotechnology, Faculty of Advanced
Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran
Email: saghfis@tbzmed.ac.ir

Introduction
The use of nanotechnology in medicine offers some exciting possibilities. Some techniques are
only imagined, while others are at various stages of testing, or actually being used today.
Nanotechnology in medicine involves applications of nanoparticles currently under
development, as well as longer range research that involves the use of manufactured nano-robots
to make repairs at the cellular level (sometimes referred to as nanomedicine). Whatever you call
it, the use of nanotechnology in the field of medicine could revolutionize the way we detect and
treat damage to the human body and disease in the future, and many techniques only imagined a
few years ago are making remarkable progress towards becoming realities. The interface
between nanosystems and biosystems is emerging as one of the broadest and most dynamic areas
of science and technology, bringing together biology, chemistry, physics and many areas of
engineering, biotechnology and medicine. Any damage at molecular or cellular level is the major
culprit for disease & ill health. Nanotechnology, “the manufacturing technology of the 21st
century," helps us to build a broad range of complex molecular machines by manipulating matter
on an atomic and molecular scale. Nanotechnology is the creation of useful materials, devices
and system through the manipulation of matter on an atomic, molecular, and supra-molecular
level in the length scale of 1-100 nanometer size. At the nano scale, the physical, chemical, and
biological properties of materials differ in fundamental and valuable ways from the properties of
individual atoms and molecules or bulk matter .It enables the alignment of atoms in the most
effective way in a very limited place. Extraordinary devices can be created by using this
technique. Molecules could be aligned in such a way as to produce desired result in the areas of
strength, ductility, reactivity, conductivity and capacity. This idea facilitates creating devices and
structures that would occupy an unimaginably small space and have novel properties and
functions because of their small size. The application of nanotechnology in the medical sector is
referred as Nanomedicine. Nanoparticles have potential applications in the field of medical
sciences including new diagnostic tools, imaging agents and methods, targeted drug delivery,
pharmaceuticals, bio implants and tissue engineering. Drugs with high toxic potential like cancer
chemotherapeutic drugs can be given with better safety profile with the utility of
nanotechnology. The aim of the nanotechnology in the medical sciences is to develop new
materials and methods to detect and treat diseases in a targeted, precise, effective and lasting
way, with the ultimate goal of making medical practice safer and less intrusive.

Nanotechnology in Medicine Application

Nanotechnology is the study of extremely small structures, having size of 0.1 to 100 nm. Nano
medicine is a relatively new field of science and technology. Brief explanation of various types
of pharmaceutical nanosystems is given. Classification of nanomaterials based on their
dimensions is given. An application of nanotechnology in various fields such as health and
medicine, electronics, energy and environment, is discussed in detail. Applications of
nanoparticles in drug delivery, protein and peptide delivery, cancer are explained. Applications
of various nanosystems in cancer therapy such as carbon nanotube, dendrimers, nano crystal,
nano wire, nano shells etc. are given. The advancement in nanotechnology helps in the treatment
of neuro degenerative disorders such as Parkinson’s disease and Alzheimer’s disease.
Applications of nano technology in tuberculosis treatment, the clinical application of
nanotechnology in operative dentistry, in ophthalmology, in surgery, visualization, tissue
engineering, antibiotic resistance, immune response are discussed in this article. Nano
pharmaceuticals can be used to detect diseases at much earlier stages.

Questions

1. How nanoparticles will produced?

2. With which conditions we can prepared monodisperse NPs?
3. How we characterized all NPs?
4. What is biomedical applications of this NPs?

Assumptions

1. This NPs will produce with cheap methods.

2. With controlling pH and temperature we will produce monodispers NPs.

3. We will characterized NPs via FT-IR, UV, NMR, DLS, SEM, TEM, XRD,…etc

4. This NPs have many applications in Medicine, Dentistry, Pharmacy, Radiology, Petroleum,
Civil, Electronic, Mechanic, Agriculture,….

Aim of this study

-Synthesis, Preparation and Characterization of Nanoparticles for Biomedical Applications.

-Engineering Hydrogel with tunable properties for soft tissue regeneration.

-Preparation of Polymeric Micro and Nanoparticles Containing Protein- Peptide and Anti Cancer
Drugs.

