David Padilla Biology of Toxins Final ProjectApril 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 tumor-forming carcinomas.

Additionally it will discuss two types of drug-delivery vehicles, Liposome-Based Particles and Virus-Like Particles, currently being developed by the Brinker Nanostructures Research Group to be employed in targeted-drug 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 DeliveryNanomedicine 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, Liposomal-Based Particles and Virus-Like Particles are the leading carriers that are in development.

Virus-Like ParticlesVirus-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 19-nucleotide 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 ParticlesLiposomes 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

ConclusionSince 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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