Size matters for in vitro gene delivery: investigating the relationships among complexation protocol, transfection medium, size and sedimentation

Although branched and linear polyethylenimines (bPEIs and lPEIs) are gold standard transfectants, a systematic analysis of the effects of the preparation protocol of polyplexes and the composition of the transfection medium on their physicochemical behaviour and effectiveness in vitro have been much neglected, undermining in some way the identification of precise structure-function relationships. This work aimed to address these issues. bPEI/DNA and lPEI/DNA, prepared using two different modes of addition of reagents, gave rise to polyplexes with exactly the same chemical composition but differing in dimensions. Upon dilution in serum-free medium, the size of any kind of polyplex promptly rose over time while remained invariably stable in complete DMEM. Of note, the bigger the dimension of polyplexes (in the nano- to micrometer range), the greater their efficiency in vitro. Besides, centrifugal sedimentation of polyplexes displaying different dimensions to speed up and enhance their settling onto cells boosted transfection efficiencies. Conversely, transgene expression was significantly blunted in cells held upside-down and transfected, definitively pointing out the impact of gravitational sedimentation of polyplexes on their transfection efficiency. Overall, much more attention must be paid to the actual polyplex size that relies on the complexation conditions and the transfection medium

Mucoadhesive chitosan-methylcellulose oral patches for the treatment of local mouth bacterial infections

: Mucoadhesive buccal patches are dosage forms promising for successful drug delivery. They show the distinctive advantages of long residence time on the oral mucosa and increased in situ drug bioavailability. In this context, electrophoretic deposition (EPD) of chitosan (CS) has been demonstrated as a simple and easily tunable technique to produce mucoadhesive buccal patches. However, CS-based buccal patches may suffer from weak mucoadhesion, which can impair their therapeutic effect. In this work, methylcellulose (MC), a widely investigated biopolymer in the biomedical area, was exploited to increase the mucoadhesive characteristic of pristine CS patches. CS-MC patches were obtained in a one-pot process via EPD, and the possibility of incorporating gentamicin sulfate (GS) as a model of a broad-spectrum antibiotic in the so-obtained patches was investigated. The resulting CS-MC patches showed high stability in a water environment and superior mucoadhesive characteristic (σadh = 0.85 ± 0.26 kPa, Wadh = 1192.28 ± 602.36 Pa mm) when compared with the CS control samples (σadh = 0.42 ± 0.22 kPa, Wadh = 343.13 ± 268.89 Pa mm), due to both the control of the patch porosity and the bioadhesive nature of MC. Furthermore, GS-loaded patches showed no in vitro cytotoxic effects by challenging L929 cells with material extracts and noteworthy antibacterial activity on both Gram-positive and Gram-negative bacterial strains

VALIDATION OF MESOSCALE METEOROLOGICAL SIMULATION OVER PO VALLEY FOR AIR QUALITY APPLICATIONS

Very high ground level concentrations of PM in winter and of ozone in summer often occur in Northern Italy, due to the high anthropogenic emissions and frequent stagnant meteorological conditions that characterize the area. These problems are not only related to urban, but also to suburban areas through the entire Po Valley. In such a situation it is important to use deterministic Chemical Transport Models, that allows to evaluate the effect of different air quality control policies on secondary pollution concentrations. Chemical Transport Models generally are part of more complex deterministic modelling systems, encompassing also emission models, meteorological models, and initial and boundary condition processors. Meteorological models are an important module of deterministic modelling systems and, due to their complexity, require high computational costs to perform simulations. In fact they solve a full set of non-hydrostatic equations that describe atmospheric dynamics and thermodynamics, and conservation equations, usually considering two-way interacting nested domains. Within the HPC-EUROPA (Pan-European Research Infrastructure on High Performance Computing) cooperation project, that allows to use clusters of CPUs all around Europe, the meteorological fields over Northern Italy were simulated using RAMS4.4 in parallel mode, creating a database for future air quality assessments. In the present work a CPUs cluster of the Italian computing centre CINECA have been used. The meteorological simulations have been performed considering three nested grids. The first grid covers an area that encompasses the entire Europe, the second grid is focused on Mediterranean sea, while the third one is limited to the Po Valley area. The spatial resolution of the three grids is respectively 128 km, 32 km and 8 km. The number of cells for the three grids is respectively 40x40, 86x86 and 102x102, with 33 vertical levels covering the domain from surface to roughly 20 km height. The entire 2004 year has been simulated through 72 simulations of 126 hours each, considering a spin-up time of 6 hours and 16 CPUs each simulation. In this paper the model configuration and the validation of the simulated meteorological fields are presented

