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The objective of this work was to prepare and characterize liposomes containing co-encapsulated ascorbic acid (AA) and ascorbyl palmitate (AP), as well as to evaluate their stability, cytotoxicity, antioxidant, and antimicrobial activity. Through the pre-formulation studies, it was possible to improve the formulation, as leaving it more stable and with a greater antioxidant activity, resulting in a formulation designated LIP-AAP, with 161 nm vesicle size, 0.215 polydispersity index, -31.7 mV zeta potential, and pH of 3.34. Encapsulation efficiencies were 37% for AA and 79% for AP, and the content was 1 mg/mL for each compound. The optimized liposomes demonstrated stability under refrigeration for 60 days, significant antioxidant activity (31.4 µMol of TE/mL), and non-toxicity, but no antimicrobial effects against bacteria and fungi were observed. These findings confirm that the co-encapsulated liposomes are potent, stable antioxidants that maintain their physical and chemical properties under optimal storage conditions.

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Synthetic plastic polymers are causing considerable emerging ecological hazards. Starch-based biofilms are a potential alternative. However, depending on the natural source and extraction method, the properties of starch can vary, affecting the physicochemical characteristics of the corresponding casted films generated from it. These differences might entail morphological changes at the nanoscale, which can be explored by inspecting their surfaces. Potato (Solanum tuberosum) is a well-known tuber containing a high amount of starch, but the properties of the biofilms extracted from it are dependent on the specific variety. In this research, four Ecuadorian potato varieties (Leona Blanca, Única, Chola, and Santa Rosa) were analyzed and blended with different glycerol concentrations. The amylose content of each extracted starch was estimated, and biofilms obtained were characterized at both macroscopic and nanoscopic levels. Macroscopic tests were conducted to evaluate their elastic properties, visible optical absorption, water vapor permeability, moisture content, and solubility. It was observed that as the glycerol percentage increased, both moisture content and soluble matter increased, while tensile strength decreased, especially in the case of the Chola variety. These results were correlated to a surface analysis using atomic force microscopy, providing a possible explanation based on the topography and phase contrast observations made at the nanoscale.

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The Hertz-Sneddon elastic indentation model is widely adopted in the biomechanical investigation of living cells and other soft materials using atomic force microscopy despite the explicit viscoelastic nature of these materials. In this work, we demonstrate that an exact analytical viscoelastic force model for power-law materials, can be interpreted as a time-dependent Hertz-Sneddon-like model. Characterizing fibroblasts (L929) and osteoblasts (OFCOLII) demonstrates the model's accuracy. Our results show that the difference between Young's modulus EY obtained by fitting force curves with the Hertz-Sneddon model and the effective Young's modulus derived from the viscoelastic force model is less than 3%, even when cells are probed at large forces where nonlinear deformation effects become significant. We also propose a measurement protocol that involves probing samples at different indentation speeds and forces, enabling the construction of the average viscoelastic relaxation function of samples by conveniently fitting the force curves with the Hertz-Sneddon model.

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Modern imaging strategies are paramount to studying living systems such as cells, bacteria, and fungi and their response to pathogens, toxicants, and nanomaterials (NMs) as modulated by exposure and environmental factors. The need to understand the processes and mechanisms of damage, healing, and cell survivability of living systems continues to motivate the development of alternative imaging strategies. Of particular interest is the use of label-free techniques (microscopy procedures that do not require sample staining) that minimize interference of biological processes by foreign marking substances and reduce intense light exposure and potential photo-toxicity effects. This review focuses on the synergic capabilities of atomic force microscopy (AFM) as a well-developed and robust imaging strategy with demonstrated applications to unravel intimate details in biomedical applications, with the label-free, fast, and enduring Holotomographic Microscopy (HTM) strategy. HTM is a technique that combines holography and tomography using a low intensity continuous illumination laser to investigate (quantitatively and non-invasively) cells, microorganisms, and thin tissue by generating three-dimensional (3D) images and monitoring in real-time inner morphological changes. We first review the operating principles that form the basis for the complementary details provided by these techniques regarding the surface and internal information provided by HTM and AFM, which are essential and complimentary for the development of several biomedical areas studying the interaction mechanisms of NMs with living organisms. First, AFM can provide superb resolution on surface morphology and biomechanical characterization. Second, the quantitative phase capabilities of HTM enable superb modeling and quantification of the volume, surface area, protein content, and mass density of the main components of cells and microorganisms, including the morphology of cells in microbiological systems. These capabilities result from directly quantifying refractive index changes without requiring fluorescent markers or chemicals. As such, HTM is ideal for long-term monitoring of living organisms in conditions close to their natural settings. We present a case-based review of the principal uses of both techniques and their essential contributions to nanomedicine and nanotoxicology (study of the harmful effects of NMs in living organisms), emphasizing cancer and infectious disease control. The synergic impact of the sequential use of these complementary strategies provides a clear drive for adopting these techniques as interdependent fundamental tools.

