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- The Bridge Between Nanotechnology and Chronobiology: Circadian Control of Cancer Therapy by Gene Delivery SystemsPublication . Neves, Ana Raquel Bastos; Costa, Diana Rita Barata; Paixão, Telma Alexandra Quintela; Fan, Donglei EmmaCancer is nowadays the second leading cause of death worldwide, making it a significant public health concern. Europe, following Asia, is, according to the Global Cancer Observatory (GLOBOCAN), the second continent where the incidence, mortality, and prevalence are highest. Statistics indicate that the number of cancer-related deaths in Europe is projected to increase 36.4% by 2050 compared to 2022. In particular, glioblastoma (GB), designated by the World Health Organization (WHO) as a grade 4 astrocytoma, represents the most prevalent (more than 50% of the total cases) and highly aggressive type of primary brain cancer in adults. This cancer is characterized by a high inter- and intratumourally genetic heterogeneity, aggressiveness, angiogenesis, invasiveness, resistance to current standard treatment protocols, poor patient prognosis (12-15 months), and low survival rate. Molecularly, several altered signalling pathways (p53 tumour suppressor pathway inclusive), gene mutations (tumour suppressor p53 gene (TP53), epidermal growth factor receptor (EGFR), isocitrate dehydrogenase (IDH)-1/2, etc.), and methylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter contribute to its hyperproliferation, growth, and chemotherapeutic resistance. For these reasons, actual care treatments, as the classical Stupp protocol, face several challenges. This protocol focuses on first removing the tumour by surgical resection, without compromising the normal neurological function, followed by radiotherapy and chemotherapy with Temozolomide (TMZ), whose effectiveness depends on the MGMT gene promotor methylation status. However, after being subjected to this, patients prognosis remains very poor, and 90% of them have a tumour recurrence within two years. Thus, the development of new and more effective therapeutic approaches is essential to fight this disease. Innovative therapeutic strategies as nanotechnology, gene therapy, and chronotherapy, have been developed and explored to improve the treatment efficacy of GB. Gene therapy consists of delivering exogenous nucleic acids to cells to activate, silence, modify, or edit certain genes and correct genetic defects that may contribute to disease progression. In this case, correcting abnormal gene expression, since several mutations have been associated with glioblastoma, seems a promising approach to treat it. However, delivering nucleic acids to cells is difficult unless delivery systems are used. Nanotechnology, with the use of non-viral vectors (cell-penetrating peptides (CPPs), liposomes, polymers, exosomes, dendrimers, etc.), leads to ground-breaking possibilities for a precise delivery of anticancer drugs and nucleic acids to specific sites, overcoming several physiological barriers, as the blood-brain barrier (BBB). Targeting tumour-specific receptors using ligands at the delivery systems surface can minimize side effects and improve therapeutic responses. Additionally, in the past few years, the study of cancer cells circadian rhythms, normally disrupted, has shown that the circadian clock as a significant role in cancer development and therapeutic efficacy. Circadian rhythm is defined as the approximately 24 h oscillation of physiological and metabolic processes, which are synchronized with the Earth's diurnal cycle. Several studies indicated that rhythms could modulate drug’s effectiveness and their side effects, and delivery systems cellular uptake and efficacy. Studies have shown that some chemotherapy drugs are better tolerated or more effective at certain times of the day. Accordingly, the main object of this doctoral thesis was to design and develop a delivery system, functionalized to target glioblastoma cells, and to evaluate the influence of circadian rhythms on the efficacy of targeting, internalization, cargo release, and, ultimately, therapeutic effect for a precise cancer therapy strategy. This approach focused on the use of a non-viral CPP, namely the WRAP5 peptide, for the construction of a delivery system bearing a transferrin (Tf) receptor ligand sequence (TfR) to co-deliver the anticancer TMZ drug and a plasmid DNA (pDNA) coding for p53 to glioblastoma cells. This system was designed to present an improved brain cell targeting ability and cellular uptake and to penetrate the brain barriers easily. The first step consisted of acquiring its physicochemical properties (size, polydisperse index, surface charge, and complexation capacity), morphology, and biocompatibility. Results revealed these properties to be under the influence of the N/P ratio, which can be optimized to improve complexes desired characteristics. These formulations demonstrated appropriate physicochemical characteristics for in vitro applications, and confocal microscopy using U87 glioblastoma cells confirmed their ability to internalize into cells and deliver the pDNA into the nucleus. Following nuclear localization, successful transcription and translation of the TP53 were observed. The resulting complexes significantly reduced the viability of glioblastoma cells. In a three-dimensional (3D) 9-day U87 