In vitro scratch wound healing assay, a simple and low-cost technique that works along with other image analysis tools, is one of the most widely used 2D methods to determine the cellular migration and proliferation in processes such as regeneration and disease. There are open-source programs such as imageJ to analyze images of in vitro scratch wound healing assays, but these tools require manual tuning of various parameters, which is time-consuming and limits image throughput. For that reason, we developed an optimized plugin for ima-geJ to automatically recognize the wound healing size, correct the average wound width by considering its inclination, and quantify other important parameters such as: area, wound area fraction, average wound width, and width deviation of the wound images obtained from a scratch/ wound healing assay. Our plugin is easy to install and can be used with different operating systems. It can be adapted to analyze both individual images and stacks. Additionally, it allows the analysis of images obtained from bright field, phase contrast, and fluorescence microscopes. In conclusion, this new imageJ plugin is a robust tool to automatically standardize and facilitate quantification of different in vitro wound parameters with high accuracy compared with other tools and manual identification.

IntroductionControlled delivery of therapeutic molecules in a localized manner has become an area of interest due to its potential to reduce drug exposure to healthy tissues and consequently to minimize undesirable side effects. We have recently introduced novel cell-penetrating vehicles by immobilizing the antimicrobial peptide Buforin II (BUF-II) on magnetite nanoparticles (MPNPs). Despite the potent translocating abilities of such nanobioconjugates, they failed to preserve the antimicrobial activity of native BUF-II. In this work, we explored immobilization on MNPs with the aid of polymer surface spacers, which has been considered as an attractive alternative for the highly efficient conjugation of various biomolecules.MethodsHere, we immobilized BUF-II on polyetheramine-modified magnetite nanoparticles to preserve its structural integrity. As a result, for the obtained nanobioconjugates the lost antimicrobial activity against gram-positive and gram-negative bacteria was only 50% with respect to the native BUF-II. The nanobioconjugates were also characterized via FTIR, DLS, TEM, and TGA. Delivery on THP-1, HaCaT, HFF, and Escherichia coli cells was conducted to confirm capability for cell membrane translocation.ResultsColocalization with Lysotracker showed an endosomal escape efficiency of about 73∓12% in THP-1 cells. Avoidance of endocytic pathways of internalization was qualitatively confirmed by a delivery assay at low temperature. Nuclear penetration of the nanobioconjugates was corroborated via confocal microscopy and showed high biocompatibility as demonstrated by hemolysis levels below 5% and acute cytotoxicity of around 15%.ConclusionThe obtained nanobioconjugates were capable of translocating the cell membrane and nuclei of different normal and cancerous cell lines without significantly decreasing viability. This makes the vehicle addressable for a number of applications ranging from antimicrobial topical treatments to the delivery of nucleotides and therapeutic molecules with difficulties to bypass cell membranes.

IntroductionOne of the major challenges of modern pharmacology is the development of systems for the delivery of therapeutic molecules in a controlled and localized manner. One strategy is to use nanostructured supports, which are well suited to carry a large number of molecules on a per mass basis. A major challenge for these supports is, however, their limited ability to bypass the cell membrane. Recent studies propose that to overcome this issue, potent translocating cell-penetrating peptides (CPPs) can be conjugated to their surfaces.MethodsHere, we conjugated the antimicrobial CPP buforin II (BUF2) to the surface of magnetite nanoparticles to enhance their cell penetration. Conjugates were characterized via Fourier transform infrared spectroscopy, dynamic light scattering, and thermogravimetric analysis, and their biocompatibility was corroborated. The conjugates were delivered in both bacterial and mammalian cells demonstrating the intracellular inclusion in THP-1 cells for the first time.ResultsDespite the promising outcome, our studies showed that the obtained conjugates failed to maintain the native antimicrobial activity of BUF2. We hypothesize that to overcome this issue, a flexible linker can be inserted prior to conjugation.ConclusionOur study highlights the potential of BUF2-magnetite conjugates as cell-penetrating vehicles for the targeted delivery of pharmacological agents. This provides support for the idea of a promising combined drug delivery and antimicrobial peptide therapy.

Outer membrane protein A (OmpA) has been extensively studied in Gram-negative bacteria due to its relevance in the adhesion of pathogens to host cells and its surfactant capabilities. It consists of a hydrophobic β-barrel domain and a hydrophilic periplasmic domain, that confers OmpA an amphiphilic structure. This study aims to elucidate the capacity of Escherichia coli OmpA to translocate liposomal membranes and serve as a potential cell-penetrating vehicle. We immobilized OmpA on magnetite nanoparticles and investigated the possible functional changes exhibited by OmpA after immobilization. Liposomal intake was addressed using egg lecithin liposomes as a model, where magnetite–OmpA nanobioconjugates were able to translocate the liposomal membrane and caused a disruptive effect when subjected to a magnetic field. Nanobioconjugates showed both low cytotoxicity and hemolytic tendency. Additional interactions within the intracellular space led to altered viability results via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confocal microscopy images revealed that immobilized nanoparticles effectively enter the cytoplasm of THP-1 and Vero cells by different routes, and, subsequently, some escape endosomes, lysosomes, and other intracellular compartments with relatively high efficiencies. This was demonstrated by co-localization analyses with LysoTracker green that showed Pearson correlations of about 80 and 28%.