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The cornea is a fundamental ocular tissue for the sense of sight. Thanks to it, the refraction of two-thirds of light manages to participate in the visual process and protect against mechanical damage. Because it is transparent, avascular, and innervated, the cornea comprises five main layers: Epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Each layer plays a key role in the functionality and maintenance of ocular tissue, providing unique ultrastructural and biomechanical properties. Bullous Keratopathy (BK) is an endothelial dysfunction that leads to corneal edema, loss of visual acuity, epithelial blisters, and severe pain, among other symptoms. The corneal layers are subject to changes in their biophysical properties promoted by Keratopathy. In this context, the Atomic Force Microscopy (AFM) technique in air was used to investigate the anterior epithelial surface and the posterior endothelial surface, healthy and with BK, using a triangular silicone tip with a nominal spring constant of 0.4 N/m. Six human corneas (n = 6) samples were used for each analyzed group. Roughness data, calculated by third-order polynomial adjustment, adhesion, and Young's modulus, were obtained to serve as a comparison and identification of morphological and biomechanical changes possibly associated with the pathology, such as craters and in the epithelial layer and exposure of a fibrotic layer due to loss of the endothelial cell wall. Endothelial cell membrane area and volume data were calculated, obtaining a relevant comparison between the control and patient. Such results may provide new data on the physical properties of the ocular tissue to understand the physiology of the cornea when it has pathology.

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We conducted a comprehensive analysis of the surface microtexture of kefir biofilms grown on Theobroma grandiflorum Shum (cupuaçu) juice using atomic force microscopy. Our goal was to investigate the unique monofractal and multifractal spatial patterns of these biofilms to complement the existing limited literature. The biofilms were prepared dispersing four different concentrations of kefir grains in cupuaçu juice. Our morphological analysis showed that the surface of the obtained biofilms is essentially formed by the presence of cupuaçu fibers and microorganisms like lactobacilli and yeast. The topographic height-based parameter analysis reveals that there is a dependence between surface roughness and the concentration of kefir grains used. The strongly anisotropic well-centralized topographical height distribution of the biofilms also exhibited a quasi-symmetrical and platykurtic pattern. The biofilms exhibit comparable levels of spatial complexity, surface percolation and surface homogeneity, which can be attributed to their similar topographic uniformity. This aspect was further supported by the presence of similar multifractality in the biofilms, suggesting that despite their varying topographic roughness, their vertical growth dynamics follow a similar pattern. Our findings demonstrate that the surface roughness of kefir biofilms cultivated on cupuaçu juice is influenced by the concentration of kefir grains in the precursor solution. However, this dependence follows a consistent pattern across different concentrations. Graphical Abstract.

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Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV) are naked plant viruses with similar characteristics; both form a T = 3 icosahedral protein capsid and are members of the bromoviridae family. It is well known that these viruses completely disassemble and liberate their genome at a pH around 7.2 and 1 M ionic strength. However, the 1 M ionic strength condition is not present inside cells, so an important question is how these viruses deliver their genome inside cells for their viral replication. There are some studies reporting the swelling of the CCMV virus using different techniques. For example, it is reported that at a pH~7.2 and low ionic strength, the swelling observed is about 10% of the initial diameter of the virus. Furthermore, different regions within the cell are known to have different pH levels and ionic strengths. In this work, we performed several experiments at low ionic strengths of 0.1, 0.2, and 0.3 and systematically increased the pH in 0.2 increments from 4.6 to 7.4. To determine the change in virus size at the different pHs and ionic strengths, we first used dynamic light scattering (DLS). Most of the experiments agree with a 10% capsid swelling under the conditions reported in previous works, but surprisingly, we found that at some particular conditions, the virus capsid swelling could be as big as 20 to 35% of the original size. These measurements were corroborated by atomic force microscopy (AFM) and transmission electron microscopy (TEM) around the conditions where the big swelling was determined by DLS. Therefore, this big swelling could be an easier mechanism that viruses use inside the cell to deliver their genome to the cell machinery for viral replication.

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IMPORTANCE: In this work, we characterized the composition, structure, and functional potential for biofilm formation of Exiguobacterium strains isolated from the Salar de Huasco in Chile in the presence of arsenic, an abundant metalloid in the Salar that exists in different oxidation states. Our results showed that the Exiguobacterium strains tested exhibit a significant capacity to form biofilms when exposed to arsenic, which would contribute to their resistance to the metalloid. The results highlight the importance of biofilm formation and the presence of specific resistance mechanisms in the ability of microorganisms to survive and thrive under adverse conditions.

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Few studies have been able to elucidate the correlation of factors determining the strength of interaction between bacterial cells and substrate at the molecular level. The aim was to answer the following question: What biophysical factors should be considered when analyzing the bacterial adhesion strength on titanium surfaces and its alloys for implants quantified by atomic force microscopy? This review followed PRISMA. The search strategy was applied in four databases. The selection process was carried out in two stages. The risk of bias was analyzed. One thousand four hundred sixty-three articles were found. After removing the duplicates, 1126 were screened by title and abstract, of which 57 were selected for full reading and 5 were included; 3 had a low risk of bias and 2 moderated risks of bias. (1) The current literature shows the preference of bacteria to adhere to surfaces of the same hydrophilicity. However, this fact was contradicted by this systematic review, which demonstrated that hydrophobic bacteria developed hydrogen bonds and adhered to hydrophilic surfaces; (2) the application of surface treatments that induce the reduction of areas favorable for bacterial adhesion interfere more in the formation of biofilm than surface roughness; and (3) bacterial colonization should be evaluated in time-dependent studies as they develop adaptation mechanisms, related to time, which are obscure in this review.