Enhancing Neuronal Cell Uptake of Therapeutic Nucleic Acids with Tetrahedral DNA Nanostructures.

The successful translation of therapeutic nucleic acids (NAs) for the treatment of neurological disorders depends on their safe and efficient delivery to neural cells, in particular neurons. DNA nanostructures can be a promising NAs delivery vehicle. Nonetheless, the potential of DNA nanostructures for neuronal cell delivery of therapeutic NAs is unexplored. Here, tetrahedral DNA nanostructures (TDN) as siRNA delivery scaffolds to neuronal cells, exploring the influence of functionalization with two different reported neuronal targeting ligands: C4-3 RNA aptamer and Tet1 peptide are investigated. Nanostructures are characterized in vitro, as well as in silico using molecular dynamic simulations to better understand the overall TDN structural stability. Enhancement of neuronal cell uptake of TDN functionalized with the C4-3 Aptamer (TDN-Apt), not only in neuronal cell lines but also in primary neuronal cell cultures is demonstrated. Additionally, TDN and TDN-Apt nanostructures carrying siRNA are shown to promote silencing in a process aided by chloroquine-induced endosomal disruption. This work presents a thorough workflow for the structural and functional characterization of the proposed TDN as a nano-scaffold for neuronal delivery of therapeutic NAs and for targeting ligands evaluation, contributing to the future development of new neuronal drug delivery systems based on DNA nanostructures.

Laminin-Inspired Cell-Instructive Microenvironments for Neural Stem Cells.

Laminin is a heterotrimeric glycoprotein with a key role in the formation and maintenance of the basement membrane architecture and properties, as well as on the modulation of several biological functions, including cell adhesion, migration, differentiation and matrix-mediated signaling. In the central nervous system (CNS), laminin is differentially expressed during development and homeostasis, with an impact on the modulation of cell function and fate. Within neurogenic niches, laminin is one of the most important and well described extracellular matrix (ECM) proteins. Specifically, efforts have been made to understand laminin assembly, domain architecture, and interaction of its different bioactive domains with cell surface receptors, soluble signaling molecules, and ECM proteins, to gain insight into the role of this ECM protein and its receptors on the modulation of neurogenesis, both in homeostasis and during repair. This is also expected to provide a rational basis for the design of biomaterial-based matrices mirroring the biological properties of the basement membrane of neural stem cell niches, for application in neural tissue repair and cell transplantation. This review provides a general overview of laminin structure and domain architecture, as well as the main biological functions mediated by this heterotrimeric glycoprotein. The expression and distribution of laminin in the CNS and, more specifically, its role within adult neural stem cell niches is summarized. Additionally, a detailed overview on the use of full-length laminin and laminin derived peptide/recombinant laminin fragments for the development of hydrogels for mimicking the neurogenic niche microenvironment is given. Finally, the main challenges associated with the development of laminin-inspired hydrogels and the hurdles to overcome for these to progress from bench to bedside are discussed.

Delivery of Splice Switching Oligonucleotides by Amphiphilic Chitosan-Based Nanoparticles.

Splice switching oligonucleotides (SSOs) are a class of single-stranded antisense oligonucleotides (ssONs) being used as gene therapeutics and demonstrating great therapeutic potential. The availability of biodegradable and biocompatible delivery vectors that could improve delivery efficiencies, reduce dosage, and, in parallel, reduce toxicity concerns could be advantageous for clinical translation. In this work we explored the use of quaternized amphiphilic chitosan-based vectors in nanocomplex formation and delivery of splice switching oligonucleotides (SSO) into cells, while providing insights regarding cellular uptake of such complexes. Results show that the chitosan amphiphilic character is important when dealing with SSOs, greatly improving colloidal stability under serum conditions, as analyzed by dynamic light scattering, and enhancing cellular association. Nanocomplexes were found to follow an endolysosomal route with a long lysosome residence time. Conjugation of a hydrophobic moiety, stearic acid, to quaternized chitosan was a necessary condition to achieve transfection, as an unmodified quaternary chitosan was completely ineffective. We thus demonstrate that amphiphilic quaternized chitosan is a biomaterial that holds promise and warrants further development as a platform for SSO delivery strategies.

Biological Response Following the Systemic Injection of PEG-PAMAM-Rhodamine Conjugates in Zebrafish.