Methodology

1) Magnetic Nanoparticles(MNPs)

Magnetic nanoparticles(MNPs), due to their distinctive physical properties and ability to role at
the cellular and molecular level of biological interactions, are a major class of nano-scale
materials with the potential to be useful in biomedical fields, such as, drug delivery,
hyperthermia treatment, and magnetic resonance imaging (MRI) of cancer cells. Several methods
have been developed to synthesize Fe 3 O 4 particles with sizes in the nanometer range. One of
the oldest techniques for the preparation of NPs is the precipitation of products from solutions.
Co-precipitation is the most widely used and most proper method for the synthesis of MNPs of
controlled sizes and magnetic properties. It is extensively used for biomedical applications,
because of the ease of application and less need for harmful materials and procedures. In this
method, MNPs are prepared from aqueous salt solutions, by the addition of a base under an inert
atmosphere at room temperatures or at high temperature. The reaction is simply as follows:

Fe2+ +2Fe3+ +8OH-→ Fe3 O4 +4H2 O

This technique is based on the resuscitation of metallic elements, from salt solution, by
surcharging NaOH solution, at room temperatures or at high temperatures. Giving to this
method, FeCl2.4H2O and FeCl3.6H2O were dissolved in deionized water, such that the ratio of
Fe3/Fe2 was 2:1, respectively. This solution was stirred under nitrogen at 85°C for 1 h. In the
next step NH3.H2O (25%) was added into the solution quickly, stirred under nitrogen for 1 h, and
then cooled at room temperature. Finally, the precipitated particles were washed three times with
water and dried under vacuum at 70°C. The structure confirmation of Fe3O4 can be studied by
FTIR spectroscopy. The X-ray diffraction was used to study the crystal structure of the Fe3O4
nanoparticles. The magnetic properties of Fe3O4 nanoparticles are determined with VSM. The
size and surface morphology of the nanoparticles can be observed by SEM.

2) Gold nanoparticles(GNPs)

GNPs have exceptional optical properties due to surface plasmon resonance (SPR) eff ects. It is
an optical phenomenon occurring from the interaction between an electromagnetic wave and the
conduction of electrons in a metal. The SPR properties of GNPs makes them quite useful in the
fields of bioimaging and biomedical therapeutics, and as biodiagnostic tools. GNPs can
accumulate in the tumor cells and show optical scattering; thus, these nanoparticles can play an
important role as a probe for the microscopy study of cancer cells. In addition, these
nanoparticles can be used in chemotherapy and for the diagnosis of cancer. The GNPs also
provide an applicable and promising scaffold for drug and gene delivery. In 2003, GNP were
applied for the first time as agents for photothermal therapy. There are some particular properties
for GNPs such as photon absorption and conversion it into thermal energy that is useful for
photothermal therapies, especially for cancer treatment.

A variety of techniques, including chemical, thermal, electrochemical and sonochemical

pathways, have so far been introduced for the synthesis of GNPs.
Generally, the preparation of GNPs by the chemical reduction method includes two main parts:
(1) Reduction by agents, for instance borohydrides, aminoboranes,citric and oxalic acids, sugars,
hydrogen peroxide.
(2) stabilization using agents, for instance trisodium citrate dihydrate, sulfur ligands (in
particular thiolates), phosphorus ligands, oxygen-based ligands, nitrogen-based ligands
(including heterocyclic compounds), dendrimers, polymers and surfactants (in particular,
cetyltrimethylammonium bromide (CTAB)).
To avoid the aggregation of the particles, some kind of stabilizing agent is usually added. One of
the most well-known techniques for the synthesis of GNPs is based on the reduction of HAuCl4
by citrate in water, which was first designed by Turkevich in 1951. In this method, the HAuCl 4
solution is boiled, and the trisodium citrate dihydrate is then rapidly added into the boiling
solution under vigorous stirring. After a few minutes, the color of the solution changes from light
yellow to wine red. This method results in GNPs measuring about 20 nm in diameter. In this
technique, citrate ions play a double role, as both stabilizing and reducing agents.
3) Silver nanoparticles (AgNPs)

