Technological Perception with Rural and Urban Differentiation and Its Influence on the Quality of Life of Older People with Age-Related Macular Degeneration.

The past decade has seen a global increase in population age, especially in developed countries, where aging involves visual diseases such as age-related macular degeneration (AMD), which severely affect quality of life (QoL) and mental health, as well as increase isolation and care costs. This study investigated how persons with AMD perceive the impact of technology use on their QoL, focusing on potential disparities between urban and rural contexts in Spain. Using a cross-sectional observational design, data from the 2020 National Statistics Institute's Disability, Personal Autonomy, and Dependency Situations Survey were analyzed, focusing on QoL aspects based on the WHO items of the WHOQOL-100 scale. The results revealed a generally positive perception of technology among participants, with urban residents perceiving technology's positive impact more favorably. Sex discrepancies in technology perception were also observed, as women exhibited a more positive outlook on technology's influence on QoL. The analysis of QoL aspects, such as 'Visibility', 'Learning', 'Mobility', and 'Domestic life', highlighted distinct challenges faced by rural and urban populations, underscoring the importance of context-specific approaches in technology interventions. However, these perceptions were intertwined with comorbidities, which can exacerbate AMD-related issues. Furthermore, this study explored the role of technology in enhancing QoL among older adults with AMD, examining how it influences daily activities and independence, particularly in the context of AMD management. This study concluded that developing more-inclusive policies tailored to the specific needs of persons with AMD, with special attention to environmental and sex differences, is imperative to enhance the positive impact of technology on their QoL.

Machine learning enables non-Gaussian investigation of changes to peripheral nerves related to electrical stimulation.

Electrical stimulation of the peripheral nervous system (PNS) is becoming increasingly important for the therapeutic treatment of numerous disorders. Thus, as peripheral nerves are increasingly the target of electrical stimulation, it is critical to determine how, and when, electrical stimulation results in anatomical changes in neural tissue. We introduce here a convolutional neural network and support vector machines for cell segmentation and analysis of histological samples of the sciatic nerve of rats stimulated with varying current intensities. We describe the methodologies and present results that highlight the validity of the approach: machine learning enabled highly efficient nerve measurement collection, while multivariate analysis revealed notable changes to nerves' anatomy, even when subjected to levels of stimulation thought to be safe according to the Shannon current limits.

Assessment of Different Niosome Formulations for Optogenetic Applications: Morphological and Electrophysiological Effects.

Gene therapy and optogenetics are becoming promising tools for treating several nervous system pathologies. Currently, most of these approaches use viral vectors to transport the genetic material inside the cells, but viruses present some potential risks, such as marked immunogenicity, insertional mutagenesis, and limited insert gene size. In this framework, non-viral nanoparticles, such as niosomes, are emerging as possible alternative tools to deliver genetic material, avoiding the aforementioned problems. To determine their suitability as vectors for optogenetic therapies in this work, we tested three different niosome formulations combined with three optogenetic plasmids in rat cortical neurons in vitro. All niosomes tested successfully expressed optogenetic channels, which were dependent on the ratio of niosome to plasmid, with higher concentrations yielding higher expression rates. However, we found changes in the dendritic morphology and electrophysiological properties of transfected cells, especially when we used higher concentrations of niosomes. Our results highlight the potential use of niosomes for optogenetic applications and suggest that special care must be taken to achieve an optimal balance of niosomes and nucleic acids to achieve the therapeutic effects envisioned by these technologies.

New Highly Fluorescent Water Soluble Imidazolium-Perylenediimides: Synthesis and Cellular Response.

