The depth and versatility of siRNA technologies enable their use in disease targets that are undruggable by small molecules or that seek to achieve a refined turn-off of the genes for any therapeutic area. Major extracellular barriers are enzymatic degradation of siRNAs by serum endonucleases and RNAases, renal clearance of the siRNA delivery system, the impermeability of biological membranes for siRNA, activation of the immune system, plasma protein sequestration, and capillary endothelium crossing. To overcome the intrinsic difficulties of the use of siRNA molecules, therapeutic applications require nanometric delivery carriers aiming to protect double-strands and deliver molecules to target cells. This review discusses the history of siRNAs, siRNA design, and delivery strategies, with a focus on progress made regarding siRNA molecules in clinical trials and how siRNA has become a valuable asset for biopharmaceutical companies.
Infections caused by SARS-CoV-2 induce a severe acute respiratory syndrome called COVID-19 and have led to more than six million deaths worldwide. Vaccination is the most effective preventative measure, and cellular and humoral immunity is crucial to developing individual protection. Here, we aim to investigate hybrid immunity against SARS-CoV-2 triggered by the ChAadOx1 nCoV-19 vaccine in a Brazilian cohort. We investigated the immune response from ChAadOx1 nCoV-19 vaccination in naïve (noCOVID-19) and previously infected individuals (COVID-19) by analyzing levels of D-dimers, total IgG, neutralizing antibodies (Nabs), IFN-γ (interferon-γ) secretion, and immunophenotyping of memory lymphocytes. No significant differences in D-dimer levels were observed 7 or 15 days after vaccination (DAV). All vaccinated individuals presented higher levels of total IgG or Nabs with a positive correlation (R = 0.88). Individuals in the COVID-19 group showed higher levels of antibody and memory B cells, with a faster antibody response starting at 7 DAV compared to noCOVID-19 at 15 DAV. Further, ChAadOx1 nCoV-19 vaccination led to enhanced IFN-γ production (15 DAV) and an increase in activated T CD4+ naïve cells in noCOVID-19 individuals in contrast with COVID-19 individuals. Hence, our data support that hybrid immunity triggered by ChAadOx1 nCoV-19 vaccination is associated with enhanced humoral response, together with a balanced cellular response.
Introduction: Cancer is the second leading cause of death from diseases in the world. Only in 2018, this disease was responsible for about 9.6 million deaths, where breast cancer is the tumor type with the highest incidence and mortality in the female population worldwide. Taking into account the adverse effects of current treatments, in addition to low efficiency and specificity, it is evident the need to develop new therapies aimed at greater specificity and less side effects.Objective: This work aims to develop cationic liposomes that carry interference RNA (siRNA) molecules for silencing target genes in breast tumors. Methodology:The selection of targets was previously outlined by our research group based on data from the interactome and transcriptome of seven tumor cell lines and one non-tumor cell line. The development of the liposome was carried out by forming a lipid film of phospholipid and cholesterol followed by hydration of the it and subsequent extrusion. The particle physicochemical characterization was evaluated by dynamic light scattering (DLS) and zeta potential; and their biological effects were assessed. First, we evaluated the effect of delivery formulations (empty cationic liposome, in the presence or absence of Polyethylene glycol -PEG) on cell viability in MCF-7 tumor cell line (human breast adenocarcinoma, Luminal A), MDA-MB-231 (human breast adenocarcinomas, triple negative) and HEP-G2 (human hepatocarcinoma). Flow cytometry methodology was used to evaluate gene silencing, where cells were treated with siRNA-carrying liposomes in different concentrations for 48 hours. Results:We performed bioassays at different times and concentrations, that shows greater cell viability in the presence of liposomes with PEG when compared in the absence of it, however, both demonstrated viability greater than 50%. Then, we proceed with the evaluation of protein silencing in flow cytometry after treatment with siRNA carrier liposomes in the presence or absence of PEG, in different concentrations for 48 hours. The silencing evaluation showed a decrease in the expression of two target proteins in cells treated with liposomes containing RNAi for these two targets. Conclusion:The liposomes were successfully obtained. The evaluation of the silencing of two target proteins revealed a decrease in their expression in relation to the control, indicating the efficiency of siRNA delivery through liposome system. Thus, our results indicate a promising profile of the target genes under study for the development of an innovative therapy in the treatment of breast cancer.
