Terapia genetica baseada em nanopartículas para tratamento de cancro de mama triplo negativo avaliadas por tecnologia de microfluidica

(O1) Develop a biocompatible engineered NP for safeguarding nucleic acid-based cargo (ASOs/siRNAs); • Synthesis of PLGA/PEG targeting polymeric conjugate that will constitute the NP matrix. Briefly, a PLGA-PEG polymer (50 KDa PLGA, 2 KDa PEG) will be chemically coupled with His using a carbodiimide reaction to form PLGA-PEG-His. Conjugation efficiency of His will be assessed. Cargo loading will be achieved using the double-emulsion solvent evaporation method. PLGA-PEG-His and oligonucleotide will be mixed with water for H-NPs nanoprecipitation and purification[3, 7, 8]. (O2) Thoroughly characterize and functionalize nanoparticles; Chemical engineering studies include: • Full physical-chemistry characterization of NPs (size and charge), while optimizing the nanocarrier size (~200 nm) to enhance cell uptake; • NP stability and interaction enhancement: addition of dioleoyl phosphatidylglycerol (DOPG) to achieve Zeta-potential +30 mV; • Corona examination: investigation of hard, medium and soft corona formation via serum fraction of platelet rich fibrin (SPRF) incubation. The composition and identification (by mass spectrometry) of the protein corona-NP will be assessed. Also, the mapping of the protein corona- NP complex interactions with array of targeted proteins will be assessed, in order to study the mechanism of binding and the effect of the corona. Circular dichroism (CD) will be used to study protein structural changes upon particle association, especially near-UV CD spectroscopy monitoring the protein tertiary structure around aromatic groups. • cargo release efficiency: under tumor-environment conditions (pH 5.6-6.8); • NP stability under varied conditions (temperature, pH, storage) by assessing particle size, surface change and gene release. Molecular and cell biology studies include • mRNA-CLU and CLU silencing • cell migration evaluation; • endocytosis and tracking evaluation: through fluorescence microscopy; • cytotoxicity evaluation and sensitivity to cytotoxic chemotherapy following exposure to doxorubicin, docetaxel or paclitaxel[4, 9] of bone-tropic TNBC cell lines with constitutive CLU expression (MDA-MB-231, MDA-MB-436, MDA-MB-468 (O3) improve an organ-on-chip system to preclinically validate nanocarriers; • study the permeability of tumor cells through the cellular barrier, and interactions between the TNBC cell models and human peripheral blood-derived osteoclasts seeded on top of a bone matrix, • NP size, circulation, distribution and cell uptake using Nanoparticle Tracking Analysis NanoSight Ns300. Confocal microscope system and imaging flow cytometer (ImageStreamX) will enable to execute complementary studies of individual cell/nano interaction dynamics at a high resolution. Moreover, molecular translocation events reflecting associated changes in intracellular signaling pathways will be assessed. Apply these methods to give input on the spatio-temporal pathway of all the nanomaterials, as well as the accompanying morphological changes in the cell. • NP biocompatibility and effect on tumor cell apoptosis, by annexin V staining and HCS apoptosis – TUNEL assay • NP effect on tumor cell migration using cell culture inserts • NP effect on tumor cell sensitization to cytotoxic chemotherapy. NP4CANC3Rs success will serve as strong indicator of progress in improving patient outcomes through gene targeted therapies while also advancing nanomedicine and bioengineered models to support other cancer thera