Photothermal therapy (PTT) has attracted increasing interest as a complementary method to be used alongside conventional therapies. Despite a great number of studies in this field, only a few have explored how temperatures affect the outcome of the PTT at nanoscale. In this work, we study the necrosis/apoptosis process of cancerous cells that occurs during PTT, using a combination of local laser heating and nanoscale fluorescence thermometry techniques. The temperature distribution within a whole cell was evaluated using fluorescence lifetime imaging microscopy during laser-induced hyperthermia. For this, gold nanorods were utilized as nanoheaters. The local near-infrared laser illumination produces a temperature gradient across the cells, which is precisely measured by nanoscale thermometry. This allows one to optimize the PTT conditions by varying concentration of gold nanorods associated with cells and laser power density. During the PTT procedure, such an approach enables an accurate determination of the percentages of apoptotic and necrotic cells using 2D and 3D models. According to the performed cell experiments, the influence of temperature increase during the PTT on cell death mechanisms has been verified and determined. Our investigations can improve the understanding of the PTT mechanisms and increase its therapeutic efficiency while avoiding any side effects.
The human ErbB3 receptor confers resistance to the pharmacological inhibition of EGFR and HER2 receptor tyrosine kinases in cancer, which makes it an important therapeutic target. Several anti-ErbB3 monoclonal antibodies that are currently being developed are all classical immunoglobulins. We took a different approach and discovered a group of novel heavy-chain antibodies targeting the extracellular domain of ErbB3 via a phage display of an antibody library from immunized llamas. We first produced three selected single-domain antibodies, named BCD090-P1, BCD090-M2, and BCD090-M456, in E. coli, as SUMO fusions that yielded up to 180 mg of recombinant protein per liter of culture. Then, we studied folding, aggregation, and disulfide bond formation, and showed their ultimate stability with half-denaturation of the strongest candidate, BCD090-P1, occurring in 8 M of urea. In surface plasmon resonance experiments, two most potent antibodies, BCD090-P1 and BCD090-M2, bound the extracellular domain of ErbB3 with 1.6 nM and 15 nM affinities for the monovalent interaction, respectively. The receptor binding was demonstrated by immunofluorescent confocal microscopy on four different ErbB3+ cancer cell lines. We observed that BCD090-P1 and BCD090-M2 bind noncompetitively to two distinct epitopes on the receptor. Both antibodies inhibited the ErbB3-driven proliferation of MCF-7 breast adenocarcinoma cells and HER2-overexpressing SK-BR-3 cells, with the EC50 in the range of 0.1–25 μg/mL. BCD090-M2 directly blocks ligand binding, whereas BCD090-P1 does not compete with the ligand and presumably acts through a distinct allosteric mechanism. We anticipate that these llama antibodies can be used to engineer new biparatopic anti-ErbB3 or bispecific anti-ErbB2/3 antibodies.
Tandem repeat proteins have composite structure and unique properties, which allow them to be used in multiple fields, such as soft photonics, drug delivery and textile industry. The recent discovery of squid ring teeth (SRT) proteins have expanded the existing repertoire of repetitive polypeptides. We chose previously unexplored squid B. magister for our research, isolated and analyzed a new protein forming its ring teeth and hooks, and amplified the corresponding gene. Finally, we used this new isolated SRT protein to fabricate transparent thin films and microspheres.
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