Development and Characterization of Cathepsin-B Cleavable Tandem Peptides to Improve Nucleic Acid Delivery

Introduction:: Ovarian cancer is the 5th leading cause of cancer-related deaths among women, with a 5-year survival rate of less than 50%. Most patients are diagnosed in stage III and stage IV, where the likelihood of chemoresistance rises, and the survival rate reduces drastically. There is a need more improved personalized and targeted medicines that can treat ovarian cancer where there is an increased expression of proteins associated with chemoresistance, tumorigenicity, and metastasis. RNA interference is a mechanism that can be utilized to target such proteins, using small interfering RNA (siRNA). However, several barriers limit the therapeutic potential of siRNAs, including immunogenicity, poor cellular uptake, lack of specificity to target cells, and inability to escape endo/lysosomal compartments. To address this, we have developed a novel series of peptide sequences with three functional domains that work together to overcome these barriers. These domains include a cell targeting region for site specific delivery of siRNA, an enzyme (Cathepsin B) cleavable region for separation of the domains once internalized into endo/lysosomal compartments, and a fusogenic region for enhanced endosomal escape. The main objective of our work is to emphasize the importance of a multifunctional peptide design and to evaluate the potential of our peptide nanocarriers in addressing the challenges associated with siRNA delivery through in vitro studies and characterization.


Materials and Methods:: Cleavable tandem peptides, GF-LD and VA-LD, were complexed with non-targeting (NT-siRNA) at N:P molar ratios 2.5:1-80:1 and peptides were subjected to agarose gel electrophoresis to analyze siRNA binding. To determine stability of the peptides, GF-LD and VA-LD siRNA complexes were incubated in either 50% fetal bovine serum (FBS) or RNAse A for 1 hour and subjected to gel electrophoresis. Using OVCAR-3 and ES-2 ovarian cancer cells, the cytocompatibility of both peptides was tested using an MTS assay and was compared to non-cleavable tandem peptide sequence, G3-LD. To investigate the potential of cathepsin B inhibition in reducing cytotoxicity at high N:P ratios, ES-2 cells were treated with cathepsin B inhibitors E64d and Z-FA-FMK for 24 hours, followed by treatment with tandem peptides for another 48 hours. Cytotoxicity was measured using an MTS assay. Fluorescence microscopy and flow cytometry was used to visualize and quantify cellular internalization. Specifically, OVCAR-3 and ES-2 ovarian cancer cell lines were treated with GF-LD and VA-LD peptides complexed with Cy5-labeled NT-siRNA with an incubation time of 4 hours. Internalization of tandem peptides in ovarian cancer cells was compared to internalization of tandem peptides in GFP expressing healthy, human embryonic kidney cells (HEK293-GFP).


Results, Conclusions, and Discussions:: This study utilized two cleavable tandem peptides, GF-LD and VA-LD, with the same cell targeting and fusogenic sequence but different cathepsin B cleavable linker sequences, and compared their functionality. The VA-LD peptide can bind siRNA at an N:P ratio of 10:1 and higher, while GF-LD can bind siRNA at an N:P ratio of 20:1 or higher, which is likely due to the slight difference in amino acid length. Both peptides can protect siRNA when incubated in simulated physiological environments that have nucleases and other degradation components. Additionally, both peptides are cytocompatible with ovarian cancer cells at N:P ratios of 40:1 and lower, but show dose-dependent cytotoxicity at higher N:P ratios ( >40:1). Interestingly, using the non-cleavable version of the tandem peptide (G3-LD), no cytotoxic effects were observed at higher N:P ratios. Inhibition of cathepsin-B restores cytocompatibility of the cleavable tandem peptides, indicating that cathepsin-B cleavage could be an indicator of cytotoxicity. his was an interesting finding that we will continue to explore in our future studies due to the fact that we did not see cytotoxicity using healthy, human embryonic kidney cells as well. Using N:P ratios of < 40:1, our cellular uptake studies showed that both VA-LD and GF-LD peptides are internalized into ovarian cancer cells with low internalization into HEK293 cells. This demonstrates that our peptides are being internalized through receptor mediated endocytosis as HEK293 cells express low level of our target receptor. Our preliminary results demonstrate that our tandem peptides can bind and protect siRNA, mediate delivery of siRNA to ovarian cancer cells, and are cytocompatible with ovarian cancer cells and healthy human embryonic kidney cells. Future work will deliver therapeutic siRNA for a gene correlated with chemoresistance in ovarian cancer and will explore bioactivity (endosomal escape ability, gene and protein knockdown) of the siRNA. The overall impact of this work would allow for the delivery system to be employed as a platform for the treatment of various diseases by delivering negatively charged nucleic acids or by replacing the peptide targeting region.


Acknowledgements (Optional): : This work was supported in part by the South Carolina Bioengineering Center for Regeneration and Formation of Tissues (SC BioCRAFT) under National Institutes of Health award #5P30GM131959-02 and National Science Foundation Early Career Development Program under NSF Award #204669 (A.A.-B.) NSF Award # EPSCoR Program under NSF Award #OIA-1655740 (A.A.-B.), Clemson’s Call Me Doctor Fellowship Program (K.L.A.), and Clemson’s Creative Inquiry Program (R.X.). Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect those of the National Institutes of Health or the National Science Foundation.

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