spheroid model, generated by two different protocols, complexes were shown to have some effect on the spheroids morphology and size after a single dose treatment. Moreover, complexes have been revealed to be biocompatible with several non-cancerous cells and with zebrafish Danio rerio embryos. Experiments with other two glioma cell lines (SNB19 and U373) also highlighted the complexes ability for internalization and p53 expression levels increase. In these cell lines, apoptosis activation by the intrinsic pathway was implicated. This thesis also aimed to elucidate the influence of circadian core clock components in the performance of developed peptide nanocomplexes, namely, TfR expression, complexes internalization, and p53 expression promotion. The obtained results demonstrated that, at specific time-points, the highest circadian activity of Period circadian regulator 2 (PER2) and TfR led to higher cellular uptake of complexes, TP53 expression induction, and consequently p53 expression. In summary, our comprehensive dataset provides strong evidence supporting the high potential of TMZ/TP53 co-delivery WRAP5 complexes for targeted cellular transfection, p53 expression, and for effectively triggering apoptotic pathways, holding promising therapeutic value toward glioblastoma. Moreover, aligning the timing of complexes administration with the circadian rhythms of GB cells may significantly enhance cellular uptake and gene/protein expression. This chronobiologically optimized approach offers a promising possibility for developing more precise and impactful treatment strategies against glioblastoma. Future preclinical research should prioritize the study of delivery systems bioavailability, tumour targeting, and pharmacokinetics at specific times of day in patient-derived 3D models and in vivo xenograft models. Additionally, it will be relevant to study the temporal expression of glioblastoma biomarkers and the effects of their knockout on survival and therapeutic outcomes. Understanding how brain barrier dynamics, such as permeability, for example, oscillate with circadian changes and impact delivery systems uptake, will bring noticeable improvements to this strategy. Ultimately, this research should instigate complexes clinical translation toward more effective glioblastoma therapy.
- Cancer gene therapy: Design, development and in vitro evaluation of a plasmid DNA delivery systemPublication . Neves, Ana Raquel Bastos; Costa, Diana Rita Barata; Sousa, Ângela Maria Almeida deCancer is one of the major causes of morbidity and mortality worldwide. It involves genetic changes that affect a variety of genes, namely tumour suppressor genes. The traditional approaches for cancer treatment include chemotherapy, surgery and radiation. Chemotherapy represents the main choice of treatment in most cases, however, traditional therapies seemed unvalued to fight against metastasis, recurrence of tumour, and the treatment of advanced cancer. Therefore, new strategies need to be developed to increase therapy efficacy. Gene therapy has brought a promising and unique approach to medicine because of its wide application in the treatment of various diseases, including hereditary diseases to acquired (infection or cancer) pathologies. p53 suppressor gene mutation or degradation is found in more than 50% of human cancers, therefore, gene therapy protocols focussed on the restauration of p53 protein are a priority in this field. For gene therapy viability in a clinical setting, the development of an efficient gene delivery system is imperative. The conception of delivery systems based on cell penetrating peptides represents an incredible asset and may deeply contribute for the evolution of cancer therapy. In this context, a new system for p53 encoding plasmid DNA (pDNA) delivery based on RALA peptide was designed and developed in order to produce a suitable intracellular delivery platform capable of gene delivery and restoration of p53 levels within the cancer cells. These carriers were characterized in terms of morphology, size, surface charge, loading and encapsulation efficiency and the fine structure was analyzed by Fourier-transformed infrared (FTIR) spectroscopy. The results showed that formed nanoparticles are suitable for cell uptake, internalization and gene release. Furthermore, stability studies demonstrated that RALA is capable of protect encapsulated DNA from serum nucleases and MTT assay showed that these systems are biocompatible. Confocal microscopy and live cell imaging experiments confirmed intracellular localization of nanoparticles, resulting in enhanced sustained pDNA uptake. Moreover, in vitro transfection of HeLa cells mediated by RALA/pDNA vectors allowed the detection of mRNA transcripts by RT-PCR and p53 protein expression by Western Blot. An ELISA kit assay quantified the produced protein levels. From these progresses, apoptosis in cancer cells was investigated. Caspase 3 activation was monitored by means of colorimetric assay and TUNEL assay enabled to confirm nuclear DNA fragmentation post transfection with the carriers. Lastly, a western blot assay with BAX antibody permitted to figure out which apoptosis pathway is responsible for cancer cells death. Taken together, the presented results revealed that the RALA/pDNA vector seems to be suitable as an innovative platform for p53 mediated cancer gene therapy.