| Description |
Drug-free macromolecular therapeutics are a new paradigm in polymer-based nanomedicines. Instead of carrying cytotoxic small molecular weight drugs, drug-free macromolecular therapeutics crosslink proteins in the cell membrane through hybridization of oligonucleotides to initiate apoptosis signaling. However, the mechanism of the nanomedicines was not fully understood. To study the mechanism and to better understand the interactions between the therapeutics and the cell membrane, super-resolution optical microscopy was used. Super-resolution imaging was performed on Raji B cells treated with the drug-free conjugates. The clustering of CD20 and lipid rafts was quantified. Lipid raft cluster size increased after treatment with drug-free conjugates. Drug-free conjugates induced apoptosis in a lipid raft-dependent mechanism where stable lipid rafts are needed for proper initiation of apoptosis. Direct stochastic optical reconstruction microscopy revealed nanoscale differences in membrane distribution of CD20 and lipid rafts. Pair-correlation analysis of super-resolution images showed lipid raft sizes of ~200 nm in cells treated with drug-free conjugates. General applicability of direct stochastic optical reconstruction microscopy to studying drug-delivery systems was also demonstrated. Two conceptually different polymer-based therapeutics were labeled with 4 different synthetic fluorophores, and three-dimensional (3D) direct stochastic optical reconstruction microscopy was conducted at different time points to track localization of the therapeutic components. An internalized polymer conjugate was localized in clusters at 4 h, but after 24 h, the polymer released into the cytosol a fluorophore attached via an enzymatically degradable peptide. Pair-correlation functions of the dye attached to the polymer and the released dye showed changes in their decay lengths between 4 h and 24 h. The pair-correlation function of the released dye showed random distribution after 24 h. Using reversible addition?fragmentation chain-transfer (RAFT) polymerization, branched and star polymers were synthesized to study the effect of architecture on apoptosis induction in Raji B cells. A new chain transfer monomer was synthesized in order to produce controlled branched polymers in RAFT polymerization. A degradable tetra-functional chain transfer agent was also synthesized. The star chain transfer agent produced degradable star polymers of high molecular weight (~170 kDa). Drug-free conjugates were synthesized to produce linear, branched, and star polymer-MORF2 conjugates. Apoptosis in Raji B cells was measured but the three different architectures induced the same levels of apoptosis as measure by annexin V and caspase 3. |
