This document discusses using gold nanospheres for drug and gene delivery. It begins by introducing nanotechnology and some of its applications in fields like bioremediation. It then discusses problems with existing drug delivery systems like solubility issues and cytotoxicity. It describes how gold nanospheres will be fabricated using a sacrificial galvanic replacement method and coated with molecules like epsin to enter cells. A genetic construct containing marker and therapeutic genes will be encapsulated. The spheres will be detected using surface enhanced Raman scattering detected by an optical probe. Overall, the document proposes using gold nanospheres as a new drug and gene delivery system to address issues with current technologies.

1. BALAGANESH KURUBA
second year , masters in biology
department of biological, physical and chemical sciences
Illinois institute of technology
Bionanotechnology
Mentor:Prof. David Gidalevitz,
Departmentof Physics, Illinois Instituteof Technology

2. CONTENTS
Introduction
Need for Drug/Gene Delivery systems
Problems faced by existing Drug delivery systems
Gold Nanospheres
Genetic construct
Fabrication
Detection
Future prospects
Conclusion

3. INTRODUCTION
• Several approaches are designed to produce materials at
“Nano” scale to increase their prowess and efficiency.
• Used in several aspects in the fields on research and
manufacture as in production of IC’s, design of motors or
transistors performing intense and complicated functions.
• Building up nanobots – surveillance, bioremediation and
bio/nuclear hazard detection.
• Nanomaterials – Imposing immense strength and
flexibility into structures and reducing the size concerns.
• Storage – Infinite opportunities in storing vast amount of
data in terms of TB/µm.

4. • Drug Delivery systems: Liposomes, Hydrogels and others.
• Diagnostic applications: Use of Quantum dots, CLIOs and
gold nanoparticles in detection of potential threats.
• Nano scaffolds: Supports by nanowires or Carbon
nanotubes for generating tissues via the implementation of
stem cells. Eg: Bone calcification, muscle tissue generation.
• High throughput Analysis: Multiplex screening of drugs/
other target molecules in ligand selection .
• Structural and behavioral studies: Nanotechnology enables
the study of surface of membranes and other membraned
structures when in the process of interaction so as to
determine the rate of dissociation of the ligand from the
receptors. Similarly, it also enables study of proteomes of
different organisms.
• APPLICATIONS IN THE FIELD OF LIFE SCIENCES.

5. PROBLEMS FACED BY EXISTING DRUG /GENE
DELIVERYSYSTEMS
• Solubility issues.
• Biocompatibility and Biodegradability of the delivery
system being used.
• Immunoglobulin conjugated delivery systems faced an
issue of short half life of Mab’s being used which
determine the specificity of the system.
• Viral vector systems though efficient the chances of
resurrection of the vector systems have its own chances
resulting in problematic conditions.
• Cytotoxicity of the nanoparticles being used is another
concern.

6. • Gold nanospheres are cluster of gold nanoparticles which
by themselves are powerful nanomaterials.
• The properties of Gold nanoparticles differ from gold
nanospheres in terms of Surface Enhanced Raman scattering.
• Surface Enhanced Raman Scattering
SERS is a productive effect
of Surface Plasmon resonance caused
due to the oscillations of adjacent atoms
induced by incident beam of predetermined
wavelength resulting in an electromagnetic
field producing SERS.
• The scattering patterns vary from
a solid Gold nanoparticle to a hollow gold nanosphere, which
forms the basis for our project.
GOLD NANOSPHERES
Fig: Illustration of Generating
SERS response. (Source [1])

7. • Gold nanospheres are produced
a result of deposition of gold nanoparticles
on a solid/solid like template (Gelatin or
Latex beads) which is dissolved so as to
retain the deposition of the particles.
• Size and thickness of the sphere
shall depend upon the diameter of the
beads used and concentration of the Gold
salt being used.
• Process of Synthesizing: Sacrificial Galvanic replacement method.
Au3+ + Ag+ Au particle + 3Ag+ ions
Fig: A Computer generated model
of golden nanorods assembling
into a hollow sphere. (Source [2])

8. According to Sacrificial Galvanic replacement method,
• Exploits the phenomenon of Redox potential between Gold
(salt solution) and silver (metallic).
• Use of capping agents such as citric acid determines the
number of nucleation sites being formed and in turn nucleation
sites determines the size of the particle being produced.
• A stabilizing agent also has to be added during the process to
stabilize the nucleated sites from cross reacting.
• As an alternate considering the economics of the process, use
of Cobalt sacrificial template is used here instead of silver.
• Capping agent : Citrate
• Reducing agent : Sodium borohydride
• Starting material: Chloroauric acid.