Numerous therapeutic and diagnostic approaches used within a clinical setting depend on the administration of compounds via systemic delivery. Biomaterials at the nanometer scale, as dendrimers, act as delivery systems by improving cargo bioavailability, circulation time, and the targeting of specific tissues. Although evaluating the efficacy of pharmacological agents based on nanobiomaterials is crucial, conducting toxicological assessments of biomaterials is essential for advancing clinical translation. Here, a zebrafish larvae model was explored to assess the biocompatibility of poly(amido amine) (PAMAM), one of the most exploited dendrimers for drug delivery. We report the impact of a systemic injection of polyethylene glycol (PEG)-modified G4 PAMAM conjugated with rhodamine (Rho) as a mimetic drug (PEG-PAMAM-Rho) on survival, animal development, inflammation, and neurotoxicity. A concentration- and time-dependent effect was observed on mortality, developmental morphology, and innate immune system activation (macrophages). Significant effects in toxicological indicators were reported in the highest tested concentration (50 mg/mL PEG-PAMAM-Rho) as early as 48 h post-injection. Additionally, a lower concentration of PEG-PAMAM-Rho (5 mg/mL) was found to be safe and subsequently tested for neurotoxicity through behavioral assays. In accordance, no significative signs of toxicity were detected. In conclusion, the dose response of the animal was assessed, and the safe dosage for future use in theragnostics was defined. Additionally, new methodologies were established that can be adapted to further studies in toxicology using other nanosystems for systemic delivery.

Dendrimers and Derivatives as Multifunctional Nanotherapeutics for Alzheimer's Disease.

Alzheimer's disease (AD) is the most prevalent form of dementia. It affects more than 30 million people worldwide and costs over US$ 1.3 trillion annually. AD is characterized by the brain accumulation of amyloid ß peptide in fibrillar structures and the accumulation of hyperphosphorylated tau aggregates in neurons, both leading to toxicity and neuronal death. At present, there are only seven drugs approved for the treatment of AD, of which only two can slow down cognitive decline. Moreover, their use is only recommended for the early stages of AD, meaning that the major portion of AD patients still have no disease-modifying treatment options. Therefore, there is an urgent need to develop efficient therapies for AD. In this context, nanobiomaterials, and dendrimers in particular, offer the possibility of developing multifunctional and multitargeted therapies. Due to their intrinsic characteristics, dendrimers are first-in-class macromolecules for drug delivery. They have a globular, well-defined, and hyperbranched structure, controllable nanosize and multivalency, which allows them to act as efficient and versatile nanocarriers of different therapeutic molecules. In addition, different types of dendrimers display antioxidant, anti-inflammatory, anti-bacterial, anti-viral, anti-prion, and most importantly for the AD field, anti-amyloidogenic properties. Therefore, dendrimers can not only be excellent nanocarriers, but also be used as drugs per se. Here, the outstanding properties of dendrimers and derivatives that make them excellent AD nanotherapeutics are reviewed and critically discussed. The biological properties of several dendritic structures (dendrimers, derivatives, and dendrimer-like polymers) that enable them to be used as drugs for AD treatment will be pointed out and the chemical and structural characteristics behind those properties will be analysed. The reported use of these nanomaterials as nanocarriers in AD preclinical research is also presented. Finally, future perspectives and challenges that need to be overcome to make their use in the clinic a reality are discussed.

Electrochemical miRNA-34a-based biosensor for the diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia type and a leading cause of death and disability in the elderly. Diagnosis is expensive and invasive, urging the development of new, affordable, and less invasive diagnostic tools. The identification of changes in the expression of non-coding RNAs prompts the development of diagnostic tools to detect disease-specific blood biomarkers. Building on this idea, this work reports a novel electrochemical microRNA (miRNA) biosensor for the diagnosis of AD, based on carbon screen-printed electrodes (C-SPEs) modified with two gold nanostructures and a complementary anti-miR-34a oligonucleotide probe. This biosensor showed good target affinity, reflected on a 100 pM to 1 µM linearity range and a limit of detection (LOD) of 39 pM in buffer and 94 aM in serum. Moreover, the biosensor's response was not affected by serum compounds, indicating selectivity for miR-34a. The biosensor also detected miR-34a in the cell culture medium of a common AD model, stimulated with a neurotoxin to increase miR-34a secretion. Overall, the proposed biosensor makes a solid case for the introduction of a novel, inexpensive, and minimally invasive tool for the early diagnosis of AD, based on the detection of a circulating miRNA overexpressed in this pathology.

