Self-assembled nanostructures for the cellular delivery of small molecules, oligonucleotides and proteins for the treatment of cancer

Abstract

We have developed chemically self-assembled nanostructures (CSANs) as a platform to achieve targeted drug delivery. CSANs are composed of dihydrofolate reductase (DHFR) and its inhibitor methotrexate. We have demonstrated that DHFR nanorings spontaneously self assemble in a controlled manner, when two fused DHFR molecules (DHFR2) are mixed with two chemically attached methotrexate (bis-MTX) molecules. Targeting CSANs selectively to the cancer cells was achieved by fusing one of the DHFR molecules with a single chain variable fragment (scFv) of an antiCD3 antibody. Delivery of antiCD3 scFv functionalized CSANs to CD+ leukemia T-cells have been demonstrated. The next step was to exploit the targeting ability of CSANs towards delivery of payload molecules such as drugs, nucleic acids and imaging agents. The research presented in the thesis focuses on evaluating the CSANs for its ability to achieve targeted drug delivery. In the first part of the thesis, we have engineered DHFR-DHFR dimer interface to drive the equilibrium towards formation of preferential heterodimers of DHFR. We have performed a series of point mutations at the dimer interface and screened mutants for the formation homodimeric and heterodimeric species. We have demonstrated that 65 % heterodimers of DHFR could be obtained by this method compared to 50 % obtained when protein mixed in the stoichiometric proportion. The second part of the thesis focuses on CSANs mediated targeted delivery of oligonucleotides. CSANs were functionalized with antisense oligonucleotides (ASO) targeting eukaryotic translation initiation factor 4E (eIF4E) via bis-MTX. Targeting was achieved using cyclic RGD peptide that binds to the alfa-v beta-3 integrin receptors. We have demonstrated that ASO bearing RGD CSANs bind to the alfa-v beta-3 integrin positive breast cancer cells, undergo receptor-mediated endocytosis and knock down the expression of eIF4E protein. In the third part, we have assembled bispecific CSANs that engage the immune cells to attack the cancerous cells. Bispecifc CSANs are composed of DHFR2antiCD3 scFv that binds to the CD3 receptor on immune T-cells and DHFR2 linked to a small peptide that target the epitopes on cancer cell surface. In the current study, we have targeted epithelial growth factor receptor (EGFR) that is over-expressed on several solid tumors. We observed about 70 % killing of EGFR positive glioblastoma cells over 24 hrs when incubated with T-cells that were functionalized with bispecific CSANs. In the last part of the thesis, we have evaluated targeted CSANs for their in-vivo biodistribution in mice bearing human tumors. Our preliminary studies demonstrate higher uptake of the CSANs by organs such as the liver, pancreas and kidneys primarily due to the macrophage uptake. To overcome this problem we have site-specifically PEGylated the CSANs using non-natural amino acid mutagenesis. In the in-vitro studies, we have demonstrated that PEGyaltion of CSANs does not affect their cancer cell binding ability but significantly reduced their macrophage uptake. In addition, we have also performed the biodistribution of PEGylated CSANs in mice tumor models.