Microfluidic-based platforms have become a hallmark for chemical and biological assays, empowering micro- and nano-reaction vessels. The fusion of microfluidic technologies (digital microfluidics, continuous-flow microfluidics, and droplet microfluidics, just to name a few) presents great potential for overcoming the inherent limitations of each approach, while also elevating their respective strengths. This work exploits the combination of digital microfluidics (DMF) and droplet microfluidics (DrMF) on a single substrate, where DMF enables droplet mixing and further acts as a controlled liquid supplier for a high-throughput nano-liter droplet generator. Droplet generation is performed at a flow-focusing region, operating on dual pressure: negative pressure applied to the aqueous phase and positive pressure applied to the oil phase. We evaluate the droplets produced with our hybrid DMF–DrMF devices in terms of droplet volume, speed, and production frequency and further compare them with standalone DrMF devices. Both types of devices enable customizable droplet production (various volumes and circulation speeds), yet hybrid DMF–DrMF devices yield more controlled droplet production while achieving throughputs that are similar to standalone DrMF devices. These hybrid devices enable the production of up to four droplets per second, which reach a maximum circulation speed close to 1540 µm/s and volumes as low as 0.5 nL.

Poly(p-xylylene) derivatives, widely known as Parylenes, have been considerably adopted by the scientific community for several applications, ranging from simple passive coatings to active device components. Here, we explore the thermal, structural, and electrical properties of Parylene C, and further present a variety of electronic devices featuring this polymer: transistors, capacitors, and digital microfluidic (DMF) devices. We evaluate transistors produced with Parylene C as a dielectric, substrate, and encapsulation layer, either semitransparent or fully transparent. Such transistors exhibit steep transfer curves and subthreshold slopes of 0.26 V/dec, negligible gate leak currents, and fair mobilities. Furthermore, we characterize MIM (metal–insulator–metal) structures with Parylene C as a dielectric and demonstrate the functionality of the polymer deposited in single and double layers under temperature and AC signal stimuli, mimicking the DMF stimuli. Applying temperature generally leads to a decrease in the capacitance of the dielectric layer, whereas applying an AC signal leads to an increase in said capacitance for double-layered Parylene C only. By applying the two stimuli, the capacitance seems to suffer from a balanced influence of both the separated stimuli. Lastly, we demonstrate that DMF devices with double-layered Parylene C allow for faster droplet motion and enable long nucleic acid amplification reactions.

Neoangiogenesis is generally correlated with poor prognosis, due to the promotion of cancer cell growth, invasion and metastasis. The progression of chronic myeloid leukemia (CML) is frequently associated with an increased vascular density in bone marrow. From a molecular point of view, the small GTP-binding protein Rab11a, involved in the endosomal slow recycling pathway, has been shown to play a crucial role for the neoangiogenic process at the bone marrow of CML patients, by controlling the secretion of exosomes by CML cells, and by regulating the recycling of vascular endothelial factor receptors. The angiogenic potential of exosomes secreted by the CML cell line K562 has been previously observed using the chorioallantoic membrane (CAM) model. Herein, gold nanoparticles (AuNPs) were functionalized with an anti-RAB11A oligonucleotide (AuNP@RAB11A) to downregulate RAB11A mRNA in K562 cell line which showed a 40% silencing of the mRNA after 6 h and 14% silencing of the protein after 12 h. Then, using the in vivo CAM model, these exosomes secreted by AuNP@RAB11A incubated K562 did not present the angiogenic potential of those secreted from untreated K562 cells. These results demonstrate the relevance of Rab11 for the neoangiogenesis mediated by tumor exosomes, whose deleterious effect may be counteracted via targeted silencing of these crucial genes; thus, decreasing the number of pro-tumoral exosomes at the tumor microenvironment.

Estradiol-BODIPY linked via an 8-carbon spacer chain and 19-nortestosterone- and testosterone-BODIPY linked via an ethynyl spacer group were evaluated for cell uptake in the breast cancer cell lines MCF-7 and MDA-MB-231 and prostate cancer cell lines PC-3 and LNCaP, as well as in normal dermal fibroblasts, using fluorescence microscopy. The highest level of internalization was observed with 11β-OMe-estradiol-BODIPY 2 and 7α-Me-19-nortestosterone-BODIPY 4 towards cells expressing their specific receptors. Blocking experiments showed changes in non-specific cell uptake in the cancer and normal cells, which likely reflect differences in the lipophilicity of the conjugates. The internalization of the conjugates was shown to be an energy-dependent process that is likely mediated by clathrin- and caveolae-endocytosis. Studies using 2D co-cultures of cancer cells and normal fibroblasts showed that the conjugates are more selective towards cancer cells. Cell viability assays showed that the conjugates are non-toxic for cancer and/or normal cells. Visible light irradiation of cells incubated with estradiol-BODIPYs 1 and 2 and 7α-Me-19-nortestosterone-BODIPY 4 induced cell death, suggesting their potential for use as PDT agents.

Introduction: The research on tumor microenvironment (TME) has recently been gaining attention due to its important role in tumor growth, progression, and response to therapy. Because of this, the development of three-dimensional cancer models that mimic the interactions in the TME and the tumor structure and complexity is of great relevance to cancer research and drug development. Methods: This study aimed to characterize colorectal cancer spheroids overtime and assess how the susceptibility or resistance to doxorubicin (Dox) or the inclusion of fibroblasts in heterotypic spheroids influence and modulate their secretory activity, namely the release of extracellular vesicles (EVs), and the response to Dox-mediated chemotherapy. Different characteristics were assessed over time, namely spheroid growth, viability, presence of hypoxia, expression of hypoxia and inflammation-associated genes and proteins. Due to the importance of EVs in biomarker discovery with impact on early diagnostics, prognostics and response to treatment, proteomic profiling of the EVs released by the different 3D spheroid models was also assessed. Response to treatment was also monitored by assessing Dox internalization and its effects on the different 3D spheroid structures and on the cell viability. Results and Discussion: The results show that distinct features are affected by both Dox resistance and the presence of fibroblasts. Fibroblasts can stabilize spheroid models, through the modulation of their growth, viability, hypoxia and inflammation levels, as well as the expressions of its associated transcripts/proteins, and promotes alterations in the protein profile exhibit by EVs. Summarily, fibroblasts can increase cell-cell and cell-extracellular matrix interactions, making the heterotypic spheroids a great model to study TME and understand TME role in chemotherapies resistance. Dox resistance induction is shown to influence the internalization of Dox, especially in homotypic spheroids, and it is also shown to influence cell viability and consequently the chemoresistance of those spheroids when exposed to Dox. Taken together these results highlight the importance of finding and characterizing different 3D models resembling more closely the in vivo interactions of tumors with their microenvironment as well as modulating drug resistance.