TASK 1. ENGINEERING LOGIC miRNA CIRCUITS TO DEVELOP A MULTICOLOR BIOBARCODE - miRNA and whole transcriptome expression of several tumor cell lines were analyzed, namely BT474 cells (Luminal HER2-); MDA-MB-231 (Triple negative); MCF-7 (Luminal A); and SK-BR-3 (Luminal B HER2+). For example, MCF-7 mimics the majority of invasive human breast cancers that express ER while triple negative MDA-MB-231 is highly aggressive and lacks ER, PR and HER2. Interestingly, it was studied that hsa-mir-125b-2 is expressed in the four tumor cell lines in different degrees, such as BT474 cells (1.2±0.3) MDA-MB-231 (0.5±0.2); MCF-7 (1.6±0.2) and SK-BR-3 (0.98±0.22). The analysis of miRNA and whole transcriptome expression was also performed for MCF-10-A cell line as control condition.
TASK 2. SYNTHESIS OF NANOPARTICLES AND HYDROGELS FOR CIRCUITS DELIVERY - Polymeric nanoparticles have been explored to efficiently deliver the plasmid to the cells. The optimization of empty polymeric NP has been achieved by changing the conditions of sonication, namely amplitude and time of sonication. Regarding the characterization of the several batches produced for the process optimization, the selected parameters were amplitude 20%, for 15 seconds, which yielded NPs with a hydrodynamic diameter of 250 nm, polydispersity index of 0.19 and surface charge close to neutrality. The modifications in PLA polymer yielded NP with negative surface charge, which would not allow the electrostatic interaction with plasmid DNA. These NP were not considered for further studies. NP containing 50% or 100% modified TPGS presented positive charges above + 5mV, regular hydrodynamic diameters, and polydispersity index values. These NPs were considered for further studies for complexation with pDNA. Particles formulated with 50% and 100% modified TPGS (TPGS@50 NPs and TPGS@100 NPs) were incubated with pDNA according to different amine to phosphates (N/P) ratios. Afterwards, gel shift assays were performed to evaluate the complexation profile of the NPs with the pDNA. The formulation with TPGS@100 NPs enabled higher complexation with pDNA. After the complexation of the TPGS@100 NPs formulated with pDNA either containing the region coding for miR125b (pDNA miR125b) or not (pDNA), the size and surface charge of the produced NPs were characterized.
TASK 3. IN VITRO DELIVERY OF miRNA CIRCUITS INTO BREAST CANCER CELLS
a. Evaluation of plasmid transfection efficiency
BT474 cell line was incubated in lipofectamine loaded with CMV-NOmiRNA-RFP circuits, ERBB2-NOmiRNA-RFP circuits, and ERBB2-miRNA-RFP circuits for 24, 48, and 72 hours post incubation. Flow cytometry results showed a significant reduction in cell viability, namely 42.7 ± 4.8 % (only cells - control), 46.8 ± 10.1 % (CMV-NOmiRNA-RFP circuits), 47.4 ± 5.3 % (ERBB2-NOmiRNA-RFP circuits), and 39.5 ± 6.1 % (ERBB2-miRNA-RFP circuits). The transfection efficiency was evaluated by the expression of the reported gene via flow cytometry, demonstrating 10.9 ± 3.0 % for CMV-NOmiRNA-RFP circuits, 0.1 ± 0.1 % for ERBB2-NOmiRNA-RFP circuits, and 2.5 ± 0.2 % for ERBB2-miRNA-RFP circuits.
MCF7 cell line was incubated in lipofectamine loaded with CMV-NOmiRNA-RFP circuits, ERBB2-NOmiRNA-RFP circuits, and ERBB2-miRNA-RFP circuits for 48 hours post incubation. Flow cytometry results showed a high cell viability, namely 81.4 ± 0.4 % (only cells - control), 86 ± 1.8 % (CMV-NOmiRNA-RFP circuits), 80.5 ± 3.9 % (ERBB2-NOmiRNA-RFP circuits), and 80.9 ± 1.8 % (ERBB2-miRNA-RFP circuits), indicating minimal toxicity. Moreover, fluorescent microscopy images and flow cytometry results demonstrated transfection of miRNA circuits into the MCF7 cells. The transfection efficiency was evaluated by the expression of the reported gene via flow cytometry, namely 11.6 ± 1.6 % for CMV-NOmiRNA-RFP circuits, 9.9 ± 1.4 % for ERBB2-NOmiRNA-RFP circuits, and 14.7 ± 1.8 % for ERBB2-miRNA-RFP circuits. Mainly due to the low levels of transfection in the different tumor cell lines, MiR compounds will be used.
b. Evaluation of nanoparticles cytotoxicity
Complexes of modified polymeric TPGS NPs with pDNA did not greatly affect cell viability, which was about 80% and comparable to viability observed for the lipofectamine control in two breast cancer human cell lines, SK-BR-3 and MDA-MB-231.
c. Recreation of the tumor microenvironment by 3D spheroids techniques
Cell commercial cell lines from the common 4 types of breast cancer were used for spheroids generation using standard organoids fabrication techniques, such as ultralow adherent and agarose embedded well-plates culture systems. The spheroids constructs were produced at different cell densities, namely 5000, 7500, and 10000 cell per spheroid. The spheroids were stable over 10 days in culture with several morphological differences. BT474 spheroids (Luminal HER2-) were the most stable constructs, showing a spherical morphology with a Ferret diameter around 400-500 μm. MDA-MB-231 spheroids (Triple negative) demonstrated a significantly reduction of their Ferret diameter, namely 1300-1400 μm at Day 1 to 500 – 600 μm at Day 4. MCF-7 spheroids (Luminal A) almost maintained their size over time in culture from 600 to 450 μm and showed a globular morphology containing small spheroids. Finally, SK-BR-3 spheroids (Luminal B HER2+) have a disk-shape with a Ferret diameter size around 1550 – 2000 μm that is very similar over cell culture time.
The viability of spheroids constructs for BT474 at different timepoints was analyzed by flow cytometry. The results showed a high cell viability over time in culture at different cell densities, showing a viability from 60 to 96%.
