Browsing by Author "Costa, Matilde Bogalheiro"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- Plasmid DNA-loaded calcium phosphate functional nanocomplexes: innovative approach against cervical cancerPublication . Costa, Matilde Bogalheiro; Sousa, Ângela Maria Almeida de; Valente, Joana Filipa Abreu Pereira; Patrício, Tatiana Marisa FernandesCancer is among the leading causes of death worldwide. By 2040 it is expected that the number of new cancer cases will rise to 29.5 million and the number of cervical cancerrelated deaths to 16.4 million. Generally, cancer incidence rate increases in countries with highest life expectancy, education level and standard of living. However, for cervical cancer the reverse happens, its incidence is higher in less developed countries, due to the lack of proper awareness, screening programs, inaccessibility to proper diagnosis and efficient treatment procedures, these facts lead to a late diagnosis and consequently low survival rates. Human Papillomavirus (HPV), a highly sexually transmissible virus, is the main agent in cervical cancer. Around 79 % of the diagnosed causes are attributed to HPV-16 and -18. The severity of symptoms is usually associated with the affinity presented by oncoproteins E6 and E7 within each HPV type toward the target protein, p53, a tumour suppressor, and pRb, a retinoblastoma protein, respectively. Thus, the deregulation of these proteins’ expression leads to invasion, proliferation, and survival of cancer cells. A plethora of strategies are being developed to detect and prevent the disease, with antiHPV vaccines being at the front. However, in less developed countries, with poor health resources, the preventative measures do not always work, and new therapies are being developed to treat this type of cancer. Gene therapy has been increasingly used since it allows the correction of a genetic effect while also facilitating for an induced cell death. Thus, plasmid DNA that encodes the p53 gene can be a promising approach to combat cervical cancer. However, pDNA use is limited since it is easily degraded by various agents, so a delivery system is needed to carry pDNA to the target cells. Calcium phosphate nanoparticle are promising delivery systems since they are composed of ions that naturally exist in the human body, and its degradation does not entail negative consequences for the patient. In this context, the present work’s focus is the development of plasmid DNA-loaded calcium phosphate nanoparticles and further functionalization with folic acid for a targeted delivery to HPV positive cancer cells. Initially, the delivery system formulation was optimized resulting in delivery systems presenting a size of 76.34 ± 34.08 nm, with a polydispersity index (PdI) of 0.40 ± 0.09 and a surface charge of – 20.90 ± 0.90 mV. pDNA inclusion was performed through an adsorption approach yielding a size of 133.13 ± 58.12 nm, PdI of 0.65 ± 0.17 and a surface charge of – 23.70 ± 1.10 mV with an encapsulation efficiency of approximately 100 %. Folic acid loading was investigated, and two methods were chosen, firstly when folic acid is added in the precursor calcium solution (CaP-NP/FACa) and also when folic acid is added through adsorption (CaP-NP/FAAd), which exhibited a folic acid loading efficiency of approximately 92 and 70 %, respectively. The CaP-NP/pDNA/FAAd achieved an efficiency of approximately 100 % for pDNA encapsulation and approximately 99.6 % for folic acid loading, with a size of 112.63 ± 19.81 nm, PdI of 0.50 ± 0.22 and a surface charge of – 27.40 ± 0.69 mV. The CaP-NP/pDNA/FACa presented an encapsulation efficiency of pDNA in the nanoparticles of 100 % and an efficiency of approximately 73 % for the loading of folic acid, with a size of 110.42 ± 65.71 nm, PdI of 0.50 ± 0.07 and a charge of – 22.20 ± 0.60 mV. Overall, the in vitro assays showed no inherent cytotoxicity for the CaP-NPs, with viability reduction only in higher concentrations (500 and 1000 µg/mL). The delivery systems did not affect the healthy cells viability but decreased the viability in the cancer cell line. Cell internalization was evaluated through confocal microscopy, which showed significant uptake in both cell lines. The formulations presenting pDNA and folic acid, in the calcium solution (CaP-NP/pDNA/FACa) and adsorbed after the nanoparticle formulation (CaP-NP/pDNA/FAAd), presented high accumulation in cancer cells. PCR evaluation seems to present increased expression values of p53 when the cancer cell line was transfected with both CaP-NP/pDNA and CaP-NP/pDNA/FACa. The present work demonstrated that CaP-NPs, loaded with pDNA and modified with folic acid, show a therapeutic effect, and represents a promising treatment for cervical cancer.