Supramolecular presentation of bioinstructive peptides on soft multilayered nanobiomaterials stimulates neurite outgrowth.

Peptide amphiphiles (PAs) have emerged as effective molecular building blocks for creating self-assembling nanobiomaterials for multiple biomedical applications. Herein, we report a straightforward approach to assemble soft bioinstructive platforms to recreate the native neural extracellular matrix (ECM) aiming for neuronal regeneration based on the electrostatic-driven supramolecular presentation of laminin-derived IKVAV-containing self-assembling PA (IKVAV-PA) on biocompatible multilayered nanoassemblies. Spectroscopic and microscopic techniques show that the co-assembly of positively charged low-molecular-weight IKVAV-PA with oppositely charged high-molecular-weight hyaluronic acid (HA) triggers the formation of ordered ß-sheet structures denoting a one-dimensional nanofibrous network. The successful functionalization of poly(L-lysine)/HA layer-by-layer nanofilms with an outer positively charged layer of self-assembling IKVAV-PA is demonstrated by the quartz crystal microbalance with dissipation monitoring and the nanofibrous morphological properties revealed by atomic force microscopy. The bioactive ECM-mimetic supramolecular nanofilms promote the enhancement of primary neuronal cells' adhesion, viability, and morphology when compared to the PA without the IKVAV sequence and PA-free biopolymeric multilayered nanofilms, and stimulate neurite outgrowth. The nanofilms hold great promise as bioinstructive platforms for enabling the assembly of customized and robust multicomponent supramolecular biomaterials for neural tissue regeneration.

It takes two to remyelinate: A bioengineered platform to study astrocyte-oligodendrocyte crosstalk and potential therapeutic targets in remyelination.

The loss of the myelin sheath insulating axons is the hallmark of demyelinating diseases. These pathologies often lead to irreversible neurological impairment and patient disability. No effective therapies are currently available to promote remyelination. Several elements contribute to the inadequacy of remyelination, thus understanding the intricacies of the cellular and signaling microenvironment of the remyelination niche might help us to devise better strategies to enhance remyelination. Here, using a new in vitro rapid myelinating artificial axon system based on engineered microfibres, we investigated how reactive astrocytes influence oligodendrocyte (OL) differentiation and myelination ability. This artificial axon culture system enables the effective uncoupling of molecular cues from the biophysical properties of the axons, allowing the detailed study of the astrocyte-OL crosstalk. Oligodendrocyte precursor cells (OPCs) were cultured on poly(trimethylene carbonate-co-ε-caprolactone) copolymer electrospun microfibres that served as surrogate axons. This platform was then combined with a previously established tissue engineered glial scar model of astrocytes embedded in 1 % (w/v) alginate matrices, in which astrocyte reactive phenotype was acquired using meningeal fibroblast conditioned medium. OPCs were shown to adhere to uncoated engineered microfibres and differentiate into myelinating OL. Reactive astrocytes were found to significantly impair OL differentiation ability, after six and eight days in a co-culture system. Differentiation impairment was seen to be correlated with astrocytic miRNA release through exosomes. We found significantly reduction on the expression of pro-myelinating miRNAs (miR-219 and miR-338) and an increase in anti-myelinating miRNA (miR-125a-3p) content between reactive and quiescent astrocytes. Additionally, we show that OPC differentiation inhibition could be reverted by rescuing the activated astrocytic phenotype with ibuprofen, a chemical inhibitor of the small rhoGTPase RhoA. Overall, these findings show that modulating astrocytic function might be an interesting therapeutic avenue for demyelinating diseases. The use of these engineered microfibres as an artificial axon culture system will enable the screening for potential therapeutic agents that promote OL differentiation and myelination while providing valuable insight on the myelination/remyelination processes.

Oral vaccination based on DNA-chitosan nanoparticles against Schistosoma mansoni infection.

