Engineering peptide based nanotherapeutics for managing metastatic breast cancer

Chakraborty, Kasturee (2021) Engineering peptide based nanotherapeutics for managing metastatic breast cancer. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Kasturee Chakraborty (14RS070)) 14RS070.pdf - Submitted Version Restricted to Repository staff only Download (15MB)

Abstract

Breast cancer remains a major concern worldwide being the second most prevalent form of malignancy. Advanced form of breast cancer often remains incurable by treatment with available therapeutic approaches like surgery, radiotherapy, chemotherapy etc. Moreover, the available chemotherapy and radiotherapy usually render into toxicity to healthy cells. Peptides have emerged as potential alternative for the treatment of breast cancer. The main aim of this thesis is to develop peptide based nanotherapeutics against triple negative breast cancer (TNBC), especially, MDA-MB-231 cell line. This study utilizes peptides as potential therapeutic drug and also uses peptides as molecular transporters for intracellular delivery of cytotoxic drugs and RNAi therapeutics like siRNA. Chapter 1 reviews the literature reports on potential use of peptides as an anticancer agents and molecular transporter. This Chapter also summarizes the advantage of nanoparticle based active targeting to improve the intracellular delivery of siRNA. Chapter 2 describes the use of membrane active ionophore peptide gramicidin A (gA) and gA inspired shorter hydrophobic octapeptide (LD8) as potential peptide-based drugs against MDA-MB-231 cell line. Both these peptides caused mitochondrial depolarization and resulted in apoptosis. We have also examined the efficacy of gA and peptide LD8 as molecular transporters for intracellular transport of hydrophobic and amphipathic cytotoxic drugs. We found that peptide LD8 being a slow acting peptide-based drug remains a safer choice for delivering other cytotoxic drugs like doxorubicin (Dox). Both Dox loaded gA (gA-Dox-NP) and Dox loaded LD8 (LD8-Dox-NP) nanostructures worked as 2-in-1 nanomedicine. They showed increased cytotoxicity against MDA-MB-231 cell line, with LD8-Dox-NP showing the most efficacy. Possibly we were first to report that membrane active peptides can be used to potentiate the effect of combination therapy involving cytotoxic drug against TNBC. Chapter 3 describes the use of natural TAT-peptide inspired designed proteolytically stable arginine enriched lipopeptides as molecular transporter for intracellular siRNA delivery. For this analysis, we have designed lipopeptides having Arg-DHis-Arg backbone. DHis residue (side chain pKa = 6.5) acts as a spacer residue to minimize adjacent Arg-Arg repulsion at physiological pH and also performs “proton sponge effect” to facilitate endosomal release. We have also incorporated both saturated lipidic moieties like stearic acid and unsaturated lipidic moieties like linoleic acid in the designed linear and cyclic peptide sequences. We have observed that cyclic peptide having Arg-DHis-Arg backbone and unsaturated linoleic acid (peptide 6) showed maximum efficacy in siRNA mediated gene silencing of erk1/2 gene. Peptide 6 showed comparable gene knockdown efficacy like commercially available transfection reagent HiPerFect (sold by multinational company Qiagen). These peptide based molecular transporters are nontoxic and cost effective compared to HiPerFect. We further amplified the efficacy of siRNA mediated gene knockdown by performing combination therapy with membrane active peptide-cytotoxic drug complex (gA-Dox-NP). Our findings suggest that combination therapy is more effective than siRNA mediated gene silencing based monotherapy. Chapter 4 describes the use of peptide based ligand coated shape and size dependent multivalent nanoparticles in siRNA delivery via active targeting. We have designed peptides having cMET receptor targeting moieties and siRNA binding moiety containing Arg-Xxx-Arg segment joined via a linker poly(ethylene glycol) (PEG). PEG increases the half life period of the nanoparticle and prevents opsonization. The peptide sequences also contains cysteine (Cys) residues to facilitate covalent conjugation with gold nanostructures. We observed that peptide conjugation with gold nanostructures leads to non toxicity of the gold nanostructures. We also found that siRNA mediated gene knockdown efficacy was also dependent on the shape and size of the nanoparticle. We observed that peptide conjugated gold nanosphere of 10 nm diameter and peptide conjugated gold nanorod of 10 nm diameter (aspect ratio 4) showed maximum Notch1 gene silencing compared to gold nanospheres of 50 nm diameter. We also observed that peptide conjugated multivalent gold nanorods show ~1.9 fold higher Notch1 gene silencing activity than free peptides and spherical gold nanoparticles. In Notch1 silenced cells, the expression of markers of epithelial mesenchymal transition (EMT), E-cadherin was found to be increased whereas, the expression of N-cadherin and MMP2 was found to be decreased. This study indicates the probable inhibition of EMT progression in Notch1 silenced cells by peptide conjugated multivalent gold nanorods. We also checked the uptake of siRNA by peptide conjugated cylindrical multiwalled carbon nanotube (MWCNT), single walled carbon nanotube (SWCNT) and spherical fullerene. For this study, we have used peptides having pyrene butyric acid which non-covalently binds with carbon nanostructures via π-π interaction. We observed that both peptide coated MWCNT and peptide coated fullerene showed similar knockdown efficiency comparable with free peptide. In case of non-covalently attached peptide-carbon nanostructure complexes, interpeptide and intrapeptide repulsion occurs among Arg residues inhibiting the attachment of multiple ligands to the carbon nanostructures. Our study demonstrates that the effect of multivalency was observed in case of covalently conjugated peptide-gold nanostructure complex and such effect was not observed when peptides were non-covalently attached with carbon nanostructures via π-π interaction.

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