The synthesis and characterization of two new water soluble 2,6-bis(imidazolylmethyl)-4-methylphenoxy-containing perylenediimides, PDI-1 and PDI-2, are described. These compounds demonstrate a high fluorescence quantum yield in water and were investigated as potential photosensitizers for generating reactive oxygen species with applications in anticancer activities. The HeLa cell line (VPH18) was used to evaluate their efficacy. Fluorescence microscopy was employed to confirm the successful internalization of PDI-1 and PDI-2, while confocal microscopy revealed the specific locations of both PDIs within the lysosomes and mitochondria. In vitro studies were conducted to evaluate the anticancer activity of PDI-1 and PDI-2. Remarkably, these photosensitizers demonstrated a significant ability to selectively eliminate cancer cells when exposed to a specific light wavelength. The water solubility, high fluorescence quantum yield, and selective cytotoxicity of these PDIs toward cancer cells highlight their potential as effective agents for targeted photodynamic therapy. In conclusion, the findings presented here provide a strong foundation for the future exploration and optimization of PDI-1 and PDI-2 as effective photosensitizers in photodynamic therapy, potentially leading to improved treatment strategies for cancer patients.

Comparative Analysis of CRISPR-Cas Systems in Pseudomonas Genomes.

Pseudomonas is a bacterial genus with some saprophytic species from land and others associated with opportunistic infections in humans and animals. Factors such as pathogenicity or metabolic aspects have been related to CRISPR-Cas, and in silico studies into it have focused more on the clinical and non-environmental setting. This work aimed to perform an in silico analysis of the CRISPR-Cas systems present in Pseudomonas genomes. It analyzed 275 complete genomic sequences of Pseudomonas taken from the NCBI database. CRISPR loci were obtained from CRISPRdb. The genes associated with CRISPR (cas) and CAS proteins, and the origin and diversity of spacer sequences, were identified and compared by BLAST. The presence of self-targeting sequences, PAMs, and the conservation of DRs were visualized using WebLogo 3.6. The CRISPR-like RNA secondary structure prediction was analyzed using RNAFold and MFold. CRISPR structures were identified in 19.6% of Pseudomonas species. In all, 113 typical CRISPR arrays with 18 putative cas were found, as were 2050 spacers, of which 52% showed homology to bacteriophages, 26% to chromosomes, and 22% to plasmids. No potential self-targeting was detected within the CRISPR array. All the found DRs can form thermodynamically stable secondary RNA structures. The comparison of the CRISPR/Cas system can help understand the environmental adaptability of each evolutionary lineage of clinically and environmentally relevant species, providing data support for bacterial typing, traceability, analysis, and exploration of unconventional CRISPR.

Long-term biophysical stability of nanodiamonds combined with lipid nanocarriers for non-viral gene delivery to the retina.

Nanodiamonds were combined with niosome, and resulting formulations were named as nanodiasomes, which were evaluated in terms of physicochemical features, cellular internalization, cell viability and transfection efficiency both in in vitro and in in vivo conditions. Such parameters were analyzed at 4 and 25 °C, and at 15 and 30 days after their elaboration. Nanodiasomes showed a particle size of 128 nm that was maintained over time inside the ± 10% of deviation, unless after 30 days of storage at 25 °C. Something similar occurred with the initial zeta potential value, 35.2 mV, being both formulations more stable at 4 °C. The incorporation of nanodiamonds into niosomes resulted in a 4-fold increase of transfection efficiency that was maintained over time at 4 and 25 °C. In vivo studies reported high transgene expression of nanodiasomes after subretinal and intravitreal administration in mice, when injected freshly prepared and after 30 days of storage at 4 °C.

Electrical Stimulation Induced Current Distribution in Peripheral Nerves Varies Significantly with the Extent of Nerve Damage: A Computational Study Utilizing Convolutional Neural Network and Realistic Nerve Models.

Electrical stimulation of the peripheral nervous system is a promising therapeutic option for several conditions; however, its effects on tissue and the safety of the stimulation remain poorly understood. In order to devise stimulation protocols that enhance therapeutic efficacy without the risk of causing tissue damage, we constructed computational models of peripheral nerve and stimulation cuffs based on extremely high-resolution cross-sectional images of the nerves using the most recent advances in computing power and machine learning techniques. We developed nerve models using nonstimulated (healthy) and over-stimulated (damaged) rat sciatic nerves to explore how nerve damage affects the induced current density distribution. Using our in-house computational, quasi-static, platform, and the Admittance Method (AM), we estimated the induced current distribution within the nerves and compared it for healthy and damaged nerves. We also estimated the extent of localized cell damage in both healthy and damaged nerve samples. When the nerve is damaged, as demonstrated principally by the decreased nerve fiber packing, the current penetrates deeper into the over-stimulated nerve than in the healthy sample. As safety limits for electrical stimulation of peripheral nerves still refer to the Shannon criterion to distinguish between safe and unsafe stimulation, the capability this work demonstrated is an important step toward the development of safety criteria that are specific to peripheral nerve and make use of the latest advances in computational bioelectromagnetics and machine learning, such as Python-based AM and CNN-based nerve image segmentation.

