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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.

The synthesis of starch-based physical hydrogels in combination with chitosan and polyvinyl alcohol, and their potential co-application with chitosan nanoparticles was evaluated. The potential of starch-chitosan hydrogel obtained by physical/chemical method for tissue engineering uses was also studied in a mouse wound healing model. Although the microscopical structure of each synthesized hydrogel suggests a possible biological application, starch-polyvinyl alcohol hydrogel exhibited rigid behavior with minor channel diameters, a lower swelling rate (less than 300%), and negatively affected cell viability in a cytotoxicity assay. Starch-chitosan hydrogel obtained by chemical crosslinking with glutaraldehyde demonstrated the higher swelling rate (about 1100%), cell viability values over 80%, and a homogeneous tri-dimensional structure; along with an excellent interaction with chitosan nanoparticles. This type of hydrogel was selected for an in vivo experiment, showing significant differences in wound healing process against a non-treated control, in terms of inflammation, exudate production and tissue recovering.

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.

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 Hbm detection in Raman spectra of oxygenated erythrocytes (oxyRBCs). Raman spectra of oxyRBCs incubated with AgNPs showed enhanced deoxyHb signals with good spectral reproducibility, supporting the Hbm detection 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 both z-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.

Detonation nanodiamonds (DNDs) are usually small particles of 4–5 nm, but in aqueous suspension, DNDs form agglomerates in sizes larger than 1 μm. We propose the use of Bead Assisted Sonic Disintegration and a carboxylation procedure, to reduce DNDs aggregates sizes to approximately 100 nm. High cost zirconium beads have been substituted by silica beads synthetized in our laboratory and less-time consuming conditions were standardized. Techniques as Dynamic Light Scattering (DLS), Fourier Transform InfraRed Spectroscopy (FTIR), Transmission Electron Microscopy (TEM) and X-ray Photoelectron Spectroscopy (XPS), have been used to characterize the resulting diamond nanoparticles. While the incubation of Red Blood Cells with partially disaggregated DNDs was used to study whether these nanodiamonds impact in a living system. Our results show the absence of a negative effect in cell viability as well as no differences between Raman spectra of hemoglobin (Hb), from control and cell + DNDs conditions.

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.

Incubated erythrocytes with and without silver nanoparticles (AgNP) were analyzed by Raman spectroscopy, resulting in two Raman spectra datasets. AgNP were added to red blood cells (RBC) in order to enhance the Raman signals. This technique is known as surface-enhanced Raman scattering (SERS). A comparison was made between the Raman spectra with and without AgNP, to test if the SERS had taken place. Since Raman and SERS spectra are considered to be cumbersome due to the noises presented, we applied denoising criteria for detection and removal of noises like cosmic rays, shot, and fluorescence contribution. After this, the principal component analysis (PCA) was performed, in order to reduce the dimensions of the spectra being studied. Only the main key components necessary for a better interpretation of these spectra were considered. All of those noises had to be removed prior to the statistical analysis, to make sure the analysis was really based on the Raman measurements and not on other effects. As a result, RBC Raman spectra with and without AgNP got denoised, obtaining an improvement in its resolution for a better signal reading and data interpretation. Also, the first principal components (PC) were selected from each dataset under scrutiny, based on the weight of their information and their spectrum readability. In conclusion, we were able to represent the given reference system with a more affordable and smaller dimension in which information loss was minimal.

Polyethylene glycol (PEG) molecules act as a reducing and stabilizing agent in the formation of silver nanoparticles. PEG undergoes thermal oxidative degradation at temperatures over 70 °C in the presence of oxygen. Here, we studied how the temperature and an oxidizing atmosphere could affect the synthesis of silver nanoparticles with PEG. We tested different AgNO3 concentrations for nanoparticles syntheses using PEG of low molecular weight, at 60 and 100 °C. At the higher temperature, the reducing action of PEG increased and the effect of PEG/Ag+ ratio on nanoparticles aggregation changed. These results suggest that different synthesis mechanisms operate at 60 and 100 °C. Thus, at 60 °C the reduction of silver ions can occur through the oxidation of the hydroxyl groups of PEG, as has been previously reported. We propose that the thermal oxidative degradation of PEG at 100 °C increases the number of both, functional groups and molecules that can reduce silver ions and stabilize silver nanoparticles. This degradation process could explain the enhancement of PEG reducing action observed by other authors when they increase the reaction temperature or use a PEG of higher molecular weight

The use of nanodiamonds in studies with living systems generally involves the modification of their surfaces with functional groups. Fluorescent molecules can be attached to these groups, so that one can know the exact position of the particles in each moment of the interaction with the cells. Here we modify the surface of detonation nanodiamonds and nitrogen-vacancy center nanodiamonds using carboxylation and hydroxylation procedures. Subsequent reactions with silicates and cysteine, before addition of fluorescein allow to obtain fluorescent nano-conjugates. We used confocal microscopy to observe the position of nanodiamonds interacting with HeLa cells. At 3 h post-incubation the green fluorescence is localized in extracellular rounded like-vesicles assemblies while at 24 h the conjugates can be observed inside the cells. The measurement of the fluorescence emitted by both conjugates allowed to find an enhanced emission of fluorescein isothiocyanate (FITC) when the nitrogen-vacancy center is present. We propose the existence of a fluorescence enhancement by electron transference process. The procedure described in this work allows the functionalization of nanodiamonds with FITC and other molecules using functional surface groups and small size mediators. Also, as was proved in our work, the nanodiamond-fluorescein conjugates can be used to track nanoparticles position within the cell. Localization studies are particularly important for drug delivery applications of nanodiamonds.