Transcript The Reduced Systemic Toxicity of Chemotherapeutic Agents

THE REDUCED SYSTEMIC TOXICITY OF
CHEMOTHERAPEUTIC AGENTS AS A
RESULT OF TARGETED-DRUG DELIVERY
David Padilla
Biology of Toxins Final Project
April 29, 2009
Objective
This project will discuss targeted-drug delivery
and how it is employed to reduce the systemic
toxicity that results from chemotherapeutic
agents in the treatment of various tumorforming carcinomas.
Additionally it will discuss two types of drugdelivery vehicles, Liposome-Based Particles
and Virus-Like Particles, currently being
developed by the Brinker Nanostructures
Research Group to be employed in targeteddrug delivery.
Introduction

Cancer (malignant neoplasm) is defined as
a disease in which cells display uncontrollable
growth, invasion of adjacent tissues and
metastasis to other organs. Carcinomas are
malignant tumors derived from epithelial cells.

In 2008 there were ~1,437,180 new cases of
various forms of cancer diagnosed and
~565,650 cancer related deaths. It is a major
public health issue that affects people’s lives
on a daily basis.
 There are several treatments (surgery,
chemotherapy, radiation, immunotherapy,
hormonal therapy, and various targeted
therapies) for different carcinomas, all of which
have associated adverse effects.
 Most therapeutic regimens involve a combination
of the treatments which are dependent on the
type of carcinoma.
 Chemotherapy is usually part of the therapeutic
regimen. Chemotherapeutic drugs are cytotoxic
agents that kill rapidly dividing cells by
interfering/inhibiting cell division. Due to this they
have many adverse effects; among the most
common are hair loss, cardiac and neurotoxicity.
Results and Discussion
 Chemotherapeutic Agent Studied
 Benefits of Targeted-Drug Delivery
 The Drug Carriers: Virus-Like Particles
and Liposome-Based Particles
The Chemotherapeutic Agent:
Doxorubicin
 Anthracyclines, such as
doxorubicin HCl, are common
chemotherapeutic agents used in
therapeutic regimens
 Doxorubicin HCl is usually
administered intravenously in a
sodium chloride solution.
 Doxorubicin is photosensitive and
has a red fluorescent emission
spectra, which makes it ideal for
use in the development of drug
carriers.
 Does not cross blood-brain barrier
 The initial distribution half-life of
doxorubicin is ~5 minutes (rapid tissue
uptake) and terminal half-life ~20-48
hours (slow elimination)
 Enzymatic reduction by CYP3A4 position
7, and cleavage of the daunosamine sugar
yields aglycones and free radicals
 Plasma clearance is ~324-809 mL/min/m2
and occurs by metabolism and biliary
excretion
 Exact mechanism of action is unknown
but it is known that it interacts with DNA
by covalent intercalation, inhibiting the
progression of topoisomerase II.
Targeted-Drug Delivery
Nanomedicine is proving to
play a large role in the future
of cancer treatment. The
targeted-drug delivery of
chemotherapeutic agents
offers the advantage of
localized drug delivery
specifically to the malignant
tumor. Direct delivery of the
drug results in reduced
systemic toxicity due to
minimal non-specific uptake
of the drug by cells other than
the target, drastically
reducing the ED50 of the
drug.
How are drug-carriers
designed?
Many factors must be taken into
consideration when designing a good drug
carrier. The carrier must have low
immunogenicity, high affinity for
extracellular or intracellular receptors
displayed by tumors, and low affinity for
receptors displayed on healthy cells.
Additionally it must be stable while
circulating in the blood but easily degraded
upon encountering its target and exhibit
some mechanism of controlled-release of the
drug.
The exploitation of materials science
applications and new nanotechnology has
given rise to the development of various
nanoparticles which are being utilized in
targeted-drug delivery. Currently, LiposomalBased Particles and Virus-Like Particles are the
leading carriers that are in development.
Virus-Like Particles
Virus-Like Particles (VLPs) are the structural
analogs of non-enveloped viruses and
bacteriophages that are composed of the virus’s
structural proteins while excluding the viral
genome and associated enzymes. By using Phage
Display, libraries of targeting ligands with with
novel binding properties can be constructed from
populations of random peptides. Different