Macrophages are essential in the initiation, maintenance, and transition of inflammatory processes like foreign body response and wound healing. These sentinel cells are very motile, susceptible, and adaptive to environmental cues. Mounting evidence suggests that physical factors also modulate macrophage activation in vitro and in vivo. 2D in vitro systems have demonstrated that constraining macrophages to small areas or channels modulates their phenotypes and changes their responses to known inflammatory agents such as lipopolysaccharide. However, macrophage responses in these 2D systems cannot be directly correlated to an in vivo response. Thus, the study of macrophage confinement in a 3D environment that can be directly used in vivo is needed and would allow the design of more translatable biomaterials for clinical applications. In this work, we explored the change in M1/M2 polarization when macrophages were confined in microporous annealed particle scaffolds (MAPS), which are granular hydrogels generated from annealed spherical microgels. We cultured primary murine macrophages within MAPS constructed of microgels with diameters of 40 μm, 70 μm, and 130 μm. Spatially confining macrophages in scaffolds with pore size on the scale of cells led to a reduced level of the inflammatory response, which was correlated with a change in cell morphology and motility.

Microporous annealed particle scaffolds (MAPS) are a new class of granular materials generated through the interlinking of tunable microgels, which produce an interconnected network of void space. These microgel building blocks can be designed with different mechanical or bio‐active parameters to facilitate cell infiltration and modulate host response. Previously, changing the chirality of the microgel crosslinking peptides from L‐ to D‐amino acids led to significant tissue regeneration and functional recovery in D‐MAPS‐treated cutaneous wounds. In this study, the immunomodulatory effect of D‐MAPS in a subcutaneous implantation model is investigated. How macrophages are the key antigen‐presenting cells to uptake and present these biomaterials to the adaptive immune system is uncovered. A robust linker‐specific IgG2b/IgG1 response to D‐MAPS is detected as early as 14 days post‐implantation. The fine balance between pro‐regenerative and pro‐inflammatory macrophage phenotypes is observed in D‐MAPS as an indicator for regenerative scaffolds. The work offers valuable insights into the temporal cellular response to synthetic porous scaffolds and establishes a foundation for further optimization of immunomodulatory pro‐regenerative outcomes.

Cancer is one of the leading causes of death both in the United States and worldwide. The dynamic microenvironment in which tumors grow consists of fibroblasts, immune cells, extracellular matrix (ECM), and cytokines that enable progression and metastasis. Novel biomaterials that mimic these complex surroundings give insight into the biological, chemical, and physical environment that cause cancer cells to metastasize and invade in to other tissues. Twodimensional (2D) cultures are useful for gaining limited information about cancer cell behavior; however, they do not accurately represent the environments that cells experience in vivo. Recent advances in the design and tunability of diverse three-dimensional (3D) biomaterials complement biological knowledge and allow for improved recapitulation of in vivo conditions. Understanding cell-ECM and cell-cell interactions that facilitate tumor survival will accelerate the design of more effective therapies. This review discusses innovative materials currently being used to study tumor and immune cell behavior and interactions, including materials that mimic the ECM composition, mechanical stiffness, and integrin binding sites of the tumor microenvironment.

In vitro scratch wound healing assay, a simple and low-cost technique that works along with other image analysis tools, is one of the most widely used 2D methods to determine the cellular migration and proliferation in processes such as regeneration and disease. There are open-source programs such as imageJ to analyze images of in vitro scratch wound healing assays, but these tools require manual tuning of various parameters, which is time-consuming and limits image throughput. For that reason, we developed an optimized plugin for imageJ to automatically recognize the wound healing size, correct the average wound width by considering its inclination, and quantify other important parameters such as: area, wound area fraction, average wound width, and width deviation of the wound images obtained from a scratch/ wound healing assay. Our plugin is easy to install and can be used with different operating systems. It can be adapted to analyze both individual images and stacks.Additionally, it allows the analysis of images obtained from bright field, phase contrast, and fluorescence microscopes. In conclusion, this new imageJ plugin is a robust tool to automatically standardize and facilitate quantification of different in vitro wound parameters with high accuracy compared with other tools and manual identification.