Silver NPs have gained considerable interest because of their unique properties, and proven
applicability in diverse areas such as medicine, catalysis, textile engineering, biotechnology,
nanobiotechnology, bio-engineering sciences, electronics, optics, and water treatment. These
NPs have significant inhibitory effects against microbial pathogens, and are widely used as
antimicrobial agents in a diverse range of products.
The flexibility of silver nanoparticle synthetic methods and facile incorporation of silver NPs
into different media have encouraged researchers to further investigate the mechanistic aspects of
antimicrobial, antiviral and anti-inflammatory effects of these NPs. Shape, size and size
distribution of silver NPs can be controlled by adjusting the reaction conditions such as reducing
agent, stabilizer or employing different synthetic methods. Therefore, it is important to elucidate
the effects of reaction conditions on morphology and size of NPs.
Different methods have been developed to obtain silver NPs of various shapes and sizes,
including laser ablation, gamma irradiation, electron irradiation, chemical reduction,
photochemical methods, microwave processing, and thermal decomposition of silver oxalate in
water and in ethylene glycol, and biological synthetic methods. Biosynthetic methods of NPs
provide a new possibility of conveniently synthesizing NPs using natural reducing and
stabilizing agents. As possible environmentally and economically friendly alternatives to
chemical and physical approaches, biosynthesis of metal and semiconductor NPs using
organisms has been suggested. Monodispersity and particle size and shape are very important
parameters in the evaluation of NPs synthesis. Therefore, the efficient control on the morphology
and monodispersity of NPs must be explored. The reaction conditions should be optimized. By
using screened organisms with high production capability and controlling the reaction
conditions, well- characterized NPs can be obtained by synthesis rates faster or compatible to
those of chemical and physical approaches. This eco-friendly method can potentially be used in
various areas, including pharmaceuticals, cosmetics, foods, and medical applications.
Silver nanoparticles (AgNPs) have been known to have inhibitory and bactericidal effects.
Resistance to antimicrobial agents by pathogenic bacteria has emerged in recent years and is a
major health problem. One use of silver ion or metallic silver as well as silver nanoparticles can
be exploited in medicine for burn treatment, dental materials, coating stainless steel materials,
water treatment, sunscreen lotions.
Several physical and chemical methods have been used for synthesizing and stabilizing silver
nanoparticles. The most popular chemical approaches, including chemical reduction using a
variety of organic and inorganic reducing agents. In general, different reducing agents, such as
sodium citrate, ascorbate, sodium borohydride (NaBH4), are used for reduction of silver ions
(Ag+) in aqueous or non-aqueous solutions. The aforementioned reducing agents reduce silver
ions (Ag+) and lead to the formation of metallic silver (Ag0), which is followed by
agglomeration into oligomeric clusters. These clusters eventually lead to formation of metallic
colloidal silver particles. It is essential to use protective agents to stabilize nanoparticles during
the course of silver nanoparticle preparation, and protect the nanoparticles that can be absorbed
on or bind onto nanoparticle surfaces, avoiding their agglomeration. The presence of surfactants
comprising functionalities (e.g. thiols, amines, acids and alcohols) for interactions with particle
surfaces can stabilize particle growth, and protect particles from sedimentation, agglomeration or
losing their surface properties. Polymeric compounds such as poly(vinyl alcohol),
poly(vinylpyrrolidone), poly(ethylene glycol), poly(methacrylicacid) and
polymethylmethacrylate have been reported to be effective protective agents to stabilize
nanoparticles.

