Near-Infrared Light Activated Formulation for the Spatially Controlled Release of CRISPR-Cas9 Ribonucleoprotein for Brain Gene Editing.

The CRISPR/Cas9 system has emerged as a promising platform for gene editing; however, the lack of an efficient and safe delivery system to introduce it into cells continues to hinder clinical translation. Here, we report a rationally designed gene-editing nanoparticle (NP) formulation for brain applications: an sgRNA:Cas9 ribonucleoprotein complex is immobilized on the NP surface by oligonucleotides that are complementary to the sgRNA. Irradiation of the formulation with a near-infrared (NIR) laser generates heat in the NP, leading to the release of the ribonucleoprotein complex. The gene-editing potential of the formulation was demonstrated in vitro at the single-cell level. The safety and gene editing of the formulation were also demonstrated in the brains of reporter mice, specifically in the subventricular zone after intracerebral administration and in the olfactory bulb after intranasal administration. The formulation presented here offers a new strategy for the spatially controlled delivery of the CRISPR system to the brain.

Extraction of Gallic Acid and Ferulic Acid for Application in Hair Supplements.

Food supplements based on antioxidants and vitamins are often prescribed to correct inefficiencies in the human diet and delay diseases such as premature aging and alopecia (temporary or permanent hair loss), given the free radical scavenging activity of these biomolecules. By reducing the concentration of reactive oxygen species (ROS), which promote abnormal hair follicle cycling and morphology, follicle inflammation and oxidative stress are reduced, minimising the effects of these health issues. Gallic acid (GA), which is significantly present in gallnuts and in pomegranate root bark, and ferulic acid (FA), commonly found in brown rice and coffee seeds, are very important antioxidants for the preservation of hair colour, strength and growth. In this work, these two secondary phenolic metabolites were successfully extracted in the Aqueous Two-Phase Systems (ATPS) {ethyl lactate (1) + trisodium citrate (2) + water (3)} and {ethyl lactate (1) + tripotassium citrate (2) + water (3)} at 298.15 K and 0.1 MPa, moving towards the application of these ternary systems in extracting antioxidants from biowaste and their a posteriori processing as food supplements for hair fortification. The studied ATPS provided biocompatible and sustainable media for the extraction of gallic acid and ferulic acid, yielding low mass losses (<3%) and contributing to an eco-friendlier production of therapeutics. The most promising results were obtained for ferulic acid, which attained maximum partition coefficients (K) of 15 ± 5 and (3 ± 2) · 101 and maximum extraction efficiencies (E) of (92.7 ± 0.4)% and (96.7 ± 0.4)% for the longest tie-lines (TLL = 69.68 and 77.66 m%) in {ethyl lactate (1) + trisodium citrate (2) + water (3)} and {ethyl lactate (1) + tripotassium citrate (2) + water (3)}, respectively. Moreover, the effect of pH on the UV-Vis absorbance spectra was studied for all the biomolecules to minimise errors in solute quantification. Both GA and FA were found to be stable at the used extractive conditions.

A high-throughput screening platform to identify nanocarriers for efficient delivery of RNA-based therapies.

RNA-based therapies are highly selective and powerful regulators of biological functions. Non-viral vectors such as nanoparticles (NPs) are very promising formulations for the delivery of RNA-based therapies but their cell targeting, cell internalization and endolysomal escape capacity is rather limited. Here, we present a methodology that combines high-throughput synthesis of light-triggerable NPs and a high-content imaging screening to identify NPs capable of efficiently delivering different type of RNAs. The NPs were generated using polymers synthesized by Michael type addition reactions and they were designed to: (i) efficiently complex coding (mRNAs) and non-coding (miRNAs and/or lncRNAs) RNA molecules, (ii) allow rapid cell uptake and cytoplasmic release of RNA molecules and (iii) target different cell types based on their composition. Furthermore, light-responsive domains were attached to the polymers by distinctive methods to provide diverse disassembly strategies. The most efficient formulations were identified using cell-based assays and high-content imaging analysis. This strategy allows precise delivery of RNA-based therapies and provides an effective design approach to address critical issues in non-viral gene delivery.

A Light-Triggerable Nanoparticle Library for the Controlled Release of Non-Coding RNAs.

RNA-based therapies offer a wide range of therapeutic interventions including the treatment of skin diseases; however, the strategies to efficiently deliver these biomolecules are still limited due to obstacles related to the cellular uptake and cytoplasmic delivery. Herein, we report the synthesis of a triggerable polymeric nanoparticle (NP) library composed of 160 formulations, presenting physico-chemical diversity and differential responsiveness to light. Six formulations were more efficient (up to 500 %) than commercially available lipofectamine in gene-knockdown activity. These formulations showed differential internalization by skin cells and the endosomal escape was rapid (minutes range). The NPs were effective in the release of siRNA and miRNA. Acute skin wounds treated with the top hit NP complexed with miRNA-150-5p healed faster than wounds treated with scrambled miRNA. Light-activatable NPs offer a new strategy to topically deliver non-coding RNAs.

