Drug Delivery Strategies
Since their discovery over two decades ago, cell-penetrating peptides (CPPs) have been used to mediate the cellular delivery of coupled cargoes with high efficiency and low toxicity, both in vitro and in vivo. The wide range of cargoes includes proteins, oligonucleotides, λ phages, liposomes, quantum dots, and 40 nm magnetic iron oxide nanoparticles. Consequently, CPPs have emerged as a powerful tool for drug delivery. One of our main research areas is focused on developing CPPs and CPP-functionalized nanoplatforms, such as nanoparticles and liposomes, for the delivery of therapeutics with high efficiency and specificity.
We use cell and molecular biology methods, along with state-of-the-art biophysical techniques. We are also actively developing novel methods and experimental approaches. An example is our recently published experimental approach that combines two powerful biophysical techniques, fluorescence-activated cell sorting (FACS) and fluorescence correlation spectroscopy (FCS), to directly, accurately and precisely measure the cellular uptake of fluorescently-labeled molecules.
This rapid and technically simple approach is highly versatile and can readily be applied to characterize all major CPP properties that normally require multiple assays, including amount taken up by cells (in moles/cell), uptake efficiency, internalization pathways, intracellular distribution, intracellular degradation and toxicity threshold. The FACS-FCS approach provides a means for quantifying any intracellular biochemical entity, whether expressed in the cell or introduced exogenously and transported across the plasma membrane.
Several degenerative diseases are characterized by self-assembly of proteins or peptides into aggregates termed amyloid, which share several physicochemical features: a fibrillar morphology, a predominantly β-sheet secondary structure, insolubility in common solvents and detergents, and protease resistance. These so-called amyloid diseases include Alzheimer’s disease, Huntington's disease, Parkinson's disease, prion diseases (Creutzfeldt-Jakob disease in humans and ‘mad cow disease’ in cattle), and type II diabetes.
It has become generally accepted that the fibers themselves are not the toxic state; rather, it is the process of amyloid formation and in particular the formation of soluble intermediate states that represent the origin of toxicity. For instance, we determined that toxicity of islet amyloid peptide (IAPP, also known as amylin) in type II diabetes is due to aggregated membrane-bound α-helical, and not β-sheet, states of the peptide. Our results suggest that upon α-helical mediated oligomerization, IAPP acquires CPP properties, facilitating access to the mitochondrial compartment, resulting in its dysfunction.
Current efforts in the lab are focused on identifying the toxic species, sub-cellular target and mechanism of cytotoxicity of amyloid beta (Aβ or Abeta; Alzheimer’s disease) and the prion protein (PrP; prion diseases). Understanding the molecular basis for cellular dysfunction in Alzheimer’s and prion diseases will facilitate design of more effective inhibitors and development of strategies for the targeted delivery of these inhibitors.
We are interested in developing tumor-specific drug delivery nanoplatforms. This approach not only prevents targeting of healthy tissue, thereby reducing harmful side effects, but also enhances the potency of the drugs by accumulating them at the desired site of action.
A major challenge associated with cancer drug delivery is that transport in solid tumors occurs largely by passive diffusion within the tumor extracellular space (ECS), also known as the tumor interstitium, which is the aqueous matrix surrounding cancer cells. This is because little convective delivery occurs in solid tumors due to elevated interstitial fluid pressure, a consequence of vessel leakiness and the absence of functional lymphatics. Slowed diffusion in the ECS, due to ECS geometry and the extracellular matrix (ECM) composition, greatly restricts drug access in tumor therapy.
We are exploring strategies to enhance diffusion of our drug delivery nanoplatforms within tumors. The efficacy of these strategies will be assessed by our Pulsed-Infusion Microfiberoptic Photodetection (PIMP) method. This novel method rapidly and accurately determines the ECS properties, while simultaneously measuring macromolecule diffusion, within living tissue. We previously successfully applied the PIMP method to various tissues in vivo, including brain, kidney and skeletal muscle.
We are also interested in developing tumor-specific therapeutics. These include small molecules, peptides and proteins. An example is our recently published peptide derived from the mitochondrial membrane-binding N-terminal domain of hexokinase II (HKII). Overexpression of mitochondria-bound HKII in cancer cells plays a paramount role in their metabolic reprogramming and protects them against apoptosis, thereby facilitating their growth and proliferation. The HKII-derived peptide, pHK-PAS, is readily taken up by cancer cells where it effectively disrupts the mitochondria-HKII association, leading to mitochondrial dysfunction and finally apoptosis. Significantly, pHK-PAS treatment of non-cancerous cells results in substantially lower cytotoxicity. Our results demonstrate the potential of the pHK-PAS CPP as a novel cancer therapeutic strategy.