4) Polymeric Nanoparticles

Since the emergence of Nanotechnology in the past decades, the development and design
of nanomaterials has become an important field of research. An emerging component in this field
is nanomedicine, wherein nanoscale materials are being developed for use as imaging agents or
for drug delivery applications. Much work is currently focused in the preparation of well-defined
nanomaterials in terms of size and shape. These factors play a significantly role in the
nanomaterial behavior in vivo. In this context, this review focuses on the toolbox of available
methods for the preparation of polymeric nanoparticles. We highlight some recent examples
from the literature that demonstrate the influence of the preparation method on the
physicochemical characteristics of the nanoparticles. Additionally, in the second part, the
characterization methods for this type of nanoparticles are discussed. Polymer nanoparticles have
attracted the interest of many research groups and have been utilized in an increasing number of
fields during the last decades. Generally, two main strategies are employed for their preparation:
the dispersion of preformed polymers and the polymerization of monomers. Various techniques
can be used to produce polymer nanoparticles, such as solvent evaporation, salting-out, dialysis,
supercritical fluid technology, micro-emulsion, mini-emulsion, surfactant-free emulsion, and
interfacial polymerization. The choice of method depends on a number of factors, such as,
particle size, particle size distribution, area of application, etc. This review covers the general
description of the preparation of polymer nanoparticles and the detailed description of the crucial
parameters involved in techniques designed to obtain the desired properties.

PNPs can be conveniently prepared either from preformed polymers or by direct polymerization
of monomers using classical polymerization or polyreactions . Methods like solvent evaporation,
salting-out, dialysis and supercritical fluid technology, involving the rapid expansion of a
supercritical solution or rapid expansion of a supercritical solution into liquid solvent, can be
utilized for the preparation of PNP from preformed polymers. On the other hand, PNPs can be
directly synthesized by the polymerization of monomers using various polymerization
techniques such as micro-emulsion, mini-emulsion, surfactant-free emulsion and interfacial
polymerization. The choice of preparation method is made on the basis of a number of factors
such as the type of polymeric system, area of application, size requirement, etc. For instance, a
polymeric system that is developed for an application in the biomedical or environmental fields
should be completely free from additives or reactants such as surfactants or traces of organic
solvents. In this case, techniques like RESS (rapid expansion of a supercritical solution) or
RESOLV (rapid expansion of a supercritical solution into a liquid solvent) can be selected, as
they do not utilize any surfactant or organic solvent during the PNP preparation. These are just a
few of many factors that have to be considered before choosing a particular technique for the
PNP preparation(10).

5) Nanofibers

Nanofibrous materials are being studied and developed because they embrace considerable
promise for variety of applications and achieve some advantages of nanostructured materials.
Nanofibrous materials can be synthesized of biocompatible and biodegradable polymers and
produced by electrospinning processes. Because of the full potential of using biomaterials in
different applications, field of nanofibers have attracted considerable interest in biotechnology
and medicine and there has been fast development in this area in recent years. There are many
ways to fabricate nanofibers, such as template synthesis, self-assembly, electrospinning and
phase separation. The electrospinning technique possesses the unique features of simplicity,
affordability, wide range of materials selection, very high surface-to-volume ratio, tunable
porosity, and flexibility to adopt over a broad range of sizes and shapes. Because of these
extremely appreciable properties, electrospinning is a most popular technique for the production
of nanofibers. The standard electrospinning machine consists of spinneret (or needle), high-
voltage power supply with a wide range of voltage, a glass syringe with a small needle, and a
metal collector. Electrospinning nanofibers can be synthesized using an electrical potential to a
polymeric solution. Needle attached to and driven by a syringe pump which is accustomed to
manage the flow rate and volume of the polymer is ejected. A polymer solution is loaded into the
syringe that ejects the polymer solution at a constant rate. After loading of polymer solution,
solution is charged and at the tip of the syringe an electrically charged polymer droplet is
formed. Because of the repulsive force between the similar charges in electrically conductive
liquid and electric field, polymer solution tends to deform the droplet into a conicalshaped
structure known as Taylor cone, Taylor who has made essential studies on the jet formation.
Initially increase of electrical potential causes elongation of semicircular outward of solution at
the tip of the needle and forms the Taylor cone. After a threshold charge density, cone becomes
unstable and emits a jet of liquid. In the presence of an electric field, the jet travels a path to the
ground and as a consequence of elongation, solvent evaporation forms a continuous slim liquid
fiber and finally charged electro-spun fibers are collected on the Collector. The electrode plate or
Collector electrode are located on a place made of acrylic acid. The electrode is usually flatted
and is used for the collection of both random and aligned fibers. There are many biodegradable
polymers can be used to produce nanofi bers such as natural, synthetic, and composite of the
two. Biodegradable polymers were used to develop nanofi bers with diff erent functions based on
the requirement. Some can be used to deliver temporary function such as agent delivery, cell
carrier, and short-time scaff olds (until new tissue become mature and independent). In this
group, polymer will replaced by native tissue. Some polymers have been used for long-term
purpose such as an implant in surgery. At some point in the procedure of electrospinning using
synthetic polymers, the charge on the polymer solution makes it promising to govern its
trajectory using an electric field. The most commonly used and studied synthetic polymers are
PCL, PLDLA ,PLLA, PLGA and copolymers such as PCL-PEG, PCL-PLLA, PLGA-PEG,
PLLA-PEG, and etc.