peptides from these libraries can then be
conjugated to VLPs in order to target various
malignant neoplasms which display receptors that
have a high affinity for the targeting ligand.
The icosahedral capsids of bacteriophages,
such as MS2 (T=3), are composed of an integer
multiple of 60 coat protein subunits reflected by
the T-number. The MS2 genome contains a 19nucleotide sequence on the 5’ end of the replicase
cistron that encodes the coat protein subunits.
MS2-VLPs are produced by expression of the 19 nucleotide sequence using
the plasmids of bacteria. Upon expression the once a sufficient
concentration of capsomere subunits has been produced MS2-VLPs
spontaneously assemble. The VLPs can be modified to display their cargo
on their surface or encapsidate it in order for efficient delivery to a target.
Standard conjugation techniques, immunosorbent assays, and growth
inhibition experiments were used to confirm that modification of these
bacteriophages can serve as a means to deliver various cargo with low
immunogenicity.
Many tumor cells display the folate receptor on their surfaces so
conjugation of the folate-ligand serves as the perfect targeting peptide in
order to ensure that the VLPs exhibit a specific affinity for the targeted
tumor. Once the VLP has attached to its target, it enters the cell via
endocytosis and disassembles as a result of the decreasing pH in each
endosomal compartment. Upon degradation the chemotherapeutic agent
is released thus delivering the drug to its target.
By labeling the target cells (in vitro) with fluorescent dyes (Hoechst
33342 and CT Green) , confocal microscopy can be used to illustrate
and confirm that the VLP reaches its target cell. The image below
shows a hepatocellular carcinoma cell from the Hep3B line which
was targeted with a MS2-VLP loaded with doxorubicin produced in
the Brinker labs.
Liposome-Based Particles
Liposomes are artificially constructed
spheres of lipid bilayer containing an aqueous
compartment. Utilizing the same idea as the
liposome, the Brinker group has developed a
liposome-based particle termed the protocell.
Protocells are porous silica nanoparticles with a
supported lipid bilayer. The protocell, like the
liposome, offers the advantage of low
immunogenicity, low toxicity, and possesses
surface features which can be easily modified.
Unlike the liposome, it also offers the benefit of
the uniform nanoporous core which allows for
more efficient loading of the cargo and
controlled release of the drug.
By employing the Evaporation Induced-Self Assembly method,
developed by the Brinker Group, the negatively-charged
mesoporous silica core was constructed. Fusion of a positively
charged liposome on a negatively charged mesoporous silica core
serves to load the core with a negatively charged dye which is
excluded from the mesopores without lipid. Sealed within the
protocell, this membrane impermeable dye was observed to be
transported across the cell membrane and slowly released within the
cell, thus proving that it is ideal for utilization in targeted-drug
delivery.
Summary and Conclusions
 Conclusion
 Acknowledgements
 References
Conclusion
Since cancer is such a serious public health problem that affects
people throughout the world, targeted-chemotherapy has the
potential to change the clinical outcome of many therapeutic
regimens. Targeted-drug delivery offers a plethora of benefits,
amongst the most important is the drastic reduction in systemic
toxicity. Delivery of the drug to the site of action allows for
lower dosages of the drug and uptake solely into tumor cells.
The result is decreased adverse effects and an increased
therapeutic index . The future direction of this research will lead
to a variety of applications. Potential applications include use in
diagnostic imaging, vaccinations (VLPs), treatment of other
malignant neoplasms, and targeted-drug delivery in a number
of pathogenic infections.
Acknowledgements
Special thanks to my mentors Carlee Ashley, Jeff Brinker, and Carol
Ashley of the Brinker Nanostructures Research Group, and to
Genevieve Phillips of the UNMHS Cancer Research Facility Confocal
Microscopy Lab.
*All confocal images were taken by David Padilla or Carlee Ashley
**All other supporting images were obtained from the cited Articles
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