RESUMEN
Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-ß such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Bleomicina/farmacología , Daño del ADN/efectos de los fármacos , Pulmón/efectos de los fármacos , Nanopartículas/uso terapéutico , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Humanos , Pulmón/metabolismo , Estrés Oxidativo/efectos de los fármacos , Péptidos/farmacologíaRESUMEN
In this work a high performance liquid chromatographic (HPLC) method has been developed and validated for the content determination of GSE4 peptide in PEI-PLGA nanoparticles. Chromatographic separation was performed on a C18 column, and a gradient elution with a mobile phase composed of methanol and 0.1% aqueous trifluoroacetic acid (TFA) solution, at a flow rate of 1ml/min, was used. GSE4 peptide identification was made by fluorescence detection at 290nm. The elution of methanol:TFA was initially maintained at (20:80, v/v) for one min and the gradient changed to (80:20, v/v) in 6min. This ratio was then followed by isocratic elution at (80:20, v/v) during another min and for further 3min it was linearly modified to (20:80, v/v). The developed method was validated according to the ICH guidelines, being specific, linear in the range 10-100µg/ml (R(2)=0.9996), precise, exhibiting good inter-day and intra-day precision reflected by the relative standard deviation values (less than 3.88%), accurate, with a recovery rate of 100.18±0.95%, and stable for 48h at 5°C or at RT when encapsulated in nanoparticles. The method was simple, fast, and successfully used to determine the peptide content in GSE4-loaded PEI-PLGA nanoparticles.