Silk fibroin nanoparticles : in vitro performance of a putative anticancer nanomedicine

Despite the advantages of nanoparticle-based carriers for anticancer drug delivery, their translation into the clinic has been limited by factors including: (i) poor endocytic uptake and intracellular routing, (ii) macrophage clearance and (iii) a disregard of the tumour microenvironment governing nanoparticle uptake. As a result, there is a continued demand to explore the performance of polymer-based nanoparticles.;The principle hypothesis of this thesis is that silk fibroin nanoparticles can be used as anticancer nanomedicines. To validate this, the mechanisms governing drug release from silk fibroin nanoparticles are explored in Chapter 3. Next, the immunogenicity of silk fibroin nanoparticles towards macrophages is assessed (Chapter 4). Finally, Chapter 5 investigates the endocytosis of silk fibroin nanoparticles in response to the cell cycle and culture substrate mechanics.;This thesis provided the first experimental proof of lysosomotropic anticancer drug delivery from silk fibroin nanoparticles in single human breast cancer cells (Totten et al. 2017. J. Drug Target. 25, pp 865-872) (Chapter 3). Drug loaded silk fibroin nanoparticles were endocytosed by MCF-7 cells and a combination of the acidic lysosomal pH and enzymatic degradation facilitated drug release and subsequent nuclear translocation of the payload within 5 hours of dosing.;Next, nanoparticle-macrophage interactions were studied (Chapter 4). Silk fibroin nanoparticles exerted similar immunogenicity to silica and poly(lactic-co-glycolic acid) nanoparticles (Saborano, Wongpinyochit, Totten, Johnston, Seib and Duarte. 2017. Adv. Healthc. Mater. 6, 1601240). This indicated that silk fibroin nanoparticles can compete with leading healthcare materials in pre-clinical and clinical use.;Further assessment into immunomodulatory potential of silk fibroin nanoparticles revealed that they drive macrophage polarisation towards a pro-inflammatory M1-like state (Totten et al. 2019. ACS Appl. Mater. Interfaces. in press). This effect could be fine-tuned with surface modification (i.e. PEGylation). This observation is important because silk fibroin nanoparticles could act both as carriers for chemotherapeutics and as synergistic attenuators of tumour-associated macrophages in the tumour site.;Finally, advanced analysis of silk fibroin nanoparticle endocytosis was conducted (Chapter 5) by assessing intracellular trafficking in a time-dependent manner. Endocytosis of silk fibroin nanoparticles by breast cancer (MCF-7) cells was influenced by cell cycle progression, but not substrate mechanics. However, substrate mechanics were found to modulate the endocytic behaviour of healthy human (MCF-10A) breast epithelial cells. This relationship warrants further investigation with regard to the cellular response of nanomedicines.;Overall, this thesis accomplished in vitro analysis of silk fibroin nanoparticle drug delivery performance, macrophage interactions and endocytic uptake. These findings indicate that silk fibroin nanoparticles are emerging as an interesting biopolymer for anticancer applications. Work presented in this thesis provides a foundation to now move to pre-clinical in vivo studies.Despite the advantages of nanoparticle-based carriers for anticancer drug delivery, their translation into the clinic has been limited by factors including: (i) poor endocytic uptake and intracellular routing, (ii) macrophage clearance and (iii) a disregard of the tumour microenvironment governing nanoparticle uptake. As a result, there is a continued demand to explore the performance of polymer-based nanoparticles.;The principle hypothesis of this thesis is that silk fibroin nanoparticles can be used as anticancer nanomedicines. To validate this, the mechanisms governing drug release from silk fibroin nanoparticles are explored in Chapter 3. Next, the immunogenicity of silk fibroin nanoparticles towards macrophages is assessed (Chapter 4). Finally, Chapter 5 investigates the endocytosis of silk fibroin nanoparticles in response to the cell cycle and culture substrate mechanics.;This thesis provided the first experimental proof of lysosomotropic anticancer drug delivery from silk fibroin nanoparticles in single human breast cancer cells (Totten et al. 2017. J. Drug Target. 25, pp 865-872) (Chapter 3). Drug loaded silk fibroin nanoparticles were endocytosed by MCF-7 cells and a combination of the acidic lysosomal pH and enzymatic degradation facilitated drug release and subsequent nuclear translocation of the payload within 5 hours of dosing.;Next, nanoparticle-macrophage interactions were studied (Chapter 4). Silk fibroin nanoparticles exerted similar immunogenicity to silica and poly(lactic-co-glycolic acid) nanoparticles (Saborano, Wongpinyochit, Totten, Johnston, Seib and Duarte. 2017. Adv. Healthc. Mater. 6, 1601240). This indicated that silk fibroin nanoparticles can compete with leading healthcare materials in pre-clinical and clinical use.;Further assessment into immunomodulatory potential of silk fibroin nanoparticles revealed that they drive macrophage polarisation towards a pro-inflammatory M1-like state (Totten et al. 2019. ACS Appl. Mater. Interfaces. in press). This effect could be fine-tuned with surface modification (i.e. PEGylation). This observation is important because silk fibroin nanoparticles could act both as carriers for chemotherapeutics and as synergistic attenuators of tumour-associated macrophages in the tumour site.;Finally, advanced analysis of silk fibroin nanoparticle endocytosis was conducted (Chapter 5) by assessing intracellular trafficking in a time-dependent manner. Endocytosis of silk fibroin nanoparticles by breast cancer (MCF-7) cells was influenced by cell cycle progression, but not substrate mechanics. However, substrate mechanics were found to modulate the endocytic behaviour of healthy human (MCF-10A) breast epithelial cells. This relationship warrants further investigation with regard to the cellular response of nanomedicines.;Overall, this thesis accomplished in vitro analysis of silk fibroin nanoparticle drug delivery performance, macrophage interactions and endocytic uptake. These findings indicate that silk fibroin nanoparticles are emerging as an interesting biopolymer for anticancer applications. Work presented in this thesis provides a foundation to now move to pre-clinical in vivo studies