Introduction: Gold nanoparticles (AuNPs) are often used as biosensors in biological markers and also in diagnostic kits. Spherical shaped AuNPs are red. These nanoparticles have high binding affinity with proteins, antibodies and antigens forming stable bioconjugates. Are widely used in lateral flow immunochromatography platform. Therefore, AuNPs are currently one of the main raw materials used to produce various diagnostic kits, including Sars-CoV-2. The assessment of their correct stability directly affects the customized production and quality of the diagnostics kits.Objectives: Evaluate the stability of in-house prepared gold nanoparticle solutions used in the manufacture of diagnostic tests using statistical methods for determining the appropriate shelf life under established storage conditions. Methodology:In-house based on adapted Turkevish method (1951), a gold nanoparticle (AuNP) solution was synthesized and characterized by ultraviolet-visible spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), dynamic scattering (DLS) and laser Doppler electrophoresis (LDE). The solution was analyzed at the time of manufacture (T0) and every 15 days for 90 days, under thermal stress conditions. The evaluation of the stability of the AuNP solution was based on the ISO Guide 35, which statistically evaluates the time that significant change occurs of the evaluation parameters, indicating the end of shelf life at a significance level of 0.05. Results:Statistical analysis shows that at T90 days, one of the evaluated parameters showed a significant change at a significance level of 0.05. |b1| > t95%, n-2 * s(b1) and, therefore, there is statistical evidence that proves that the final solution lost stability in this time. Arrhenius equation was used to determine the shelf life. Where, Storage=25°C, Stress=40°C, Activation Energy (Ea)=3, and then: Thermal Kinetic Ratio (Qt) = 5.2. That is, 2.5 months at 40°C is equivalent to 13 months at 25°C. Conclusion:AuNPs produced in-house have a shelf life of 1 month at room temperature. Based on the analysis of the main control parameters and statistical application, the validity attributable to the AuNP solution under study is 13 months at 25°C, bringing great savings to the production process, without loss of quality. New strategies for evaluating the stability of solutions should be considered in the future.
This work aims to evaluate the biosimilar and biobetters of rituximab developed in Bio-Manguinhos regarding their ability to bind to the CD20 antigen and the in vitro biological activity. Methodology: Through flow cytometry assays it was possible to analyze the binding properties of the constructs to the CD20 antigen on the surface of leukemia cells (K562 CD20+) and the presence of NKG2DL in the constructs. Potential ADCC and CDC were evaluated using the CytoTox96 kit, a non-radioactive cytotoxicity colorimetric assay capable of measuring the lactate dehydrogenase in the medium, and NK cells expanded in vitro as effector cells. Results: Flow cytometry assays have demonstrated that the constructs produced in Bio-Manguinhos are capable of binding to CD20+ cells and that NKG2DLs are present in the biobetters constructs. ADCC and CDC assays demonstrated that the presence of NKG2DLs in the constructs improved the in vitro biological activity of the mAb, evidencing a higher percentage of cell lysis of the biobetter when compared to the biosimilar and rituximab (MabThera) antibodies. Conclusion: The conclusion is that the antibodies developed are capable of binding to CD20 expressing cells and the biobetters presented improved biological activity in vitro when compared to the biosimilar and reference rituximab, suggesting that the addition of the NK-cell ligand to the anti-CD20 mAb may enhance the therapeutic efficacy of rituximab and other therapeutic antibodies. To reinforce this hypothesis, we have established an in vivo model consisting in xenografts of CD20+ the human lymphoma cell line (RAJI) in immunodeficient mice. The immunodeficient animals will be treated with primary human NK cells and/or the mAbs of interest to evaluate the in vivo enhancing function of the new conformations of the anti CD20 mAbs.
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