6) Biosensor designs

A biosensor is an analytical device that incorporates a biological sensing element with a

transducer, to produce a signal used for the analyte concentration. Depending on the nature of the
biological process, biosensing devices may be divided into two general classes: biocatalytic
sensors (based primarily on immobilized enzymes), and affinity devices (using antibodies,
receptors and nucleic acids). There are many possible classification designs. Biosensors and
biochips are categorized either by their bioreceptor or their transducer type. The sampling part of
a biosensor consists of a biosensitive layer, and this layer can either have bioreceptors or be
made of bioreceptors covalently joined to the transducer. A biosensor is a simple device that is
made up of two elements:

1) A bioreceptor that is an immobilized sensitive biological component (e.g. enzyme, DNA

probe, antibody) recognizing the analyte (e.g. enzyme substrate, complementary DNA, antigen).
However, though antibodies and oligonucleotides are widely engaged, enzymes are still the most
commonly used biosensing elements in biosensors.

2) A transducer is used to turn a (bio) chemical signal, caused by the interplay of the analyte with
the bioreceptor, into an electronic one. The intensity of the generated signal is directly or
inversely proportional to the analyte concentration.

Biosensors contain three parts:

1) The sensitive elements (biologically derived material).
2) The transducer or detector element, to transform the detected signal into a readable, quantified
output.
3) The signal processor that represents the transformed signal in a user-friendly way In
biosensors, distinct biological elements may be associated with different kinds of transducers, if
the reaction of the biological element with the substrate can be monitored.

a) Optical measurements: Optical transducers used in biosensors include fluorescence,

interferometry, and spectroscopy of optical wave guides and surface plasmon resonances (SPR).
SPR is used to detect nanomolar levels of PCR products from genetically modified organisms, to
resolve human gene mutations

b) Electrochemical

c) Mass-sensitive measurements: This type of transducer is also classified as being a label-free

method. Piezoelectric sensors comprise a quartz crystal coated with a gold electrode, and are
applied as microbalances (QCM) sensitive to changes in the mass on the sensor surface.
7) 3DBioprinting for tissue engineering study

Compared to traditional tissue engineering methods, the technologies utilized by 3Dbioprinting

systems allow for greater precision in the spatial relationship between the individual elements of
the desired tissue. 3Dbioprinting holds great promise for regenerative medicine applications. The
bioprinting process occurs in three distinct phases. First, the pre-processing phase includes all
the planning details that precede production of bioprinted tissue. This phase includes imaging
(CT, MRI, etc.) to analyze the anatomical structure of the target tissue and subsequent CAD to
translate the imaging data into a blueprint for bioprinting. Specialized software programs (e.g.
AutoCAD, SOLIDWORKS, and CATIA) transform imaging data into cross-sectional layers of
appropriate scale such that the bioprinting device will be able to add them in a layer-by-layer
fashion.
The processing phase occurs next and involves all steps involved in the actual construction and
manufacturing of the bioprinted tissue. omplexity at this stage arises in choosing a specific
printing method and formulating a combination of materials (bioink, scaffold, and other
additives). Finally, the post-processingphase involves all steps that must occur before bioprinted
tissue is fully mature and ready for in vivo usage. For most 3D bioprinting applications, this
usually takes places within a bioreactor.

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List of Abbreviations Used

MNPs Magnetic Nanoparticles

MRI Magnetic Resonance Imaging

XRD X-Ray Diffraction

SEM Scanning Electron Microscopy

VSM Vibrating Sample Magnetometer

GNPs Gold Nanoparticles

AgNPs Silver nanoparticles

NPs Nanoparticles

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