RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(β-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.

The combination of hydrogels and magnetic nanoparticles, scarcely explored to date, offers a wide range of possibilities for innovative therapies. Herein, we have designed hybrid 3D matrices integrating natural polymers, such as collagen, chitosan (CHI) and hyaluronic acid (HA), to provide soft and flexible 3D networks mimicking the extracellular matrix of natural tissues, and iron oxide nanoparticles (IONPs) that deliver localized heat when exposed to an alternating magnetic field (AMF). First, colloidally stable nanoparticles with a hydrodynamic radius of ∼20 nm were synthesized and coated with either CHI (NPCHI) or HA (NPHA). Then, collagen hydrogels were homogeneously loaded with these coated-IONPs resulting in soft (E0 ∼ 2.6 kPa), biodegradable and magnetically responsive matrices. Polymer-coated IONPs in suspension preserved primary neural cell viability and neural differentiation even at the highest dose (0.1 mg Fe/mL), regardless of the coating, even boosting neuronal interconnectivity at lower doses. Magnetic hydrogels maintained high neural cell viability and sustained the formation of highly interconnected and differentiated neuronal networks. Interestingly, those hydrogels loaded with the highest dose of NPHA (0.25 mgFe/mg polymer) significantly impaired non-neuronal differentiation with respect to those with NPCHI. When evaluated under AMF, cell viability slightly diminished in comparison with control hydrogels magnetically stimulated, but not compared to their counterparts without stimulation. Neuronal differentiation under AMF was only affected on collagen hydrogels with the highest dose of NPHA, while non-neuronal differentiation regained control values. Taken together, NPCHI-loaded hydrogels displayed a superior performance, maybe benefited from their higher nanomechanical fluidity. STATEMENT OF SIGNIFICANCE: Hydrogels and magnetic nanoparticles are undoubtedly useful biomaterials for biomedical applications. Nonetheless, the combination of both has been scarcely explored to date. In this study, we have designed hybrid 3D matrices integrating both components as promising magnetically responsive platforms for neural therapeutics. The resulting collagen scaffolds were soft (E0 ∼ 2.6 kPa) and biodegradable hydrogels with capacity to respond to external magnetic stimuli. Primary neural cells proved to grow on these substrates, preserving high viability and neuronal differentiation percentages even under the application of a high-frequency alternating magnetic field. Importantly, those hydrogels loaded with chitosan-coated iron oxide nanoparticles displayed a superior performance, likely related to their higher nanomechanical fluidity.

Single-cell RNA sequencing (scRNA-seq) provides a powerful tool to evaluate the transcriptomic landscape and heterogeneity of thousands of cells in parallel. However, complex study designs or the unavailability of in-house instruments require the temporal disconnection between sample preparation and library construction, raising the need for efficient sample preservation methods which are compatible with scRNA-seq downstream analysis. Several studies evaluated the effect of methanol fixation as preservation method, yet none of them deeply assessed its effect on adult primary dissociated brain tissue. Here, we evaluated its effect on murine dentate gyrus (DG) single cell suspensions and on subsequent scRNA-seq downstream analysis by performing SOrting and Robot-assisted Transcriptome SEQuencing (SORT-seq), a partially robotized version of the CEL-seq2 protocol. Our results show that MeOH fixation preserves RNA integrity and has no apparent effects on cDNA library construction. They also suggest that fixation protects from sorting-induced cell stress and increases the proportion of high-quality cells. Despite evidence of mRNA leakage in fixed cells, their relative gene expression levels correlate well with those of fresh cells and fixation does not significantly affect the variance of the dataset. Moreover, it allows the identification of all major DG cell populations, including neural precursors, granule neurons and different glial cell types, with a tendency to preserve more neurons that are underrepresented in fresh samples. Overall, our data show that MeOH fixation is suitable for preserving primary neural cells for subsequent single-cell RNA profiling, helping to overcome challenges arising from complex workflows, improve experimental flexibility and facilitate scientific collaboration.

Neural injuries disrupt the normal functions of the nervous system, whose complexities limit current treatment options. Because of their enhanced therapeutic effects, neurospheres have the potential to advance the field of regenerative medicine and neural tissue engineering. Methodological steps can pose challenges for implementing neurosphere assemblies; for example, conventional static cultures hinder yield and throughput, while the presence of the necrotic core, time-consuming methodology, and high variability can slow their progression to clinical application. Here we demonstrate the optimization of primary neural cell-derived neurospheres, developed using a high-throughput, stress-free, 3D bioreactor. This process provides a necessary baseline for future studies that could develop co-cultured assemblies of stem cells combined with endothelial cells, and/or biomaterials and nanomaterials for clinical therapeutic use. Neurosphere size and neurite spreading were evaluated under various conditions using Image J software. Primary neural cells obtained from the hippocampi of three-day-old rat pups, when incubated for 24 h in a reactor coated with 2% Pluronic and seeded on Poly-D-Lysine-coated plates establish neurospheres suitable for therapeutic use within five days. Most notably, neurospheres maintained high cell viability of ≥84% and expressed the neural marker MAP2, neural marker β-Tubulin III, and glial marker GFAP at all time points when evaluated over seven days. Establishing these factors reduces the variability in developing neurospheres, while increasing the ease and output of the culture process and maintaining viable cellular constructs.