2012 Nanobiotechnology Seminar Series

Jan 12, 2012
Munzer Auditorium

Julia Ljubimova, MD, PhD
Professor
Director of Nanomedicine Research Center
Deptartment of Nuerosurgery
Cedars-Sinai Medical Center
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A new nanobioconjugate drug delivery platform poly(b-L-malic acid) (PMLA) having built-in membranolytic activity for cancer intracellular drug delivery and cell/tissue targeting is introduced for treatment of primary brain and brain metastasis in vivo. Our goal was to engineer the new drug variants for systemic treatment of brain tumors and HER2-positive brain metastases to enhance the efficacy of trastuzumab/Herceptin® for crossing blood brain barrier/blood tumor barrier (BBB/BTB). Methods: Nanobioconjugates were characterized as to their absolute molecular weight, size, and ζ potential. Membranolytic activity was measured using artificial liposome assays. Primary human glioma U87MG, T98G and breast tumor MDA-MB-231 and MDA-MB-474 cell lines were used for cytotoxicity assays and tumor treatment. In vivo imaging analysis and confocal microscopy were used to confirm tumor targeting and tissues distribution. Results: Membranolytic activity was measured for two PMLA conjugates, P-LLL and P-LOEt [PMLA modified with pendant 40% trileucine (LLL) and 40% leucine ethyl ester (LOEt), respectively]. Only P-LLL induced significant liposome leakage at endosomal gradient pH (pH 5–6), but not at physiological pH 7.4. In vivo imaging revealed enhanced drug accumulation in tumors treated with the lead drug. The lead version bearing both Herceptin and antisense to HER2 produced strong life longevity, compared to Herceptin alone (P<0.001). For brain cancer treatment, the drug variant crossed BTB and reduced intracranial brain tumor size 10-fold. Summary: The new versions of Polycefin family nanobioconjugate drugs could be promising next generation of multifunctional vectors for treating brain and breast primary and metastatic cancers.

My lab focuses on identifying novel receptor-ligand or co-receptor interactions. Now that the human genome is complete, one of the next steps is to understand the interactions between all transmembrane and secreted proteins, which represent about one-third of all human genes. This knowledge would greatly aid in characterizing the biological function of many receptors/ligand pairs and their potential as therapeutic targets.  Surprisingly, a significant number of these receptors still remain orphans. In order to identify candidate partners, we have utilized Genentech's SPDI (Secreted Protein Discovery Initiative) protein library, consisting of over 1,000 purified, secreted proteins.  In the past, we have used surface plasmon resonance (SPR) and Bio-layer interferometry (BLI) technology to screen hundreds of interactions per day.   More recently, we have adapted and validated the assay in a protein microarray format, enabling the screening of thousands of extracellular interactions per day.  To date this work has led to the identification of several novel interactions that are now being evaluated for their therapeutic potential.  Development of our screening platforms and examples of interaction identified will be discussed. 

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability, and a high capacity for disparate cargos. We recently developed a new class of hierarchical nanocarriers termed protocells that synergistically combine features of mesoporous silica nanoparticles and liposomes. Fusion of liposomes to a spherical, high-surface-area, mesoporous silica core followed by modification of the resulting supported lipid bilayer (SLB) with multiple copies of a targeting peptide, an endosomolytic peptide, and PEG results in a nanocarrier construct (the ‘protocell’) that, compared with liposomes, the most extensively studied class of nanocarriers, improves on capacity, selectivity, and stability and enables targeted delivery and controlled release of high concentrations of multicomponent cargos (chemotherapeutic drugs, siRNA, dsDNA, toxins, etc.) within the cytosol or nucleus of cancer cells. Specifically, owing to its high surface area (>1000 square meters per gram), the mesoporous silica core possesses a higher capacity for therapeutic and diagnostic agents than similarly sized liposomes. Furthermore, owing to the substrate–membrane adhesion energy, the mesoporous silica core suppresses large-scale membrane bilayer fluctuations, resulting in greater stability than unsupported liposomal bilayers. In addition to conferring higher stability, the nanoporous support also results in enhanced lateral bilayer fluidity compared with that of either liposomes or SLBs formed on non-porous particles. We show the enhanced fluidity yet stability of the SLB enables dynamic reconfiguration of the surface allowing membrane bound ligands to engage in complex multivalent interactions with the target cell at very low targeting peptide densities. The synergistic combination of materials and biophysical properties organized over several hierarchical length scales enables high delivery efficiency and enhanced targeting specificity with a minimal number of targeting ligands, features crucial to maximizing specific binding, minimizing nonspecific binding, reducing dosage, and mitigating immunogenicity. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a million-fold improvement over comparable liposomes.

Twenty years is the estimated time scale to develop nanotechnology from basic interdisciplinary concepts in 2000 to create a general purpose technology with mass and sustainable use by 2020 (“Nanotechnology Research Direction” NSTC 1999).   This presentation outlines the outcomes in the last ten years, what has worked and was not, the current status, and most importantly how we prepare now for the future (see “Nanotechnology Research Directions for Societal Needs in 2020”  Springer 2011 www.wtec.org/nano2/).   There is an increased focus on nanoscale science and engineering integration, convergence with biology and other scientific domains, and establishing a general-purpose technology.  Use of “direct” investigative tools and fundamental knowledge progress through breakthroughs remain essential in still formative phase of development of nanotechnology in 2012.   The labor and markets are estimated to double each three years, reaching a $3 trillion market encompassing 6 million jobs by 2020.  It will be imperative over the next decade to focus on four distinct aspects of nanotechnology development: better comprehension of nature and communication leading to knowledge progress; technology, economic and societal solutions leading to material progress; international collaboration on sustainable development and quality of life leading to global progress; and people working together for equitable governance leading to moral progress.