Raman spectroscopy and silver nanoparticles for efficient detection of membrane proteins in living cells.

Molecular fingerprints revealed by Raman techniques show great potential for biomedical applications, like disease diagnostic through Raman detection of tumor markers and other molecules in the cell membrane. However, SERS substrates used in membrane molecule studies produce enhanced Raman spectra of high variability and challenging band assignments that limit their application. In this work, these drawbacks are addressed to detect membrane-associated hemoglobin (Hbm) in human erythrocytes through Raman spectroscopy. These cells are incubated with silver nanoparticles (AgNPs) in PBS before Raman measurements. Our results showed that AgNPs form large aggregates in PBS that adhered to the erythrocyte membrane, which enhances Raman scattering by molecules around the membrane, like Hbm. Also, deoxyHb markers may allow Hbmdetection in Raman spectra of oxygenated erythrocytes (oxyRBCs). Raman spectra of oxyRBCs incubated with AgNPs showed enhanced deoxyHb signals with good spectral reproducibility, supporting the Hbmdetection through deoxyHb markers. Instead, Raman spectra of oxyRBCs showed oxyHb bands associated with free cytoplasmic hemoglobin. Other factors influencing Raman detection of membrane proteins are discussed, like bothz-position and dimension of the sample volume. The results encourage membrane protein studies in living cells using Raman spectroscopy, leading to the characterization and diagnostic of different pathologies through a non-invasive technique.

Nanodiamonds and gold nanoparticles to obtain a hybrid nanostructure with potential applications in biomedicine.

Using detonation nanodiamonds and fluorescent nitrogen-vacancy center nanodiamonds, linked to gold nanoparticles, we synthesized two hybrid nanostructures (HGDs) that were subsequently conjugated with a fluorophore. An amplification effect induced by the gold nanoparticles increased the emission spectrum of the fluorophore, maximizing the possibilities for imaging applications of these HGDs. The incubation of the nanostructures with HeLa cells produced no alteration of cell viability after 3 h and showed the presence of nanostructures in the cell cytoplasm at 24 h. These observations also indicate the potential biomedical use of the proposed HGDs.

Long-term release of bioactive interferon-alpha from PLGA-chitosan microparticles: in vitro and in vivo studies.

Effective cytokine treatments often require high- and multiple-dose due to the short half-life of these molecules. Here, porcine interferon-alpha (IFNα) is encapsulated in PLGA-chitosan microparticles (IFNα-MPs) to accomplish both slow drug release and drug protection from degradation. A procedure that combines emulsion and spray-drying techniques yielded almost spherical microspheres with an average diameter of 3.00 ± 1.50 µm. SEM, Microtrac, and Z-potential analyses of three IFNα-MP batches showed similar results, indicating the process is reproducible. These studies supported molecular evidence obtained in FTIR analysis, which indicated a compact structure of IFNα-MPs. Consistently, IFNα release kinetics assessed in vitro followed a zero-order behavior typical of sustained release from a polymeric matrix. This study showed that IFNα-MPs released bioactive molecules for at least 15 days, achieving IFNα protection. In addition, pigs treated with IFNα-MPs exhibited overexpression of IFNα-stimulated genes 16 days after treatment. Instead, the expression levels of these genes decreased after day 4th in pigs treated with non-encapsulated IFNα. In vitro and in vivo experiments demonstrated that the formulation improved the prophylactic and therapeutic potential of IFNα, accomplishing molecule protection and long-term release for at least two weeks. The procedure used to obtain IFNα-MPs is reproducible, scalable, and suitable for encapsulating other drugs.

Polymeric nanoencapsulation of alpha interferon increases drug bioavailability and induces a sustained antiviral response in vivo.

Polymeric nanoparticulate systems allow the encapsulation of bio-active substances, giving them protection against external agents and increasing the drug's bioavailability. The use of biocompatible and biodegradable polymers usually guarantees the harmless character of the formulation, and a controlled drug release is also assured. A relatively easy procedure to obtain polymeric formulations of bioactive agents is ionotropic gelation, which allows the synthesis of chitosan (CS) - sodium tri-polyphosphate nanoparticles (NPs) loading encapsulated proteins. In this work, Bovine serum albumin (BSA) model protein and a recombinant porcine alpha interferon variant were used to obtain nanoparticulate formulations. The internalization of the encapsulated material by cells was studied using a BSA-fluorescein system; the fluorescent conjugate was observable inside the cells after 20 h of incubation. The therapeutic CS-alpha interferon formulation showed a maximum of protein released in vitro at around 90 h. This system was found to be safe in a cytotoxicity assay, while biological activity experiments in vitro showed antiviral protection of cells in the presence of encapsulated porcine alpha interferon. In vivo experiments in pigs revealed a significant and sustained antiviral response through overexpression of the antiviral markers OAS2 and PKR. This proves the preservation of porcine alpha interferon biological activity, and also that a lasting response was obtained. This procedure is an effective and safe method to formulate drugs in nanoparticulate systems, representing a significant contribution to the search for more effective drug delivery strategies.