Design and validation of nanotechnological strategies for HER2-positive breast cancer treatment

Resumen

Among the different molecular subtypes of breast cancer that are distinguished, the HER2-positive subtype is characterized, as its name indicates, by keeping the epidermal growth factor receptor-2 overexpressed. This overexpression, which occurs in 15-20% of breast cancer cases diagnosed, is related to a higher rate of cell proliferation and metastasis, so that it has long being associated with inferior outcomes for patients. Nevertheless, in recent decades, HER2-overexpression has also allowed the development of targeted therapies, which have contributed to considerably increase the disease-free survival rate of patients with HER2-positive breast cancer. Thus, today, one of the standard first-line treatments for this type of cancer consists of the combination of paclitaxel (an anti-mitotic drug) and trastuzumab (an anti-HER2 monoclonal antibody), since several trials have shown that a synergist effect occurs between this taxane and antibody. However, although paclitaxel is one the most successful anti-tumour drugs available these days and trastuzumab has proven to be very effective in inducing tumour regression, their administration entails severe side toxicity, and treatment resistances appear frequently. Both, the apparition of adverse effects and drug resistances is mainly caused by the low bioavailability and the lack of specificity of conventional antineoplastic agents. For this reason, in the past few years, nanomedicine has aroused special interest in cancer chemotherapy, and has been applied to develop drug delivery systems to improve drug biodistribution, therapeutic activity and selectivity. In this way, pursuing these goals, three main different nanotechnological strategies were developed in this doctoral thesis to improve the current drawbacks of HER2-positive breast cancer treatment. The first strategy developed was focused on the design of polymeric nanoparticles, made of alginate and piperazine, that served as a targeted vehicle for paclitaxel and trastuzumab. In order to select the most suitable nanoparticles for this application, alginate and piperazine solutions were mixed in different ratios. The resulting nanosystems, obtained thanks to the electrostatic interactions that take place between both compounds at acidic pH, were characterised. Then, the smallest nanoparticles (160 nm) were conjugated with trastuzumab and paclitaxel, previously included into β-cyclodextrins. This conjugation was carried out by means of the carbodiimide chemistry and, once the loaded nanoparticles were also characterized, their internalization in HER2-positive breast cancer cells was analysed. Subsequently, their efficacy and specificity were validated in vitro. Co-cultures of stromal and HER2-overexpressing breast cancer cells were treated with the conjugated nanoparticles, and conventional viability assays were performed with cell lines showing different HER2 expression levels. Results achieved in both types of experiments showed that the nanosystem developed was as effective as equivalent concentrations of paclitaxel-β-cyclodextrin complexes and even more effective than equivalent concentrations of paclitaxel. Furthermore, it reduced the viability of normal cells significantly less than the parent drug. At last, HER2-positive tumour spheroids were also developed to ascertain whether the paclitaxel-trastuzumab nanosystem obtained maintained its anti-tumour activity in these 3D biostructures mimicking human tumours. Alive/death confocal assays and cell counting were performed, and results achieved demonstrated that conjugated alginate-piperazine nanoparticles kept their anti-tumour activity when validated in 3D cell cultures. Next, the second strategy consisted of developing new drug delivery systems based on polydopamine nanoparticles. Polydopamine, a synthetic melanin analogue, has acquired a relevant role in cancer nanomedicine in recent years thanks to its outstanding physicochemical properties. Nonetheless, despite the interest that this polymer has aroused in the scientific community, its antineoplastic activity had not been studied in vitro in depth yet. This is why such study was done in this thesis prior developing any polydopamine-based drug delivery system. Thereby, for this, polydopamine nanoparticles were synthesized by dopamine solution oxidation in alkaline media containing different types of alcohols (ethanol, methanol, 2-propanol, 1-propanol and 2-methyl-2-propanol). Besides, with the aim of modulating the size (100 - 450 nm) of the polydopamine nanoparticles prepared, the NH4OH concentration used was modified in the synthesis media. Then, the cytotoxicity of the different nanosystems obtained was determined by performing viability assays with several malignant and normal cell lines. A new protocol was stablished to subtract polydopamine absorbance when carrying out MTT experiments. By following this procedure, it was noticed that polydopamine nanoparticle size conditioned their cytotoxicity: the smaller the diameter, the greater the anti-tumour activity of the nanoparticles, which was more marked to malignant cells than to stromal cells. Likewise, it was observed that polydopamine nanoparticle cytotoxicity was also conditioned by the type of alcohol employed to synthesize them, which also influenced the well-known ability of these nanoparticles to chelate Fe3+. As a consequence, and since it was proven that polydopamine nanoparticles were internalized in the cellular endo/lysosomes using confocal microscopy, it was hypothesized that their cytotoxicity may be related to their ability to chelate the Fe3+ existing in these organelles. This hypothesis was later verified, when further viability assays were performed after treating different cell lines with polydopamine nanoparticles plus an iron chelator or an antioxidant compound. Both compounds antagonized polydopamine nanoparticle toxicity, so it was revealed that a disequilibrium of the intracellular Fe homeostasis could be