BMP-2 and type I collagen preservation in human deciduous teeth after demineralization

Background: Great interest has recently been focused on tooth and tooth derivatives as suitable substrates for the treatment of alveolar bone defects. Here, we propose the use of demineralized baby teeth (BT) as potential grafting materials for bone augmentation procedures. Methods: Particles of human BT (Ø < 1 mm) were demineralized by means of a chemical/thermal treatment. Demineralized BT particles were thoroughly characterized by scanning electron microscopy/energy dispersive X-ray analyses to evaluate the effects of the demineralization on BT topography and mineral phase composition, and by enzyme-linked immunosorbent assays (ELISA) to quantify collagen and bone morphogenetic protein-2 (BMP-2) protein contents. The response of SAOS-2 cells to exogenous BMP-2 stimulation was evaluated to identify the minimum BMP-2 concentration able to induce osteodifferentiation in vitro (alkaline phosphatase (ALP) activity). Results: The demineralization treatment led to a dramatic decrease in relative Ca and P content (%) of ≈75% with respect to the native BT particles, while preserving native protein conformation and activity. Interestingly, the demineralization process led to a rise in the bioavailability of BMP-2 in BT particles, as compared to the untreated counterparts. The BMP-2 content found in demineralized BT was also proved to be very effective in enhancing ALP activity, thus in the osteodifferentiation of SAOS-2 cells in vitro, as confirmed by cell experiments performed upon exogenously added BMP-2. Conclusions: In this study we demonstrate that the BMP-2 content found in demineralized BT is very effective in inducing cell osteodifferentiation, and strengthens the idea that BTs are very attractive bioactive materials for bone-grafting procedures

Characterization of fine metal particles derived from shredded WEEE using a hyperspectral image system: Preliminary results

Waste of electric and electronic equipment (WEEE) is the fastest-growing waste stream in Europe. The large amount of electric and electronic products introduced every year in the market makes WEEE disposal a relevant problem. On the other hand, the high abundance of key metals included in WEEE has increased the industrial interest in WEEE recycling. However, the high variability of materials used to produce electric and electronic equipment makes key metalsâ recovery a complex task: the separation process requires flexible systems, which are not currently implemented in recycling plants. In this context, hyperspectral sensors and imaging systems represent a suitable technology to improve WEEE recycling rates and the quality of the output products. This work introduces the preliminary tests using a hyperspectral system, integrated in an automatic WEEE recycling pilot plant, for the characterization of mixtures of fine particles derived from WEEE shredding. Several combinations of classification algorithms and techniques for signal enhancement of reflectance spectra were implemented and compared. The methodology introduced in this study has shown characterization accuracies greater than 95%

Demineralized dentin and enamel matrices as suitable substrates for bone regeneration

Background: In recent decades, tooth derivatives such as dentin (D) and enamel (E) have been considered as potential graft biomaterials to treat bone defects. This study aimed to investigate the effects of demineralization on the physical-chemical and biological behavior of D and E. Methods: Human D and E were minced into particles (Ã\u98<1 mm), demineralized and sterilized. Thorough physicalchemical and biochemical characterizations of native and demineralized materials were performed by SEM and EDS analysis and ELISA kits to determine mineral, collagen type I and BMP-2 contents. In addition, MG63 and SAOS-2 cells were seeded on tooth-derived materials and Bio-Oss®, and a comparison of cell responses in terms of adhesion and proliferation was carried out. Results: The sterilization process, as a combination of chemical and thermal treatments, was found to be effective for all materials. On the other hand, D demineralization allowed preserving the collagen content, while increasing BMP-2 bioavailability. D and demineralized D (dD) displayed excellent biocompatibility, even greater than Bio-Oss®. Conversely, the high mineral content displayed by E, as confirmed by EDS analysis, inhibited cell proliferation. Of note, even though the demineralization process was somehow less effective in E than in D, demineralized E (dE) displayed increased BMP-2 bioavailability and improved performance in vitro compared with native E. Conclusions: Our results substantiate the idea that the demineralization process lead to an increase of BMP-2 bioavailability, thus paving the way toward development of more effective, osteoinductive tooth-derived materials for bone regeneration and replacement