The development of a vaccine would be essential for the control of schistosomiasis, which is recognized as the most important human helminth infection in terms of morbidity and mortality. A new approach of oral vaccination with DNA-chitosan nanoparticles appears interesting because of their great stability and the ease of target accessibility, besides chitosan immunostimulatory properties. Here we described that chitosan nanoparticles loaded with plasmid DNA encoding Rho1-GTPase protein of Schistosoma mansoni, prepared at different molar ratios of primary amines to DNA phosphate anion (N/P), were able to complex electrostatically with DNA and condense it into positively charged nanostructures. Nanoparticles were able to maintain zeta potential and size characteristics in media that simulate gastric (SGF) and intestinal fluids (SIF). Further in vivo studies showed that oral immunization was not able to induce high levels of specific antibodies but induced high levels of the modulatory cytokine IL-10. This resulted in a significative reduce of liver pathology, although it could not protect mice of infection challenge with S. mansoni worms. Mice immunized only with chitosan nanoparticles presented 47% of protection against parasite infection, suggesting an important role of chitosan in inducing a protective immune response against schistosomiasis, which will be more explored in further studies.

Engineering a Novel Self-Assembled Multi-siRNA Nanocaged Architecture with Controlled Enzyme-Mediated siRNA Release.

The RNA interference (RNAi) chemical and structural design space has evolved since its original definitions. Although this has led to the development of RNAi molecules that are starting to address the issues of silencing efficiency and delivery to target organs and cells, there is an on-going interest to improve upon their properties to attain wider therapeutic applicability. Taking advantage of the flexibility given by DNA and RNA structural and chemical properties, we here investigated unconventional RNAi encoding structures, designated by caged-siRNA structures (CsiRNAs), to explore novel features that could translate into advantageous properties for cellular delivery and intracellular activity. Using the principles of controlled nucleic acid self-assembly, branched DNA-RNA hybrid intermediates were formed, ultimately leading to the assembly of a "closed" structure encompassing multiple RNAi units. The RNAi active regions are further triggered by an encoded RNAse H-mediated release mechanism, while the overall structure possesses easily addressable anchors for hybridization-based functionalization with active biological moieties. We confirmed the production of correct structures and demonstrated that the encoded RNAi sequences maintain gene silencing activity even within this novel unconventional nanoarchitecture, aided by the intracellularly triggered RNAse H release mechanism. With this design, functionalization is easily achieved with no negative effects on the silencing activity, warranting further development of these novel molecular structures as a multi-RNAi platform for therapeutic delivery.

A paradigm shift: Bioengineering meets mechanobiology towards overcoming remyelination failure.

Therapeutic strategies aimed at overcoming the loss of myelin sheath in central nervous system demyelinating diseases are often unsuccessful due to nescience underlying the mechanisms of remyelination failure. The environment surrounding a demyelination lesion is seen as a hostile terrain, characterized by factors that can inhibit myelin production by oligodendrocytes (OLs). The formation of a glial scar containing reactive astrocytes producing high amounts of altered matrix proteins can compromise OL remyelination. Allied to glial scar, mechanical properties of the tissue are altered. The paradigms in the remyelination failure are changing. We point mechanobiology as a missing key towards unravelling the nature of (de)myelination. Mechanical cues as stiffness, axonal tension or physical constraints are emerging as dictators of tissue homeostasis and pathology. Here we delve into an in-depth characterization of the preeminent models to study mechanobiology events of (de)myelination and remyelination. Alternatives to in vivo systems are provided, either through the exploration of simpler animal models, creation of in vitro models using tissue engineered approaches or through in silico tools. We discuss how bioengineering is being explored to generate relevant models to dissect new mechanobiology mechanisms and identify novel therapeutic targets, being expected to profoundly impact the treatment of demyelinating diseases.

Molecular recognition force spectroscopy: a new tool to tailor targeted nanoparticles.

The density of targeting moieties in a nanoparticle-based gene-delivery system has been shown to play a fundamental role in its vectoring performance. Here, molecular recognition force spectroscopy is proposed as a novel screening tool to optimize the density of targeting moieties of functionalized nanoparticles towards attaining cell-specific interaction. By tailoring the nanoparticle formulation, the unbinding event probability between nanoparticles tethered to an atomic force microscopy tip and neuronal cells is directly correlated to the nanoparticle gene-vectoring capacity. Additionally, new insights into protein-receptor interaction are revealed. This novel approach opens new avenues in the field of nanomedicine.

Gene delivery into mesenchymal stem cells: a biomimetic approach using RGD nanoclusters based on poly(amidoamine) dendrimers.

