Develop new strategies for intracellular protein delivery.
Compared to nucleic acid delivery, intracellular delivery of proteins is more challenging, because i) unlike the negatively charged backbone of DNA, protein amino acids carry various charges, ii) proteins must maintain their native 3D structure in order to remain functional—a conformation often destroyed by complexation. Recently, my group has developed a universally effective strategy to deliver therapeutic proteins into cytoplasm using a combination of the reversible modification of proteins and cationic lipid-based nanoparticles. We have modified the lysine residues of proteins, converting the positive lysine into negative carboxylates using acid hydrides or carboxylic-acid-containing reagents. Such chemical modification lowers the pI of the protein and facilitates its complexation with positively charged nanoparticles. We have demonstrated the application of such a novel protein delivery system for suppressing tumor growth both in vitro and in vivo. In order to further selectively target tumors, we have developed a chemical method to modify the protein lysine with a chemical moiety that is cleavable in response to increased levels of reactive oxygen species (ROS). The chemical modification blocks the protein lysines, temporarily deactivating the protein. Exposing the modified protein to an appropriate microenvironment, such as increased reactive oxygen species (ROS) in cancer cells, efficiently cleaves the modified chemical moiety and restores the protein activity. We have demonstrated that the modified protein is not toxic to normal cells, but selectively kills cancer cells.
Scheme illustration of Intracellular ROS activated RNase A-NBC to inhibit tumor cell growth
2. Generated a new class of bioreducible lipid-based nanoparticles using combinatorial library strategy for drug delivery.
Our group has recently synthesized a combinatorial library of bioreducible lipid-like materials containing reducible disulfide bonds. These materials are biodegradable and stimulus-responsive delivery nanovectors, capable of delivery and release of their cargo in response to the physiological environment. Because this combinatorially-synthesized lipid library comprises many molecules with slight variations in structure, we have been able to correlate molecular features to the transport efficiency of biological cargos.
Combinatorial Library of Synthetic Bioreducible Lipid-based Nanoparticles
3. Mechanical sectioning-based approach for nanofabrication.
We used mechanical sectioning-based fabrication methods to biological tissues, a technique as we called “Bioskiving”. This technique has been used to create both flat and tubular scaffolds out of decelllularized tendon. Bioskiving involves taking a block of tendon (such as bovine Achilles tendon), decellularizing it in a sodium dodecyl sulfate solution, and then sectioning the block into thin sheets (5-300μm thick) using a microtome. These sheets can then be stacked and wrapped around rods of various diameters and crosslinked to form tubular conduits. These conduits can also be constructed with luminal fillers as we have previously shown. The benefit of bioskiving is that it does not require denaturation and reconstitution of the collagen which maintains the native triple helical structure as well as the proteoglycan content. This proves useful for both retaining the collagen’s mechanical strength as well as the biological activity for cell interaction. The unidirectionally aligned collagen nanofibers (derived from sections of decellularized tendon) could offer good mechanical properties to constructs, such as prosthetic grafts. Furthermore, the unidirectionally aligned collagen in tendon provides nanotopographic cues which can provide contact guidance for oriented cell growth. These novel constructs are currently being investigated for various biomedical applications, including prosthetic or tissue engineering-based blood vessels, and nerve guidance conduits.
Bioskiving: fabrication of novel tissue constructs from decellularized tissue using sectioning, stacking and rolling.