Nanodiamond Integration into Niosomes as an Emerging and Efficient Gene Therapy Nanoplatform for Central Nervous System Diseases.

Nanodiamonds (NDs) are promising materials for gene delivery because of their unique physicochemical and biological features, along with their possibility of combination with other nonviral systems. Our aim was to evaluate the biophysical performance of NDs as helper components of niosomes, named nanodiasomes, to address a potential nonviral gene delivery nanoplatform for therapeutic applications in central nervous system (CNS) diseases. Nanodiasomes, niosomes, and their corresponding complexes, obtained after genetic material addition at different ratios (w/w), were evaluated in terms of physicochemical properties, cellular uptake, intracellular disposition, biocompatibility, and transfection efficiency in HEK-293 cells. Nanodiasomes, niosomes, and complexes fulfilled the physicochemical features for gene therapy applications. Biologically, the incorporation of NDs into niosomes enhanced 75% transfection efficiency (p < 0.001) and biocompatibility (p < 0.05) to values over 90%, accompanied by a higher cellular uptake (p < 0.05). Intracellular trafficking analysis showed higher endocytosis via clathrins (p < 0.05) in nanodiaplexes compared with nioplexes, followed by higher lysosomal colocalization (p < 0.05), that coexisted with endosomal escape properties, whereas endocytosis mediated by caveolae was the most efficient pathway in the case of nanodiaplexes. Moreover, studies in CNS primary cells revealed that nanodiaplexes successfully transfected neuronal and retinal cells. This proof-of-concept study points out that ND integration into niosomes represents an encouraging nonviral nanoplatform strategy for the treatment of CNS diseases by gene therapy.

Sphingolipid extracts enhance gene delivery of cationic lipid vesicles into retina and brain.

The aim was to evaluate relevant biophysic processes related to the physicochemical features and gene transfection mechanism when sphingolipids are incorporated into a cationic niosome formulation for non-viral gene delivery to central nervous system. For that, two formulations named niosphingosomes and niosomes devoid of sphingolipid extracts, as control, were developed by the oil-in water emulsion technique. Both formulations and the corresponding complexes, obtained upon the addition of the reporter EGFP plasmid, were physicochemically and biologically characterized and evaluated. Compared to niosomes, niosphingosomes, and the corresponding complexes decreased particle size and increased superficial charge. Although there were not significant differences in the cellular uptake, cell viability and transfection efficiency increased when human retinal pigment epithelial (ARPE-19) cells were exposed to niosphingoplexes. Endocytosis via caveolae decreased in the case of niosphingoplexes, which showed higher co-localization with lysosomal compartment, and endosomal escape properties. Moreover, niosphingoplexes transfected not only primary central nervous system cells, but also different cells in mouse retina, depending on the administration route, and brain cortex. These preliminary results suggest that niosphingosomes represent a promising non-viral vector formulation purposed for the treatment of both retinal and brain diseases by gene therapy approach.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium in a Swine Model of Geographic Atrophy.