responsible for the customizable, intrinsic anti-tumour activity of these nanoparticles. Once at this point, a second drug delivery system was designed: polydopamine nanoparticles (150 nm) loaded with Fe3+ and doxorubicin. On one hand, Fe3+ was loaded to polydopamine nanoparticles in order to target HER2-positive breast tumour cells, which overexpress the transferrin receptor-1, and to enhance the ferroptosis process that these nanoparticles may produce by themselves. Since the pH determines if Fe3+ is a free cation or is forming Fe(OH)3, Fe3+ was loaded to polydopamine nanoparticles at different pH values to find the nanosystem with the greatest antineoplastic activity and selectivity. The different Fe3+-loaded nanoparticles obtained were characterized and, when MTT assays were performed with them, it was proven that the anti-tumour activity and specificity of polydopamine nanoparticles were more remarkable when charged with more free Fe3+ than Fe(OH)3. On the other hand, as doxorubicin is capable of inducing cell apoptosis and also ferroptosis as a side effect, this drug was loaded to polydopamine nanoparticles to achieve a synergist effect with the Fe3+ charged while reducing its side toxicity. Different concentrations of doxorubicin were adsorbed directly to polydopamine nanoparticles loaded with or without Fe3+, which were or not later isolated in order to perform two different types of viability assays. When the latter were carried out, polydopamine nanoparticles transporting doxorubicin showed great anti-tumour activity and less toxicity to stromal cells than the parent drug. Furthermore, when nanoparticles were charged with both Fe3+ and doxorubicin, synergy occurred between them. In addition, the importance of the pH value at which the Fe3+-loading was carried out was again revealed, since those nanoparticles charged with more free Fe3+ instead of Fe(OH)3 were more efficacious, even despite having charged less amount of doxorubicin. In this way, this drug delivery system could be tailored-made by adjusting the Fe3+-loading pH and the amount of doxorubicin charged depending on the potential clinical objectives pursued (increased therapeutic activity vs. reduced side toxicity). Subsequently, taking into account the good results that were achieved in vitro with the alginate-piperazine nanoparticles conjugated with paclitaxel and trastuzumab, loading this drug and antibody to polydopamine nanoparticles (180 nm) for the first time was determined in order to develop an even more efficient drug delivery system thanks to polydopamine advantages. Since it was previously observed that 2-propanol was the alcohol that conferred the highest anti-tumour activity to polydopamine nanoparticles, these were synthesized with this alcohol. Next, paclitaxel was directly adsorbed in polydopamine nanoparticles, without the need to include it previously in β-cyclodextrins. Trastuzumab was both directly loaded and covalently bound through the carbodiimide chemistry to compare the results obtained following both strategies. The anti-tumour activity and selectivity of the resulting loaded polydopamine nanoparticles were evaluated by means of MTT assays performed again with HER2-positive breast cancer cells and stromal cells. As a result, it was observed that the two types of conjugated polydopamine nanoparticles had great therapeutic activity, and that they were more effective than the alginate-piperazine nanosystem developed previously. Furthermore, loaded polydopamine nanoparticles proved to be less toxic to normal cells than equivalent concentrations of paclitaxel. Of the two types of loaded nanoparticles designed, the one in which trastuzumab was covalently bound showed to be slightly more effective and selective, so it was chosen to perform further studies. Thereby, results achieved in the MTT experiments were corroborated with the performance of alive/death confocal assays. Likewise, as was done with the previous paclitaxel-trastuzumab nanocarrier, the antineoplastic activity of the polydopamine nanoparticles selected was validated by employing HER2-positive breast tumour spheroids. The interaction between the nanoparticles and the 3D biostructures was analysed by scanning electron microscopy, further alive/death assays and cell counting. Results achieved with the different techniques allowed to reach the same conclusion: this third drug delivery system developed also maintained their efficacy in 3D cell cultures. In the end, to finish this thesis, a third strategy was carried out to improve HER2-positive breast cancer treatment, developing macroscopic gellan gum hydrogels loaded with paclitaxel for local, post-surgery chemotherapy applications. These hydrogels were synthesized in both acetate buffer and phosphate buffer and were crosslinked at different degrees with L-cysteine. Once obtained, hydrogels were extensively characterized by means of rheological analysis, swelling capacity studies, morphological and chemical analysis, thermogravimetric analysis and compression measurements. The results obtained after the characterization process led to select the hydrogels with an intermediate crosslinking degree to be loaded with paclitaxel, as they were shown to have a more suitable pore size for the desired application. Thus, once their biodegradability and biocompatibility were verified, these hydrogels were loaded with paclitaxel-β-cyclodextrin complexes, again developed to improve the solubility of the taxane. Paclitaxel release from the hydrogels was studied and, finally, the anti-tumour effect of the loaded gels was validated in vitro. Both MTT assays and alive/death confocal assays were performed with different HER2-positive breast cancer cell lines, and results obtained proved that loaded gellan gum hydrogels had great anti-tumour activity. In addition, they proved to be a good strategy to achieve a paclitaxel sustained release, which in fact may be tailored to potentially complement other systemic anti-tumour therapies by modifying the synthesis medium of the hydrogels and their degree of crosslinking.