Silica-coated magnetic particles for efficient RNA extraction for SARS-CoV-2 detection.

Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.

Rheological, Physical, and Spectroscopical Characterization of Gamma-Irradiated Albumin Nanoparticles Loaded with Anthocyanin.

Anthocyanins are the main active compounds in blueberry. However, they have poor oxidation stability. If anthocyanins are encapsulated in protein nanoparticles, their oxidation resistance could be increased as a result of the slowing down of the oxidation process. This work describes the advantages of using a γ-irradiated bovine serum albumin nanoparticle bound to anthocyanins. The interaction was characterized biophysically, mainly by rheology. By computational calculation and simulation based on model nanoparticles, we estimated the number of molecules forming the albumin nanoparticles, which allowed us to infer the ratio of anthocyanin/nanoparticles. Measurements by UV-vis spectroscopy, FTIR spectroscopy, fluorescence spectroscopy, dynamic light scattering (DLS), ζ potential, electron transmission microscopy, and rheology at room (25 °C) and physiological (37 °C) temperatures were performed. The spectroscopy measurements allowed identifying additional hydrophobic sites created during the irradiation process of the nanoparticle. On the basis of the rheological studies, it was observed that the BSA-NP trend is a Newtonian flow behavior type for all the temperatures selected, and there is a direct correlation between dynamic viscosity and temperature values. Furthermore, when anthocyanins are added, the system increases its resistance to the flow as reflected in the morphological changes observed by TEM, thus confirming the relationship between viscosity values and aggregate formation.

BSA-capped gold nanoclusters as potential theragnostic for skin diseases: Photoactivation, skin penetration, in vitro, and in vivo toxicity.

BSA-capped gold nanoclusters are promising theragnostic systems that can be excited to render both fluorescence emission and reactive oxygen species. Although their synthesis and photoluminescence properties are already well described, more accurate information about their use as photosensitizers is required in order to advance towards health applications. In this work, we have obtained BSA-capped gold nanoclusters and characterized their photophysics by different techniques. Singlet oxygen production was detected upon irradiation, which was enough to produce toxicity on two cell lines. Remarkably, an internal energy transfer, probably due to the presence of smaller nanoclusters and the contribution of oxidized residues of BSA in the system, caused fluorescence emission near 640 nm after excitation in the UV range. Additionally, the system was capable of penetrating human skin beyond the stratum corneum, which enhances the potential of these nanoclusters as bifunctional photodynamic therapy effectors and biomarkers with application in a diversity of skin diseases. In the absence of radiation, BSA-capped gold nanoclusters did not cause toxicity in vitro, while their toxic effect on an in vivo model as zebrafish was determined.

Comparative skin penetration profiles of formulations including ultradeformable liposomes as potential nanocosmeceutical carriers.

BACKGROUND: Ultradeformable liposomes are promising carriers for cosmeceutical actives as they can be loaded with molecules of different polarities, and they present unique penetration properties. AIMS: While those features have already been tested, we wanted to know whether their special penetration properties could be maintained after incorporation in diverse cosmetic vehicles, including commercial products already in the market. METHODS: Ultradeformable liposomes loaded with a lipophilic and a hydrophilic fluorescent probe were prepared by lipid film resuspension, followed by extrusion and incorporation to different vehicles and commercial products. Penetration was determined in human and pig skin by incubation, with the Saarbrücken penetration model, followed by the recovery of the probes or by fluorescence microscopy. RESULTS: The incorporation of ultradeformable liposomes to cosmetic vehicles did not alter their penetration in most of the cases for human skin explants. Pig skin penetration presented significant differences compared with human explants. CONCLUSIONS: Ultradeformable liposomes could be useful as versatile cosmeceutical carriers in final product formulations.

Comparative toxicity of PEG and folate-derived blue-emitting silicon nanoparticles: in vitro and in vivo studies.

AIM: Amino functionalization is a first step modification aiming to achieve biomedical applications of silicon nanoparticles, for example, for photodynamic therapy or radiotherapy. Nevertheless, toxicity and low quantum yields due to the positive charge of amino groups emerge as a problem that could be solved with subsequent derivatizations. MATERIALS & METHODS: Folic and PEG-conjugated nanoparticles were obtained from amino-functionalized silicon nanoparticle (NH2SiNP). Cytotoxicity was determined on a tumor cell line at low and high concentrations. Four end points of in vivo toxicity were evaluated on zebrafish (Danio rerio). RESULTS: Folic acid functionalization reduced the cytotoxicity in comparison to amino and PEG-functionalized nanoparticles. In zebrafish, folic functionalization lowered toxicity in general while PEG increased it. CONCLUSION: Functionalization of NH2SiNP with folic acid reduced the toxic effects in vitro and in vivo. This could be useful for therapeutic applications. PEG functionalization did not lower the toxicity.

Toward biomedical application of amino-functionalized silicon nanoparticles.

Silicon blue-emitting nanoparticles (NPs) are promising effectors for photodynamic therapy and radiotherapy, because of their production of reactive oxygen species (ROS) upon irradiation. RESULTS: Amino-functionalized silicon NPs (NH2SiNP) were intrinsically nontoxic below 100 µg/ml in vitro (on two tumor cell lines) and in vivo (zebrafish larvae and embryos). NH2SiNP showed a moderate effect as a photosensitizer for photodynamic therapy and reduced ROS generation in radiotherapy, which could be indicative of a ROS scavenging effect. Encapsulation of NH2SiNP into ultradeformable liposomes improved their skin penetration after topical application, reaching the viable epidermis where neoplastic events occur. CONCLUSION: Subsequent derivatizations after amino-functionalization and incorporation to nanodrug delivery systems could expand the spectrum of the biomedical application of these kind of silicon NPs.

Polyethylene glycol-coated blue-emitting silicon dots with improved properties for uses in aqueous and biological environments.

Grafting of polyethylene glycol (PEG) to ultrasmall photoluminescent silicon dots (SiDs) is expected to improve and expand the applications of these particles to aqueous environments and biological systems. Herein we report a novel one-pot synthesis of robust, highly water compatible PEG-coated SiDs (denoted as PEG-SiDs) of (3.3 ± 0.5) nm size. The nanoparticles' synthesis is based on the liquid phase oxidation of magnesium silicide using PEG as reaction media and leading to high PEG density grafting. PEG-SiDs enhanced photophysical, photosensitising, and solution properties in aqueous environments are described and compared to those of 2 nm size PEG-coated SiDs with low PEG density grafting (denoted as PEG-NHSiDs) obtained from a multistep synthesis strategy. PEG-SiDs form highly dispersed suspensions in water showing stable photoluminescence and quantum yields of Φ = 0.13 ± 0.04 at 370 nm excitation in air-saturated suspensions. These particles exhibited the capacity of photosensitising the formation of singlet molecular oxygen, not observed for PEG-NHSiDs. PEG robust shielding of the silicon core luminescent properties is further demonstrated in bio-imaging experiments stressing the strong interaction between PEG-SiDs and Staphylococcus aureus smears by observing the photoluminescence of particles. PEG-SiDs were found to be nontoxic to S. aureus cells at concentrations of 100 mg ml-1, though a bacteriostatic effect on S. aureus biofilms was observed upon UV-A irradiation under conditions where light alone has no effect.