9. GENETICCONSTRUCT
• The Drug/Gene to be delivered can be encapsulated
into the spheres by incorporating the drug in the medium
prior to fabrication of the surface.
• The Important step in Gene delivery being designing
the construct so as to contain effective elements for insertion
of Gene of Interest.
• Our Construct shall also include the gene for Green
fluoroscence protein ( for in vitro studies) and gene for trp b –
Beta subunit of trp synthase.
• Purpose of incorporating marker genes is to validate
the process of integration of Gene of Interest into the human
genome.

10. VIRAL ELEMENTS TO BE INCORPORATED INTO THE GENETIC
CONSTRUCT
• Promoter and a Poly Adenylation
signal.
• Viral Packaging signal ( Ѱ or €)
• Reverse Transcription signals-
• Polypurine tract (PPT).
• t – RNA binding site (PBS)
The Insert is designed
depending upon the kind of viral
system in use. As some use splicing
patterns to express a gene in that
case the insert should be contained
with introns, else the introns should
be got rid of.
Fig: Schematic representation of the
construct with necessary viral elements
as indicated. (Source [3])

11. FABRICATION
• The Gold Nanospheres are subjected for acetate wash prior
to the process of fabrication so that the surface can readily accept
the proteins/ ligand molecules being fabricated with.
• Two are the kinds of molecules to be fabricated on the
surface.
• Ligand/Mab/Protein molecule specific to
the marker on the infected cell .
• The Molecule “Epsin”: mediating
Clathrin - mediated endocytosis.
Upon Gaining entry, the nanosphere
shall open up based upon the osmotic
potential of increased potassium ion
concentration in the cells. Or it is feasible
to open up the sphere by mild
application of heat. Fig: Illustration of Clathrin mediated
Endocytosis. Source [4])

12. SERS DETECTION
• SERS can be detected by conventional methodologies
involve use of high end lasers and analytical systems.
• A Novel technique has been designed where in an
optical probe emits a beam of predetermined wavelength
and followed by which the receptor collects the emitted
signals from SERS active molecules.
• 1- Optic fiber probe; 2 – Core; 3- Shell; 4- First tapered
region; 5-Second tapered region; 6-third tapered region.
Fig: Schematic representation of SERS detecting probe. (Source [5])

13. REFERENCES: LITERATURE
• 1. Aggelliki K. "Quantum Dots Advantages and Disadvantages."
•2. Sperling R A and et al., "Biological Applications of Gold Nanoparticles". Chem Soc Rev 2008.
•3. Hainfield JF and et al., "Gold nanoparticles: a new X ray contrast agent". British Journal of
Radiology 2006.
•4. Pissuwan D and et al., "Therapeutic possibilities of plasmonically heated Gold
Nanoparticles." Trends Biotechnol 2006.
•5. Cullen T Vogelson. " Advances in Drug delivery systems." ACS publications April 2001.
•6. Ghosh P and et al., " Gold Nanoparticles in Delivery applications" . Adv Drug Delivery April
2008.
•7. Chen C Kwak and et al., "Packaging of viral capsid using gold nanoparticles". Journal of
Nanoscience and Nanotechnology 2008.
•8. Quinten M. Appl. Physics B.2001, 73, 317.
•9. Chen J Y and et al., Nano letters 2005.
•10. Chithrani and et al., Nanotechnology Letters 2006.
•11. Coffin JM and et al., "Principles of Retroviral Vector Design." Retroviruses NCBI Book shelf.
•12. Piazzi Leonardo da Vinci. "Optical probe for detecting sers-active molecules and process for
its manufacture".Standish C Hartman and Richard C Mulligan. “Two dominant-acting selectable
markers for gene transfer studies in mammalian cells”. Cell Biology Nov 1988.

14. REFERENCES: IMAGES.
• Surface Enhanced Raman Spectroscopy. Semrok corp.
• Chad Mirkin. “Coaxing Gold nanorods into orienting in
nanospheres”. Northwestern University. Ist July 2009.
•Coffin JM and et al., "Principles of Retroviral Vector Design."
Retroviruses NCBI Book shelf.
•Mathew T Drake and et al., “Epsin Binds to Clathrin by
Associating Directly with the Clathrin-terminal Domain”. The
Journal of Biological Chemistry , March 3 2000.
•. Piazzi Leonardo da Vinci. "Optical probe for detecting sers-
active molecules and process for its manufacture".

15. THANK YOU