We have synthesized polyglutamate (PGA)-based architectures as novel hydrophilic and biodegradable carriers using BTA-derivatized cores as macroinitiators to yield star-shaped PGAs. We developed methodologies and synthesized several families of star-PGAs with different branching and degree of polymerization in a controlled manner with low polydispersity. We performed physicochemical characterization of polymers, derivatives, and conjugates using advanced spectroscopic techniques and developed a self-assembly theory of star-polyelectrolytes in dilute aqueous solutions. We discovered carrier candidates with a not previously described self-assembly behavior. Indeed, this success has been translated into a patent (WO/2017/025298-licensed and in exploitation), a high impact factor publication highlighted as cover page (AdvMat2017), papers describing the formation of star-shaped PGAs with complex architectures (PolymChem2020 & ChemSci2021 Submit.), and two PhD theses with an Extraordinary Award (A Duro-Castaño) and an excellent grade (O Zagorodko).We also note the publication of reviews (BiomatSci2015, MatHorizons2019, WIREsNanoNanobi2019), with the two latter demonstrating the applicability of our polypeptide carriers in immunotherapy and neurological disorders (cover page).The lymphotropism of our polypeptide carriers, apart from apparent applications in the treatment of metastasis, has led to their exploration in immunotherapy. Indeed, this data provided the basis for the ERCPoC Polymmune on melanoma and La Caixa NanoPanther project on pancreatic cancer. After derivatization, we have also explored our carriers for brain metastasis (PhD Thesis F Rodríguez-Otormín, June2021) and CNS pathologies (SciAdv2021). Adequate drug selection and rationale linking chemistries also represent crucial parts of the conjugate design. We have developed and fully characterized 2D and 3D cultures of representative BCa cell lines and performed high throughput screening (HTS) of drug combinations by measuring cell death and exosome release. Exosome characterization via NTA and proteomic analysis has contributed to the identification of biomarkers. We also established an HTS process that combined InCell and AlphaScreen technologies as an easier means of discovering new exosome biogenesis/release inhibitors (manuscript in prep). We have identified synergistic drug combinations for each BCa subtype. We note that our screening platform forms part of the ERIC-EUOpenscreen. We developed effective biodegradable linking chemistries for selected drug combinations to control targeting and release kinetics depending on microenvironmental factors. This work has derived a patent (PCT/EP2020/058940),high-impact articles (Biomaterials 2018, AdvFuncMat 2018), excellent graded PhD thesis (JJ Arroyo-Crespo 2018), several reviews (ADDR2020; IntRevCellMolBiol2019, MacromolBiosci2017) and the current development of at least three more articles regarding different drug combinations and the impact of experimental design in their development. With our first family of combination conjugates, we sought to explore accurate SAR to identify physicochemical descriptors and functional biomarkers through evaluations in the above-described models. We have found that trafficking proteins, intracellular pH, cathepsin B levels, and redox potential correlate with conjugate activity. We have also established a biobank of patient-derived BCa organoids and have started to evaluate selected conjugates (PhD thesis P Boix). Exosomal protein analysis provided interesting biomarkers to aid conjugate development for BCa treatment. We also performed in-depth physico-chemical characterization steps to understand our systems' behavior in complex physiological fluids (PhD thesis S Đorđević). We note relevant reviews (CurrPharmDes2016 cover page, JPersMed2018, ADDR2021). We have evaluated safety/biodistribution/PK/pharmacological activity of selected conjugates in two metastatic orthotopic TNBC models (IntJCancer 2019) for anticancer and antimetastatic activity including brain metastasis through a collaboration with Valiente group at CNIO (PhD thesis F Rodriguez). Our attempts to generate PDX models have not succeeded to this point, although our conjugates' success in other in vivo and 3D models provides a solid platform for their successful evaluation in the future. Our results have been disseminated via over 100 oral and poster presentations (vicentresearchlab.com) YouTube, social media, and other significant interactions, such as Pint of Science and European Researchers Night events.