A Polymeric Nanoparticle Formulation for Targeted mRNA Delivery to Fibroblasts.

Messenger RNA (mRNA)-based therapies offer enhanced control over the production of therapeutic proteins for many diseases. Their clinical implementation warrants formulations capable of delivering them safely and effectively to target sites. Owing to their chemical versatility, polymeric nanoparticles can be designed by combinatorial synthesis of different ionizable, cationic, and aromatic moieties to modulate cell targeting, using inexpensive formulation steps. Herein, 152 formulations are evaluated by high-throughput screening using a reporter fibroblast model sensitive to functional delivery of mRNA encoding Cre recombinase. Using in vitro and in vivo models, a polymeric nanoformulation based on the combination of 3 specific monomers is identified to transfect fibroblasts much more effectively than other cell types populating the skin, with superior performance than lipid-based transfection agents in the delivery of Cas9 mRNA and guide RNA. This tropism can be explained by receptor-mediated endocytosis, involving CD26 and FAP, which are overexpressed in profibrotic fibroblasts. Structure-activity analysis reveals that efficient mRNA delivery required the combination of high buffering capacity and low mRNA binding affinity for rapid release upon endosomal escape. These results highlight the use of high-throughput screening to rapidly identify chemical features towards the design of highly efficient mRNA delivery systems targeting fibrotic diseases.

Engineering optical tools for remotely controlled brain stimulation and regeneration.

Neurological disorders are one of the world's leading medical and societal challenges due to the lack of efficacy of the first line treatment. Although pharmacological and non-pharmacological interventions have been employed with the aim of regulating neuronal activity and survival, they have failed to avoid symptom relapse and disease progression in the vast majority of patients. In the last 5 years, advanced drug delivery systems delivering bioactive molecules and neuromodulation strategies have been developed to promote tissue regeneration and remodel neuronal circuitry. However, both approaches still have limited spatial and temporal precision over the desired target regions. While external stimuli such as electromagnetic fields and ultrasound have been employed in the clinic for non-invasive neuromodulation, they do not have the capability of offering single-cell spatial resolution as light stimulation. Herein, we review the latest progress in this area of study and discuss the prospects of using light-responsive nanomaterials to achieve on-demand delivery of drugs and neuromodulation, with the aim of achieving brain stimulation and regeneration.

Efficient spatially targeted gene editing using a near-infrared activatable protein-conjugated nanoparticle for brain applications.

Spatial control of gene expression is critical to modulate cellular functions and deconstruct the function of individual genes in biological processes. Light-responsive gene-editing formulations have been recently developed; however, they have shown limited applicability in vivo due to poor tissue penetration, limited cellular transfection and the difficulty in evaluating the activity of the edited cells. Here, we report a formulation composed of upconversion nanoparticles conjugated with Cre recombinase enzyme through a photocleavable linker, and a lysosomotropic agent that facilitates endolysosomal escape. This formulation allows in vitro spatial control in gene editing after activation with near-infrared light. We further demonstrate the potential of this formulation in vivo through three different paradigms: (i) gene editing in neurogenic niches, (ii) gene editing in the ventral tegmental area to facilitate monitoring of edited cells by precise optogenetic control of reward and reinforcement, and (iii) gene editing in a localized brain region via a noninvasive administration route (i.e., intranasal).

Medical sharps in Portugal: a cross-sectional survey of disposal practices among the diabetic population.

OBJECTIVE: We aim to determine the disposal site for biohazardous materials resulting from diabetes surveillance and therapy. DESIGN: Cross-sectional study. SETTING: Five Portuguese primary care facilities. PARTICIPANTS: We randomly sampled diabetic patients representative of five primary care facilities. Inclusion criteria consisted in patients≥18 years old with an active diagnosis of diabetes mellitus (DM). Patients unable to provide written informed consent were excluded. OUTCOME MEASURE: Sociodemographic variables, diabetes duration, type of treatment, medical sharps disposal practices and whether adequate disposal information were provided. RESULTS: A total of 1436 diabetics were included. Overall, 53.8% of diabetics conducted regular capillary glicemia measurements, although 45.3% of them had no medical indication. Statistically significant predictors of adequate disposal were not having an active professional status (p=0.011) and having a DM duration between 5 and 10 years (p=0.014). Only being professionally inactive remained an independent predictor after multivariate logistic regression. Less than a fifth of patients on injectable therapy report having been advised by healthcare staff regarding sharps disposal. Over a fifth of the latter report having received wrong advice. The majority of diabetics dispose of biohazardous materials in unsorted household waste (68.1% of needles/devices with needles and 71.6% of lancets). Other incorrect disposal sites identified were recycling bins, toilet and home accumulation. Only 19.1% of the needles/devices with needles and 13.1% of the lancets were disposed of at healthcare facilities. CONCLUSIONS: Most diabetics have unsafe disposal practices for their biohazardous materials, mostly in unsorted household waste. We identified that being unemployed independently predicts adequate disposal of medical sharps and found evidence of low patient literacy on the topic, as well as poor patient education. Therefore, educating and raising awareness among healthcare professionals is crucial to address this public health issue.