BACKGROUND: The aim of this study was to test the feasibility and safety of subretinal transplantation of human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells into the healthy margins and within areas of degenerative retina in a swine model of geographic atrophy (GA). METHODS: Well-delimited selective outer retinal damage was induced by subretinal injection of NaIO3 into one eye in minipigs (n = 10). Thirty days later, a suspension of hiPSC-derived RPE cells expressing green fluorescent protein was injected into the subretinal space, into the healthy margins, and within areas of degenerative retina. In vivo follow-up was performed by multimodal imaging. Post-mortem retinas were analyzed by immunohistochemistry and histology. RESULTS: In vitro differentiated hiPSC-RPE cells showed a typical epithelial morphology, expressed RPE-related genes, and had phagocytic ability. Engrafted hiPSC-RPE cells were detected in 60% of the eyes, forming mature epithelium in healthy retina extending towards the border of the atrophy. Histological analysis revealed RPE interaction with host photoreceptors in the healthy retina. Engrafted cells in the atrophic zone were found in a patchy distribution but failed to form an epithelial-like layer. CONCLUSIONS: These results might support the use of hiPSC-RPE cells to treat atrophic GA by providing a housekeeping function to aid the overwhelmed remnant RPE, which might improve its survival and therefore slow down the progression of GA.

Evaluation and Optimization of Poly-d-Lysine as a Non-Natural Cationic Polypeptide for Gene Transfer in Neuroblastoma Cells.

Cationic polypeptides and cationic polymers have cell-penetrating capacities and have been used in gene transfer studies. In this study, we investigate the capability of a polymer of d-lysine (PDL), a chiral form of α-Poly-lysine, as a possible nonviral vector for releasing genetic materials to neuroblastoma cells and evaluate its stability against proteases. We tested and compared its transfection effectiveness in vitro as a vehicle for the EGFP plasmid DNA (pDNA) reporter in the SH-SY5Y human neuroblastoma, HeLa, and 3T3 cell lines. Using fluorescent microscopy and flow cytometry, we demonstrated high transfection efficiencies based on EGFP fluorescence in SH-SY5Y cells, compared with HeLa and 3T3. Our results reveal PDL as an efficient vector for gene delivery specifically in the SH-SY5Y cell line and suggest that PDL can be used as a synthetic cell-penetrating polypeptide for gene therapy in neuroblastoma cells.

Development of a Prodrug of Camptothecin for Enhanced Treatment of Glioblastoma Multiforme.

A novel therapeutic approach for glioblastoma multiforme (GBM) therapy has been carried out through in vitro and in vivo testing by using the prodrug camptothecin-20-O-(5-aminolevulinate) (CPT-ALA). The incorporation of ALA to CPT may promote uptake of the cytotoxic molecule by glioblastoma cells where the heme synthesis pathway is active, improving the therapeutic action and reducing the side effects over healthy tissue. The antitumor properties of CPT-ALA have been tested on different GBM cell lines (U87, U251, and C6) as well as in an orthotopic GBM model in rat, where potential toxicity in central nervous system cells was analyzed. In vitro results indicated no significant differences in the cytotoxic effect over the different GBM cell lines for CPT and CPT-ALA, albeit cell mortality induced by CPT over normal cell lines was significantly higher than CPT-ALA. Moreover, intracranial GBM in rat was significantly reduced (30% volume) with 2 weeks of CPT-ALA treatment with no significant side effects or alterations to the well-being of the animals tested. 5-ALA moiety enhances CPT diffusion into tumors due to solubility improvement and its metabolic-based targeting, increasing the CPT cytotoxic effect on malignant cells while reducing CPT diffusion to other proliferative healthy tissue. We demonstrate that CPT-ALA blocks proliferation of GBM cells, reducing the infiltrative capacity of GBM and promoting the success of surgical removal, which improves life expectancy by reducing tumor recurrence.

Brain Angiogenesis Induced by Nonviral Gene Therapy with Potential Therapeutic Benefits for Central Nervous System Diseases.

Gene therapy employing nanocarriers represents a promising strategy to treat central nervous system (CNS) diseases, where brain microvasculature is frequently compromised. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule; however, its in vivo administration to the CNS by nonviral gene therapy has not been conducted. Hence, we prepared and physicochemically characterized four cationic niosome formulations (1-4), which were combined with pVEGF-GFP to explore their capacity to transfer the VEGF gene to CNS cells and achieve angiogenesis in the brain. Experiments in primary neuronal cells showed successful and safe transfection with niosome 4, producing double levels of biologically active VEGF in comparison to the rest of the formulations. Intracortical administration of niosome 4 based nioplexes in mouse brain validated the ability of this nonviral vector to deliver the VEGF gene to CNS cells, inducing brain angiogenesis and emerging as a promising therapeutic approach for the treatment of CNS diseases.