Mapping of Asbestos Cement Roofs and Their Weathering Status Using Hyperspectral Aerial Images

and (ii) the development of a spectral index related to the roof weathering status. Aerial images were collected through the Multispectral Infrared and Visible Imaging Spectrometer (MIVIS) sensor, which acquires data in 102 channels from the visible to the thermal infrared spectral range. An image based supervised classification was performed using the Spectral Angle Mapper (SAM) algorithm. The SAM was trained through a set of pixels selected on roofs of different materials. The map showed an average producer's accuracy (PA) of 86% and a user's accuracy (UA) of 89% for the asbestos cement class. A novel spectral index, the "Index of Surface Deterioration" (ISD), was defined based on measurements collected with a portable spectroradiometer on asbestos cement roofs that were characterized by different weathering statuses. The ISD was then calculated on the MIVIS images, allowing the distinction of two weathering classes (i.e., high and low). The asbestos cement map was handled in a Geographic Information System (GIS) in order to supply the municipalities with the cadastral references of each property having an asbestos cement roof. This tool can be purposed for municipalities as an aid to prioritize asbestos removal, based on roof weathering status

Vibropolyfection: coupling polymer-mediated gene delivery to mechanical stimulation to enhance transfection of adherent cells

Background: With the success of recent non-viral gene delivery-based COVID-19 vaccines, nanovectors have gained some public acceptance and come to the forefront of advanced therapies. Unfortunately, the relatively low ability of the vectors to overcome cellular barriers adversely affects their effectiveness. Scientists have thus been striving to develop ever more effective gene delivery vectors, but the results are still far from satisfactory. Therefore, developing novel strategies is probably the only way forward to bring about genuine change. Herein, we devise a brand-new gene delivery strategy to boost dramatically the transfection efficiency of two gold standard nucleic acid (NA)/polymer nanoparticles (polyplexes) in vitro. Results: We conceived a device to generate milli-to-nanoscale vibrational cues as a function of the frequency set, and deliver vertical uniaxial displacements to adherent cells in culture. A short-lived high-frequency vibrational load (t= 5 min, f= 1,000 Hz) caused abrupt and extensive plasmalemma outgrowths but was safe for cells as neither cell proliferation rate nor viability was affected. Cells took about 1 hr to revert to quasi-naIve morphology through plasma membrane remodeling. In turn, this eventually triggered the mechano-activated clathrin-mediated endocytic pathway and made cells more apt to internalize polyplexes, resulting in transfection efficiencies increased from 10-to100-fold. Noteworthy, these results were obtained transfecting three cell lines and hard-to-transfect primary cells. Conclusions: In this work, we focus on a new technology to enhance the intracellular delivery of NAs and improve the transfection efficiency of non-viral vectors through priming adherent cells with a short vibrational stimulation. This study paves the way for capitalizing on physical cell stimulation(s) to significantly raise the effectiveness of gene delivery vectors in vitro and ex vivo

A new microfluidic platform for the highly reproducible preparation of non-viral gene delivery complexes

Transfection describes the delivery of exogenous nucleic acids (NAs) to cells utilizing non-viral means. In the last few decades, scientists have been doing their utmost to design ever more effective transfection reagents. These are eventually mixed with NAs to give rise to gene delivery complexes, which must undergo characterization, testing, and further refinement through the sequential reiteration of these steps. Unfortunately, although microfluidics offers distinct advantages over the canonical approaches to preparing particles, the systems available do not address the most frequent and practical quest for the simultaneous generation of multiple polymer-to-NA ratios (N/Ps). Herein, we developed a user-friendly microfluidic cartridge to repeatably prepare non-viral gene delivery particles and screen across a range of seven N/Ps at once or significant volumes of polyplexes at a given N/P. The microchip is equipped with a chaotic serial dilution generator for the automatic linear dilution of the polymer to the downstream area, which encompasses the NA divider to dispense equal amounts of DNA to the mixing area, enabling the formation of particles at seven N/Ps eventually collected in individual built-in tanks. This is the first example of a stand-alone microfluidic cartridge for the fast and repeatable preparation of non-viral gene delivery complexes at different N/Ps and their storage