Hyperbranched Polymeric Nanoparticles as Delivery Vehicles for Cancer Therapeutics
Polymeric nanoparticles that deliver imaging and therapeutic agents to areas of disease are of great interest in medicine, particularly for cancer diagnosis and treatment. The Perez Laboratory has developed biodegradable hyperbranched polymeric nanoparticles that can encapsulate therapeutic and imaging cargos within internal pockets created by the three-dimensional hyperbranched structure. These nanoparticles can be conjugated with various targeting ligands to facilitate their delivery to cancer cells via cell surface receptors overexpressed in cancer. Among these receptors are the prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer, and the epidermal growth factor receptor (EGFR), which is overexpressed in breast cancer and other tumors. The Perez Laboratory, in collaboration with Annette Khaled, PhD, at the University of Central Florida, Burnett School of Biomedical Sciences, College of Medicine, has recently developed polymeric nanoparticles that encapsulate a cancer-specific therapeutic peptide (CT20p) that triggers cancer cell death with minimal damage to normal cells.
When CT20p was encapsulated within the polymeric nanoparticles, targeted cell death was achieved only in cancer cells, with minimal effects on normal cells and tissues. In cancer cells, CT20p causes major disruptions in cytoskeletal dynamics and cell morphology within two to six hours that results in cell detachment by 24 hours. Importantly, these results were not observed with normal cells even when the nanoparticles were internalized by the cells, indicating that the lethal activity of the peptide was cancer cell specific. These findings revealed that CT20p could have the potential to impair cancer cell invasiveness (metastasis) through its actions on the cytoskeleton, which causes detachment-induced cancer cell death.
The Perez Lab is studying the ability of these nanoparticles to target the delivery of CT20p to prostate cancer via PSMA. In particular, we are interested in studying the ability of the peptide to treat metastatic prostate cancer. We will modify these nanoparticles to introduce imaging capabilities and be able to track their localization via positron emission tomography or magnetic resonance imaging. In addition, pre-clinical pharmacokinetics and biodistribution studies will be performed to assess the potential clinical translation of the nanoparticle formulation. The Perez Laboratory will also study the mechanism of action of this therapeutic peptide and will pursue its application in the treatment of other tumors such as metastatic breast cancer and gliomas.
Activatable Molecular Imaging Agents
Activatable nanoagents are those that enhance their signal output (magnetic or fluorescent) upon interacting with specific changes in the biological environment (pH, enzymatic activity) of areas of disease. A major focus of the Perez Laboratory is the development of activatable imaging agents for cellular- and animal-imaging applications. In particular, we have developed various iron oxide nanoparticle formulations that change their magnetic water relaxation properties upon changes in pH. These nanoagents have important applications for the imaging of acidic tumors, and for cell internalization and lysosomal localization. The main idea is to generate a nanoagent that only produces a signal upon interaction with the designated cellular target, minimizing background signal and increasing signal-to-noise ratio.
A key activatable nanoagent developed in our lab is based on iron oxide nanoparticles (IONPs). Even though IONPs have been extensively studied as MRI contrast agents, their use as drug delivery agents is rather new. Particularly, the changes in MR signal upon drug release is a phenomenon not previously observed until recently described by our lab. We have developed a method that encapsulates drugs and fluorescent dyes within the hydrophobic pockets of the polymeric coating surrounding the iron oxide nanoparticle. The cargo within the polymeric coating is stable at physiological pH, but it is released at lower pH (5¬-6). In the past, drugs have been directly conjugated to the nanoparticle, affecting drug release and efficacy. In our method, the drug is never conjugated to the polymer surrounding the nanoparticle but rather is entrapped via hydrophobic interactions with internal hydrophobic pockets within the polymer. Furthermore, studies performed using gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) — a paramagnetic chelate used for MRI imaging — as cargo within IONPs show that the MR signal of the Gd-DTPA is quenched when in closed proximity to the iron oxide core.
The Perez Laboratory will conduct additional studies to validate the use of these activatable nanoagents in various tumor models such as prostate, breast and brain tumors.
Small-Molecule-Based Activatable Prodrug for the Detection and Treatment of Tumors
The Perez Lab has designed an activatable fluorescent prodrug for the dual targeted imaging and treatment of folate receptor tumors. The activatable probe consists of the therapeutic drug doxorubicin conjugated to folic acid (folate). We made the serendipitous discovery and were the first to report that both the cytotoxicity and fluorescence properties of doxorubicin were quenched (“off” state) when connected with folic acid. The probe with the disulfide linker (Doxo-S-S-Fol) gets activated (“on” state) by intracellular glutathione (GSH), leading to fluorescence emission (optical imaging) and target-specific cytotoxicity (cancer treatment). In contrast, the noncleavable probe (Doxo-C-C-Fol) remains quenched (off) showing no migration to the nucleus and therefore no toxicity to the cancer cells. Results also confirmed that the sustained cytotoxicity of the released Doxo-SH derivative is compared to free Doxo. In our novel design, the folic acid acted as both a targeting ligand for the folate receptor as well as a quencher for doxorubicin’s fluorescence.
The Perez Lab will continue this work to try to understand the quenching mechanism of doxorubicin fluorescence by folic acid. A series of various doxorubicin conjugates will be synthesized and their fluorescence properties examined. In the near future, other activatable prodrugs will be designed with the goal of reducing systemic toxicity and allowing for in vivo drug localization and activation.
Binding Magnetic Relaxation Nanosensors for the Assessment of Molecular Interactions
The screening of targeting ligands toward molecular targets associated with cancer is of key importance for the development of efficient diagnostics, imaging and therapeutic modalities. Current modalities are predominantly composed of antibodies that, although highly specific, have serious drawbacks such as instability at higher temperatures, antibody denaturation and slow kinetics. For these reasons, it would be ideal to use stable targeting ligands, such as small molecules for targeting applications. When attached to nanoparticles, these small molecules can exhibit a variety of enhanced biological functions such as high binding affinities to proteins, toxins, and cell surface receptors, as well as chemical stability due to the presence of multiple small molecule ligands (multivalency) on the surface of the nanoparticle.
For these studies, our lab has developed binding magnetic relaxation (bMR) nanosensors that change their water relaxation upon binding of molecular targets to small molecules on the nanoparticle surface. Using this method, we have identified small molecules to bind to various toxins and cellular receptors for potential use in diagnostic and therapeutic applications. For example, in a recent paper, we have identified that sulindac, an FDA-approved nonsteroidal anti-inflammatory drug, binds to and inhibits the anthrax lethal factor toxin in solution. Most importantly, the inhibition of sulindac toward the toxin is enhanced when conjugated to iron oxide nanoparticles. These results are important as they set precedent for the use of this nano-based technology to screen for small molecules as drugs to treat a variety of other diseases.
This technology will be applied to screen for small molecules as therapeutic agents for cancer and other diseases such as Alzheimer ’s disease. Results from these studies could yield more robust, stable, targeted and translatable nanotherapeutics. In the near future the Perez Laboratory would like to examine the interactions of particular nanoparticle-small molecule conjugates with proteins and cell surface receptors overexpressed in cancer to identify new interactions. Furthermore, bifunctional and trifunctional ligands can be introduced via click chemistry to the nanoparticle to modulate the valency and small-molecule density in the nanoparticle surface in order to assess binding kinetics and specificity to a molecular target, such as a cancer cell surface receptor. The aim is to achieve the same level of specificity that is observed with antibodies, but with an array of small molecules on the nanoparticle surface.