Niosome-Based Approach for In Situ Gene Delivery to Retina and Brain Cortex as Immune-Privileged Tissues.

Non-viral vectors have emerged as a promising alternative to viral gene delivery systems due to their safer profile. Among non-viral vectors, recently, niosomes have shown favorable properties for gene delivery, including low toxicity, high stability, and easy production. The three main components of niosome formulations include a cationic lipid that is responsible for the electrostatic interactions with the negatively charged genetic material, a non-ionic surfactant that enhances the long-term stability of the niosome, and a helper component that can be added to improve its physicochemical properties and biological performance. This review is aimed at providing recent information about niosome-based non-viral vectors for gene delivery purposes. Specially, we will discuss the composition, preparation methods, physicochemical properties, and biological evaluation of niosomes and corresponding nioplexes that result from the addition of the genetic material onto their cationic surface. Next, we will focus on the in situ application of such niosomes to deliver the genetic material into immune-privileged tissues such as the brain cortex and the retina. Finally, as future perspectives, non-invasive administration routes and different targeting strategies will be discussed.

A Chronic Ocular-Hypertensive Rat Model induced by Injection of the Sclerosant Agent Polidocanol in the Aqueous Humor Outflow Pathway.

BACKGROUND: To induce a moderate chronic ocular hypertension (OHT) by injecting polidocanol, a foamed sclerosant drug, in the aqueous humor outflow pathway. METHODS: Intraocular pressure (IOP) was monitored for up to 6 months. Pattern and full-field electroretinogram (PERG and ERG) were recorded and retinal ganglion cells (RGC) and retinal nerve fiber layer (RNFL) thickness were assessed in vivo with optical coherence tomography (OCT) and ex vivo using Brn3a immunohistochemistry. RESULTS: In the first 3 weeks post-injection, a significant IOP elevation was observed in the treated eyes (18.47 ± 3.36 mmHg) when compared with the control fellow eyes (12.52 ± 2.84 mmHg) (p < 0.05). At 8 weeks, 65% (11/17) of intervention eyes had developed an IOP increase >25% over the baseline. PERG responses were seen to be significantly reduced in the hypertensive eyes (2.25 ± 0.24 µV) compared to control eyes (1.44 ± 0.19 µV) (p < 0.01) at week 3, whereas the ERG components (photoreceptor a-wave and bipolar cell b-wave) remained unaltered. By week 24, RNFL thinning and cell loss in the ganglion cell layer was first detected (2/13, 15.3%) as assessed by OCT and light microscopy. CONCLUSIONS: This novel OHT rat model, with moderate levels of chronically elevated IOP, and abnormal PERG shows selective functional impairment of RGC.

Gene delivery to the rat retina by non-viral vectors based on chloroquine-containing cationic niosomes.

The incorporation of chloroquine within nano formulations, rather than as a co-treatment of the cells, could open a new avenue for in vivo retinal gene delivery. In this manuscript, we evaluated the incorporation of chloroquine diphosphate into the cationic niosome formulation composed of poloxamer 188, polysorbate 80 non-ionic surfactants, and 2,3-di (tetradecyloxy) propan-1-amine (hydrochloride salt) cationic lipid, to transfect rat retina. Niosome formulations without and with chloroquine diphosphate (DPP80, and DPP80-CQ, respectively) were prepared by the reverse phase evaporation technique and characterized in terms of size, PDI, zeta potential, and morphology. After the incorporation of the pCMS-EGFP plasmid, the resultant nioplexes -at different cationic lipid/DNA mass ratios- were further evaluated to compact, liberate, and secure the DNA against enzymatic digestion. In vitro procedures were achieved in ARPE-19 cells to assess transfection efficacy and intracellular transportation. Both nioplexes formulations transfected efficiently ARPE-19 cells, although the cell viability was clearly better in the case of DPP80-CQ nioplexes. After subretinal and intravitreal injections, DPP80 nioplexes were not able to transfect the rat retina. However, chloroquine containing vector showed protein expression in many retinal cells, depending on the administration route. These data provide new insights for retinal gene delivery based on chloroquine-containing niosome non-viral vectors.