A light-triggerable formulation to control the stability of pro-angiogenic transcription factor hypoxia inducible factor-1α (HIF-1α).

The control of vascular remodeling mediated by transcription factor HIF-1α is critical in the treatment of several diseases including cancer, retinopathies, chronic wounds, and ischemic heart disease, among others. Gene silencing using a small interfering RNA (siRNA) is a promising therapeutic strategy to regulate HIF-1α; however, the delivery systems developed so far have limited endothelial targeting and efficiency. Herein, we have synthesized a light-triggerable polymeric nanoparticle (NP) library composed of 110 formulations which showed variable morphology, charge and disassembly rates after UV exposure. More than 35% of the formulations of the library were more efficient in gene knockdown than the siRNA delivered by a commercial transfection agent (lipofectamine RNAiMAX). The most efficient siRNA delivery formulations were tested against different cell types to identify one with preferential targeting to endothelial cells. Using a two-step methodology, we have identified a formulation that shows exquisite targeting to endothelial cells and is able to deliver more efficiently the siRNA that modulates HIF-1α than commercial transfection agents. Overall, the strategy reported here increases the specificity for tissue regulation and the efficiency for the intracellular delivery of siRNAs.

Stem cells as vehicles and targets of nanoparticles.

Modulation of endogenous adult stem cell niches represents a promising strategy for regeneration of tissues and to correct cell abnormalities, including cancer. Recent advances show the possibility to target endogenous stem cells or their progenies by using nanoparticles conjugated with specific biomolecules. In addition, the targeting of the stem cell niche can be accomplished by using stem cells loaded with nanoparticles. This review examines principles for the targeting of endogenous stem cells as well as factors for the modulation of stem cells.

Interference of cold agglutinin autoantibodies in erythrogram interpretation: a case report and literature review / Interferências das crioaglutininas na interpretação do eritrograma: relato de caso com revisão da literatura

ABSTRACT The erythrogram is one of the components of the blood count that includes red blood cell (RBC) quantification and evaluation. A correct interpretation and validation of the results obtained in an erythrogram require experience and critical awareness of health professionals. It is imperative to evaluate the interference of physiological variables, collection procedures, manipulation of samples and endogenous variables (such as the presence of cold agglutinin autoantibodies), since these may falsify the results obtained. Cold agglutinin autoantibodies are predominantly immunoglobulin type M (IgM), which cause agglutination of RBC at temperatures below 37°C, and may appear in cases of autoimmune hemolytic anemia and atypical pneumonia, among other pathologies. The presence of erythrocyte agglutination interferes with RBC and reticulocyte counts, determination of the globular volume and the blood count indices. A set of laboratorial procedures may be performed in order to eliminate the interference of these agglutinins in the results of the erythrogram. If these procedures do not correct the values obtained, the only result of the erythrogram that can be validated is hemoglobin, since the remaining results are falsified due to the presence of cold agglutinin autoantibodies.

RESUMO O eritrograma é um dos componentes do hemograma que inclui a quantificação e a avaliação eritrocitária. Uma correta interpretação e validação dos resultados obtidos em um eritrograma requer experiência e sentido crítico dos profissionais de saúde. Torna-se imperativo avaliar a interferência de variáveis fisiológicas e de colheita, a manipulação das amostras e as variáveis endógenas (como a presença de crioaglutininas), uma vez que estas podem falsear os resultados obtidos. As crioaglutininas são autoanticorpos predominantemente do tipo imunoglobulina da classe M (IgM), as quais provocam aglutinação dos eritrócitos a temperaturas inferiores a 37°C, podendo aparecer em casos de anemia hemolítica autoimune e pneumonias atípicas, entre outras patologias. A presença de aglutinação eritrocitária interfere na contagem de eritrócitos, reticulócitos, determinação do volume globular e dos índices hematimétricos. Laboratorialmente, existe um conjunto de procedimentos que podem ser executados de modo a eliminar a interferência dessas aglutininas nos resultados do eritrograma. Caso esses procedimentos não corrijam os valores obtidos, o único resultado do eritrograma que poderá ser validado é o da hemoglobina, visto que os resultados restantes estão falseados devido à presença de crioaglutininas.