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Nucleic acid based nanosystem for Human Papillomavirus related lesions

Publication . Nunes, Jéssica Lopes; Cruz, Carla Patrícia Alves Freire Madeira; Oliveira, Paula Alexandra Martins de

Human Papillomaviruses (HPVs) are among the most common sexually transmitted infectious agents. The infectious virus particles (virions) invade and replicate in dividing cells, hijack their host cellular process, and new virions are released in terminally differentiated cells, in the uppermost layers of the epithelium. The immune system usually clears the virus, but on some occasions the infection remains and can induce proliferation alterations, leading to the development of warts. Although, high-risk HPVs, namely HPV16 and HPV18, can also cause malignant transformations, being considered etiological agents of cervical or head and neck cancers. Currently, there are HPV screening and vaccination programs that contributed for a reduction in the infections prevalence by up to 85% for the genotypes covered by the vaccines, and a 50% reduction in the number of high grade lesions related to this virus, particularly in countries with high vaccination rates (≥50%). Although, they are still limited in some countries, as well as in men, which together with the prophylactic nature contribute for HPV remaining a worldwide health issue. Adding to this, the available therapeutic approaches have limited efficacy and cause several side effects, with a great impact on patients’ quality of life. Therefore, there is still investigation for the development of new treatment options for HPV-related cancers. In the pursuit of new therapeutic options, nanoparticles are being proposed as efficient delivery method. Many of them take advantage of passive targeting, which facilitates nanoparticles deposition in tumor cells, owing to some characteristics of the tumor milieu, such as the enhanced permeability of the vasculature. Although, this permeability varies between individuals, and internalization by other organs can occur, reducing the therapeutic efficacy and inducing nonspecific effects. Thus, targeting strategies are being employed to enhance the nanoparticles selectivity towards cancer cells, reducing potential off-target effects. To achieve this, biorecognition molecules are attached to the nanoparticles surface. For instance, nucleic acids can be used, such as the internalizing RNA previously developed by Magalhães et al., 2012, that shown promising results as a strategy for the development and enhancement of novel RNA-based transfection methods. On the other hand, DNA aptamers are also a promising approach due to their high selectivity towards a specific target that can be a protein or a whole cell. Particularly, G-quadruplex aptamers are emerging, due to their increased stability and higher negative charge density comparingly to duplex DNA, favoring the interactions with cationic proteins. The most well-known and used example is AS1411 with nucleolin as target, a protein that is overexpressed in cancer cells surface. To achieve better structural or antiproliferative results, derivatives of AS1411 were more recently proposed. Thus, in this thesis, new nanotherapeutic approaches for HPV-related cancers were investigated and functionalization with different targeting moieties (AS1411 and derivatives or an internalizing RNA) was tested. The nanosystems were developed for a more selective delivery of different small molecules, including the drugs imiquimod or dexamethasone and the potential therapeutic molecule C8 (10-(8-(4-iodobenzamide) octyl)-3,6-bis (dimethylamine) acridinium iodide). Firstly, the targeting capacity of AS1411 derivatives (namely AT11, AT11-Bo and AT11-L0) was investigated and these sequences were used for a more selective delivery of Zn(II) phthalocyanine (ZnPc) derivatives. The supramolecular aggregates composed by the aptamers and ZnPc derivatives were more cytotoxic in HPV cancer cells (HeLa cells), relatively to the non-malignant NHDF cells (<40% versus ~80%). These findings support the development of new strategies using these sequences for an improved anticancer effect of the therapeutic molecules. Then, different nanosystems were tested in HPV-integrated cervical, namely HeLa (HPV18+), CaSki (HPV16+ and HPV18+) and SiHa (HPV16+), or head and neck UPCI-SCC-154 (HPV16+) cancer cell lines and, in the final part of the work, in HPV16 transgenic mice. At first, gold nanoparticles functionalized with AS1411, with a known improved cancer cells uptake, were tested as a potential drug delivery system of imiquimod or C8. The obtained drug delivery systems were produced, characterized, evaluated and incorporated in a gel formulation, prepared based on the universal placebo formulation. As proposed, the AS1411-functionalized gold nanoparticles were indeed capable of improving the selectivity (decreasing the therapeutic molecules cytotoxicity in the non-malignant cells, NHDF cells; cell viabilities >60%) and to potentiate their anticancer effect, especially in HeLa cells (cell viabilities <30%). Additionally, when in a formulation, the C8 ligand was able to permeate and be retained in ex vivo porcine vaginal tissues. To achieve higher biocompatibility, the work was further moved to lipid nanoparticles, aiming to improve the potential clinical applicability of the produced drug delivery systems. First, liposomes produced by the thin film hydration method were obtained and functionalized with a promising internalizing RNA. The RNA was firstly biophysically characterized to analyze the effect of intermolecular interactions with C8 or dexamethasone, in which it was observed that these molecules do not induce alterations in its structure and thermal stability. Then, the produced liposomes were also characterized and biologically evaluated. Overall, the liposomes improved the selectivity of the used therapeutic molecules towards the tongue cancer cell line, UPCI-SCC-154 (~13/37% versus >85%). Although, this production method resulted in low amounts of liposomal suspension (<1 mL of suspension at <1 mg/mL of lipids, per batch). For this reason, we changed to the ethanol injection method, which resulted in larger volumes of liposomal suspension (~10 mL of suspension at 3.56 mg/mL of lipids) and faster (from ~3 days to ~1 h up to the obtention of the functionalized liposomes), being more compatible with a bulk manufacturing process. Thus, liposomes were produced and functionalized by a post-insertion method with AT11 and were used to deliver imiquimod or C8. The obtained liposomes shown to be cancer-selective and with an effective anticancer effect in the UPCI-SCC-154 cell line, reducing the viability, proliferation, migration, invasion and inducing cancer cells death. The AT11-functionalized liposomes with C8 were then incorporated in gel formulations, prepared based on the universal placebo, with or without essential oils (Thymus vulgaris or Origanum vulgare). The formulations presented suitable features for application (including high viscosities, 968-1318 mPa.s, and osmolality <1200 mOsmol/kg), the essential oils potentiate the anticancer effect in cervical cancer cell lines (HeLa, SiHa and CaSki) and after application in healthy or HPV16 transgenic mice, it was confirmed that the produced formulation was safe and with potential therapeutic effect in HPV-associated lesions. Overall, different nanotherapeutic strategies were proposed to enhance drug delivery towards HPV-related cervical and head and neck cancers, which highlight the great potential of nanomedicine in cancer treatment. While there is still considerable progress to be made, this work provides valuable insights into potential new targeting moieties and therapeutic molecules.

2025-01-23Doctoral thesis

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