Poly(amidoamine) dendrimers (generations 5 and 6) with amine termini were conjugated with peptides containing the arginine-glycine-aspartic acid (RGD) sequence having in view their application as gene delivery vectors. The idea behind the work was to take advantage of the cationic nature of dendrimers and of the integrin targeting capabilities of the RGD motif to improve gene delivery. Dendrimers were used as scaffolds for RGD clustering and, by controlling the number of peptides (4, 8, and 16) linked to each dendrimer, it was possible to evaluate the effect of RGD density on the gene delivery process. The new vectors were characterized in respect to their ability to neutralize and compact plasmid DNA (pDNA). The complexes formed by the vectors and pDNA were studied concerning their size, zeta potential, capacity of being internalized by cells and ability of transferring genes. Transfection efficiency was analyzed, first, by using a pDNA encoding for Enhanced Green Fluorescent Protein and Firefly Luciferase and, second, by using a pDNA encoding for Bone Morphogenetic Protein-2. Gene expression in mesenchymal stem cells was enhanced using the new vectors in comparison to native dendrimers and was shown to be dependent on the electrostatic interaction established between the dendrimer moiety and the cell surface, as well as on the RGD density of nanoclusters. The use of dendrimer scaffolds for RGD cluster formation is a new approach that can be extended beyond gene delivery applications, whenever RGD clustering is important for modulating cellular responses.

Chitosan/siRNA nanoparticles biofunctionalize nerve implants and enable neurite outgrowth.

Microstructured 20 µm thick polymer filaments used as nerve implants were loaded with chitosan/siRNA nanoparticles to promote nerve regeneration and ensure local delivery of nanotherapeutics. The stable nanoparticles were rapidly internalized by cells and did not affect cell viability. Target mRNA was successfully reduced by 65-75% and neurite outgrowth was enhanced even in an inhibitory environment. This work, thus, supports the application of nanobiofunctionalized implants as a novel approach for spinal cord and nerve repair.

Data-Driven Modeling of the Cellular Pharmacokinetics of Degradable Chitosan-Based Nanoparticles.

Nanoparticle drug delivery vehicles introduce multiple pharmacokinetic processes, with the delivery, accumulation, and stability of the therapeutic molecule influenced by nanoscale processes. Therefore, considering the complexity of the multiple interactions, the use of data-driven models has critical importance in understanding the interplay between controlling processes. We demonstrate data simulation techniques to reproduce the time-dependent dose of trimethyl chitosan nanoparticles in an ND7/23 neuronal cell line, used as an in vitro model of native peripheral sensory neurons. Derived analytical expressions of the mean dose per cell accurately capture the pharmacokinetics by including a declining delivery rate and an intracellular particle degradation process. Comparison with experiment indicates a supply time constant, τ = 2 h. and a degradation rate constant, b = 0.71 h-1. Modeling the dose heterogeneity uses simulated data distributions, with time dependence incorporated by transforming data-bin values. The simulations mimic the dynamic nature of cell-to-cell dose variation and explain the observed trend of increasing numbers of high-dose cells at early time points, followed by a shift in distribution peak to lower dose between 4 to 8 h and a static dose profile beyond 8 h.

Receptor-mediated gene delivery using PAMAM dendrimers conjugated with peptides recognized by mesenchymal stem cells.

As mesenchymal stem cells (MSCs) can differentiate into multiple cell types, the delivery of exogenous genes to this type of cell can be an important tool in tissue regeneration and engineering. However transfection of MSCs using nonviral gene delivery vectors is difficult, the development of more efficient and safe DNA vehicles being necessary. Moreover, specific transfection of MSCs may be required to avoid unwanted side effects in other tissues. In this study, a novel family of gene delivery vectors based on poly(amidoamine) (PAMAM) dendrimers functionalized with peptides displaying high affinity toward MSCs was prepared. The vectors were characterized with respect to their ability to neutralize, bind and compact plasmid DNA (pDNA). The complexes formed between the vectors and pDNA were analyzed concerning their size, zeta-potential, capacity of being internalized by cells and transfection efficiency. These new vectors exhibited low cytotoxicity, receptor-mediated gene delivery into MSCs and transfection efficiencies superior to those presented by native dendrimers and by partially degraded dendrimers.

Breaking Barriers: Bioinspired Strategies for Targeted Neuronal Delivery to the Central Nervous System.

