Research

Pharmacological activity of a therapeutic agent is an outcome of two independent variables. Equilibrium constant can reflect how well an agent fits its target. Prerequisite for a successful therapeutic outcome from this molecular interaction is a sufficient amount of free drug molecules, i.e., thermodynamic activity, in the vicinity of the target site for a desired period of time. While the former constitutes the backbone of classical medicinal chemistry, my research program focuses exclusively on the drug delivery from the bench to the target site in vivo.

With rapidly advancing knowledge in cell/molecular biology and better understanding of pathogenesis of a disease state at the molecular level, contemporary and future medicine deals with various macromolecular agents of high therapeutic potential. It ranges from peptidomimetics, proteins, oligonucleotides, to genes. This trend has inevitably resulted in a great challenge in terms of in vivo, particularly in vivo cellular, delivery. My lab has been addressing some of the basic issues in contemporary molecular pharmaceutics that are essential in developing biotechnology-derived macromolecular therapeutics.

Cellular Delivery of Antisense/siRNA Oligonucleotides

Our approaches have focused on how to devise a means by which these macromolecules can be discharged from endosome to cytosol during early stage of endocytic uptake. In one of the two approaches we exploit the acidity of endosome lumen. Weak bases of pK_a close to 6 are incorporated into polymeric carriers of gene. As they are protonated, charge balance dictates flux of chloride ion to endosome lumen. Since the polymer cannot transport across endosomal membrane, osmotic pressure increases due to influx of water from cytosol. This so-called proton sponge effect eventually leads to endosome swelling and rupture. In our second approach, we introduce into liposomes loaded with oligomer so-called Gemini or bisdetergent that produces two molecules surfactant upon hydrolysis in endosome. Destabilization of liposome and permeabilization of endosomal membrane should ensue. Net result will be transfer of oligomer to cytosol.  

Antibodies as a Carrier of CpG Oligonucleotides to Solid Tumors

Due to their prolonged t_1/2, low clearance and volume of distribution, and high serum concentration, naturally occurring antibodies (especially IgG isotype) are an ideal high-affinity low-capacity drug carrier for potent drugs that exhibit poor systemic pharmacokinetics. An example of such an agent is immunostimulatory oligonucleotides containing CpG motif. An important requirement in this approach is that the immune complex (IC) be in a 1:1 stoichio-metric ratio.   To this end, high-affinity monomeric (1:1) ICs between anti-dinitrophenyl (DNP) IgG and DNP-derivatized CpG-ODNs were formulated by modulating the valence, inter-epitope (DNP) linker length and flexibility of CpG-ODNs. Systemically-administered ICs are expected to preferentially accumulate in peritumoral areas due to the enhanced permeability and retention effect (EPR) exhibited by solid tumors.  Systemic administration of long-circulating ICs could result in a reduced therapeutic dose and dosing frequency. It could also allow for the treatment of occluded or metastasized tumors. Once at the tumor periphery, ICs are expected to be actively taken up by peritumoral dendritic cells and macrophages via FcR-mediated endocytosis.  In the endosome, IC dissociation is mediated by the acidic endosomal pH, effectively freeing CpG-ODN to bind its endosomal receptor, TLR9 and mount a powerful anti-tumor immune response.

Nanogels in Nucleic Acid Delivery

Ongoing efforts are aimed at delivering plasmid DNA and siRNA in vivo by means of nanogels of 50 – 100 nm. These nanoparticles are prepared from water-in-oil inverse micelles in the presence of genetic materials. During radical-mediated polymerization, we can incorporate chemical mechanisms that will trigger release of the entrapped gene once the nanogels enter the cell. Luciferase expression both at the cellular level in vitro and in mice serves as a measure of successful transfection.

Albumin as a Drug Carrier (Re-visited)

Fatty acids bind albumin with an association constant comparable to that involved in antigen-antibody interactions. This high-affinity binding is a result of not only hydrophobic interaction between the binding pocket and inserting long alkyl chain but also electrostatic attraction between carboxylate anion and the positively charged periphery of the binding pocket. Our novel chemical conjugation efforts attempt to maintain and maximize these interactions. Interestingly both IgG and albumin are ligands of so-called Brambell receptor (also known as FcRn receptor) and hence share equally long circulatory t_1/2.  What is applicable to IgG discussed above is thus also applicable to albumin.