Non-viral vectors based on cationic niosomes and minicircle DNA technology enhance gene delivery efficiency for biomedical applications in retinal disorders.

Low transfection efficiency is a major challenge to overcome in non-viral approaches to reach clinical practice. Our aim was to explore new strategies to achieve more efficient non-viral gene therapies for clinical applications and in particular, for retinal diseases. Cationic niosomes and three GFP-encoding genetic materials consisting on minicircle (2.3 kb), its parental plasmid (3.5 kb) and a larger plasmid (5.5 kb) were combined to form nioplexes. Once fully physicochemically characterized, in vitro experiments in ARPE-19 retina epithelial cells showed that transfection efficiency of minicircle nioplexes doubled that of plasmids ones, maintaining good cell viability in all cases. Transfections in retinal primary cells and injections of nioplexes in rat retinas confirmed the higher capacity of cationic niosomes vectoring minicircle to deliver the genetic material into retina cells. Therefore, nioplexes based on cationic niosomes vectoring minicircle DNA represent a potential tool for the treatment of inherited retinal diseases.

Gene transfer to rat cerebral cortex mediated by polysorbate 80 and poloxamer 188 nonionic surfactant vesicles.

BACKGROUND: Gene therapy can be an intriguing therapeutic option in wide-ranging neurological disorders. Though nonviral gene carriers represent a safer delivery system to their viral counterparts, a thorough design of such vehicles is crucial to enhance their transfection properties. PURPOSE: This study evaluated the effects of combined use of two nonionic surfactants, poloxamer 188 (P) and polysorbate 80 (P80) into nanovesicles - based on 2,3-di(tetradecyloxy)propan-1-amine cationic lipid (D) - destined for gene delivery to central nervous system cells. METHODS: Niosome formulations without and with poloxamer 188 (DP80 and DPP80, respectively) were prepared by the reverse-phase evaporation technique and characterized in terms of size, surface charge, and morphology. After the addition of pCMS-EGFP plasmid, the binding efficiency to the niosomes was evaluated in agarose gel electrophoresis assays. Additionally, transfection efficiency of complexes was also evaluated in in vitro and in vivo conditions. RESULTS: In vitro experiments on NT2 cells revealed that the complexes based on a surfactant combination (DPP80) enhanced cellular uptake and viability when compared with the DP80 counterparts. Interestingly, DPP80 complexes showed protein expression in glial cells after administration into the cerebral cortices of rats. CONCLUSION: These data provide new insights for glia-centered approach for gene therapy of nervous system disorders using cationic nanovesicles, where nonionic surfactants play a pivotal role.

Non-viral vectors based on cationic niosomes as efficient gene delivery vehicles to central nervous system cells into the brain.

Development of safe and efficient non-viral vectors to deliver DNA into the CNS represents a huge challenge to face many neurological disorders. We elaborated niosomes based on DOTMA cationic lipid, lycopene "helper" lipid and polysorbate 60 as non-ionic surfactants for gene delivery to the CNS. Niosomes, and their corresponding nioplexes obtained after the addition of the pCMS-EGFP plasmid, were characterized in terms of size, charge, morphology and capacity to condense, release and protect DNA. In vitro experiments were performed in NT2 cells to evaluate transfection efficiency, viability, cellular uptake and intracellular distribution. Additionally, transfection in primary cortex cells were performed prior to brain administration into rat cerebral cortex. Data obtained showed that nioplexes exhibited not only adequate physicochemical properties for gene delivery applications, but also relevant transfection efficiencies (17%), without hampering viability (90%). Interestingly, In vivo experiments depicted promising protein expression in both cortical glial cells and blood vessels.