Central nervous system (CNS) disorders encompass a vast spectrum of pathological conditions and represent a growing concern worldwide. Despite the high social and clinical interest in trying to solve these pathologies, there are many challenges to bridge in order to achieve an effective therapy. One of the main obstacles to advancements in this field that has hampered many of the therapeutic strategies proposed to date is the presence of the CNS barriers that restrict the access to the brain. However, adequate brain biodistribution and neuronal cells specific accumulation in the targeted site also represent major hurdles to the attainment of a successful CNS treatment. Over the last few years, nanotechnology has taken a step forward towards the development of therapeutics in neurologic diseases and different approaches have been developed to surpass these obstacles. The versatility of the designed nanocarriers in terms of physical and chemical properties, and the possibility to functionalize them with specific moieties, have resulted in improved neurotargeted delivery profiles. With the concomitant progress in biology research, many of these strategies have been inspired by nature and have taken advantage of physiological processes to achieve brain delivery. Here, the different nanosystems and targeting moieties used to achieve a neuronal delivery reported in the open literature are comprehensively reviewed and critically discussed, with emphasis on the most recent bioinspired advances in the field. Finally, we express our view on the paramount challenges in targeted neuronal delivery that need to be overcome for these promising therapeutics to move from the bench to the bedside.

Molecular Recognition Force Spectroscopy for Probing Cell Targeted Nanoparticles In Vitro.

In the development and design of cell targeted nanoparticle-based systems the density of targeting moieties plays a fundamental role in allowing maximal cell-specific interaction. Here, we describe the use of molecular recognition force spectroscopy as a valuable tool for the characterization and optimization of targeted nanoparticles toward attaining cell-specific interaction. By tailoring the density of targeting moieties at the nanoparticle surface, one can correlate the unbinding event probability between nanoparticles tethered to an atomic force microscopy tip and cells to the nanoparticle vectoring capacity. This novel approach allows for a rapid and cost-effective design of targeted nanomedicines reducing the need for long and tedious in vitro tests.

Delivery of Antisense Oligonucleotides Mediated by a Hydrogel System: In Vitro and In Vivo Application in the Context of Spinal Cord Injury.

Biomaterials-based hydrogels are attractive drug-eluting vehicles in the context of RNA therapeutics, such as those utilizing antisense oligonucleotide or RNA interference based drugs, as they can potentially reduce systemic toxicity and enhance in vivo efficacy by increasing in situ concentrations. Here we describe the preparation of antisense oligonucleotide-loaded fibrin hydrogels exploring their applications in the context of the nervous system utilizing an organotypic dorsal root ganglion explant in vitro system and an in vivo model of spinal cord injury.

Engineering hydrogels with affinity-bound laminin as 3D neural stem cell culture systems.

Laminin incorporation into biological or synthetic hydrogels has been explored to recapitulate the dynamic nature and biological complexity of neural stem cell (NSC) niches. However, the strategies currently explored for laminin immobilization within three-dimensional (3D) matrices do not address a critical aspect influencing cell-matrix interactions, which is the control over laminin conformation and orientation upon immobilization. This is a key feature for the preservation of the protein bioactivity. In this work, we explored an affinity-based approach to mediate the site-selective immobilization of laminin into a degradable synthetic hydrogel. Specifically, a four-arm maleimide terminated poly(ethylene glycol) (PEG-4MAL) macromer was functionalized with a mono-PEGylated recombinant human N-terminal agrin (NtA) domain, to promote high affinity binding of laminin. Different NtA concentrations (10, 50 and 100 µM) were used to investigate the impact of NtA density on laminin incorporation, hydrogel biophysical properties, and biological outcome. Laminin was efficiently incorporated for all the conditions tested (laminin incorporation >95%), and the developed hydrogels revealed mechanical properties (average storage modulus (G') ranging from 187 to 256 Pa) within the values preferred for NSC proliferation and neurite branching and extension. Affinity-bound laminin PEG-4MAL hydrogels better preserve laminin bioactivity, compared to unmodified hydrogels and hydrogels containing physically entrapped laminin, this effect being dependent on NtA concentration. This was evidenced by the 10 µM NtA-functionalized PEG-4MAL gels incorporating laminin that support enhanced human NSC proliferation and neurite extension, compared to the latter. Overall, this work highlights the potential of the proposed engineered matrices to be used as defined 3D platforms for the establishment of artificial NSC niches and as extracellular matrix-mimetic microenvironments to support human NSC transplantation.