Encapsulação de R-ficoeritrina em nanopartícula polimérica de policaprolactona para estabilização e aplicação biológica como biomarcador em células cancerígenas

Abstract

R-phycoerythrin (R-PE) is a soluble protein belonging to the phycobiliprotein class, a multimeric fluorescent protein (240kDa) whose fluorescence occurs by the presence of disc-like subunits containing up to 10 chromophores. The instability of the R-PE is one of the factors that make its application difficult, requiring the use of techniques that increase its stability. This protein can be extracted from red algae species of the rhodophyte family. Solieria filiformis is a species of red macroalgae found along the Brazilian coast. In this study we report the extraction and purification of R-PE from Solieria filiformis as well as its encapsulation in polycaprolactone (PCL) nanoparticles (NP) (R-PE / PCL nanoparticles) and its in vitro application. The R-PE was obtained from the algae Solieria filiformis (SISGEN A41C95F) collected on the beach of Flecheiras-CE. In the extraction process, mechanical rupture was used, followed by precipitation of the proteins with 90% ammonium sulfate, dialyzed and purified by ion exchange chromatography using an anionic resin, resulting in a purity index (PI) of 4.3 and a yield of 2.57 mg/g of wet seaweed. Intending to increase the stability of R-PE and enable the application of its flowering, formulations of polymeric nanoparticles were prepared by the double emulsification method and solvent evaporation using polycaprolactone. Nanoparticles containing 60 mg of PCL, 2mL of poly(polyvinyl alcohol) (PVA) 1% and 2 mg R-PE presented nanometric particle size with low polydispersity and encapsulation efficiency equivalent to 50.0 ± 7.3%. Fourier transform FITR infrared spectroscopy analyzes did not reveal any spectral difference between NP and R-PE / PCL, indicating that R-PE is molecularly dispersed in the polymer matrix. Differential scanning calorimetry (DSC) shows that R-PE has two peaks, one at 76.34°C and a second at 327°C. For R-PE/PCL nanoparticles, the widened and shifted peak at 307°C indicates the presence of the protein in the nanoparticle. The structural composition of R-PE and R-PE/PCL were analyzed by circular dichroism (DC) spectrum and it was possible to identify the presence of beta sheet and alpha helix. Characterization by scanning electron microscopy showed homogeneous nanoparticles, with no change in particle morphology after R-PE loading and indicated particle sizes according to dynamic light scattering results. Furthermore, in vitro, degradation to nonencapsulated R-PE was demonstrated, proving the increased stability of the protein once encapsulated. Finally, confocal microscopy and in vitro flow cytometry studies demonstrated the potential of R-PE/PCL nanoparticles for fluorescent labeling in PC-3 prostate cancer and 4T1 breast cancer cells, with fluorescence intensity increasing over time. until 6 am. These results support the potential of R-PE/PCL nanoparticles as an innovative tool for biological application in cancer detection.