Due to the inherent risk of a further pandemic influenza outbreak, there is a need and growing interest in investigating combinations of prophylactic vaccines and novel adjuvants, particularly to achieve antigen dose sparing and improved immunogenicity. Influenza is a highly variable virus, where the specific vaccine target is constantly changing, representing a major challenge to influenza vaccine development. Currently, commercial inactivated influenza vaccines have a poor CD8+ T response, which impacts cross-reactivity and the duration of response. Adjuvanted influenza vaccines can increase immune responses, thereby achieving better protection and cross-reactivity to help contain the spread of the disease. An early exploration of a hybrid cholesterol-PLGA nanoparticle delivery system containing the saponin tomatine and a NOD2 (nucleotide-binding oligomerization domain 2) agonist called SG101 was conducted. This combination was preliminarily evaluated for its ability to induce cellular immunity when combined with whole inactivated virus (WIV) influenza vaccine. After the adjuvants were manufactured using a single emulsion process, two formulations with different drug loadings were selected and physico-chemically characterized, showing sizes between 224 ± 32 and 309 ± 45 nm and different morphologies. After ensuring the lack of in vitro toxicity and hemolytic activity, a pilot in vivo assay evaluated the hybrid nanoparticle formulation for its ability to induce humoral and cellular immunity when combined with whole inactivated virus (WIV) H5N1 influenza vaccine by intramuscular administration in mice. Hemagglutinin inhibition (HAI) titers for adjuvanted groups showed no significant difference compared to the group vaccinated with the antigen alone. It was similar for CD4+ and CD8+ T cell responses, although the high drug loading formulation induced higher titers of IFNγ-positive CD8+ T cells. These proof-of-concept results encourage further investigations to develop the hybrid formulation with increased or different loading ratios, to investigate manufacturing optimization, and to evaluate the role of the individual immunostimulatory compounds in immune responses.

The hanging drop method is a cost-effective approach for 3D spheroid culture. However, obtaining numerous spheroids in a limited area becomes challenging due to the risk of droplet coalescence, primarly during Petri dish handling. In this study, we describe a general method to fabricate a 3D printing-based support called SpheroMold that facilitates Petri dish handling and enhances spheroid production per unit area. As a proof-of-concept, we designed a digital negative mold which comprised 37 pegs within a 13.52 cm2 area, and then printed it using stereolithography; the density of pegs can be adjusted according to user requirements. The SpheroMold was created by pouring the base and curing agent (10:1) (Sylgard® 184 silicone) into the mold, curing it at 80°C, and then attaching it to the lid of a Petri dish. Our SpheroMold effectively prevented droplet coalescence during Petri dish inversion, enabling the production of numerous 3D spheroids while simplifying manipulation. Unlike conventional techniques, our design also facilitated a larger volume of culture medium per drop compared to a standard Petri dish, potentially decreasing the necessity for frequent medium exchange to sustain cellular health and reducing labor intensity.

This paper details the comprehensive design and prototyping of a 3D-printed wearable device tailored for mouse models which addresses the need for non-invasive applications in spinal cord studies and therapeutic treatments. Our work was prompted by the increasing demand for wearable devices in preclinical research on freely behaving rodent models of spinal cord injury. We present an innovative solution that employs compliant 3D-printed structures for stable device placement on the backs of both healthy and spinal cord-injured mice. In our trial, the device was represented by two magnets that applied passive magnetic stimulation to the injury site. This device was designed to be combined with the use of magnetic nanoparticles to render neurons or neural cells sensitive to an exogenous magnetic field, resulting in the stimulation of axon growth in response to a pulling force. We show different design iterations, emphasizing the challenges faced and the solutions proposed during the design process. The iterative design process involved multiple phases, from the magnet holder (MH) to the wearable device configurations. The latter included different approaches: a “Fitbit”, “Belt”, “Bib”, and ultimately a “Cape”. Each design iteration was accompanied by a testing protocol involving healthy and injured mice, with qualitative assessments focusing on animal wellbeing. Follow-up lasted for at least 21 consecutive days, thus allowing animal welfare to be accurately monitored. The final Cape design was our best compromise between the need for a thin structure that would not hinder movement and the resistance required to maintain the structure at the correct position while withstanding biting and mechanical stress. The detailed account of the iterative design process and testing procedures provides valuable insights for researchers and practitioners engaged in the development of wearable devices for mice, particularly in the context of spinal cord studies and therapeutic treatments. Finally, in addition to describing the design of a 3D-printed wearable holder, we also outline some general guidelines for the design of wearable devices.

The core/shell 3D printing process using CPC and alginate is intended to create biodegradable scaffolds that have a similar stability to bone tissue and also offer sufficient and continuous antibiotic release. In this way, a patient-specific and patient-friendly process will be established, which should optimally support the human organism in its regeneration. To generate the best possible strength values, the printed scaffolds underwent various post-treatments and were then tested in a material test. The test methods included self-setting, storage in a drying cabinet with a water-saturated atmosphere at 37°C, followed by incubation in PBS, freeze-drying, and coating the samples with alginate. Additionally, a degradation test at pH 7.4 and pH 5 was carried out to test stability under in vitro conditions. It was shown that the untreated and freeze-dried samples failed at a maximum load of 30–700 N, while the remaining scaffolds could withstand a load of at least 2,000 N. At this failure load, most of the test series showed an average deformation of 43.95%. All samples, therefore, remained below the strength of cancellous bone. However, based on a 20% load after surgery, the coated scaffolds represented the best possible alternative, with a Young’s modulus of around 1.71 MPa. We were able to demonstrate that self-setting occurs in core-shell printed CPC/alginate scaffolds after only 1 day, and that mass production is possible. By coating with alginate, the compressive strength could be increased without the need for additional post-treatment. The mechanical strength was sufficient to be available as a scaffold for bone regeneration and additionally as a drug delivery device for future applications and experiments.

Lipid nanoparticles (LNPs) have emerged as the platform of choice for mRNA delivery. Polyethylene glycol (PEG) is considered a key component of currently approved LNP-based delivery systems as it ensures particle stability and shapes various facets of LNP behavior in biological systems. Whilst PEG has numerous characteristics that are favorable for delivery systems, there is a growing body of evidence that suggests that it is immunogenic. Thus, next-generation mRNA therapeutics are likely to benefit from the identification of PEG alternatives. Towards this end, we have assessed the suitability of poly(2-methyl-2-oxazoline) (PMOZ) for LNP-based mRNA delivery. We compared the properties and bioactivities of PMOZ-containing LNPs to that of a standard composition that includes PEG. Decreasing the percentage of PMOZ in formulations improved transfection efficiency and enhanced the immunostimulatory potential. Reducing the PMOZ density was shown to enhanced antigen-specific T-cell responses in vivo. Interestingly, we found that this was not the case for antibody responses. A direct comparison between LNPs that contain the same amount of PEG or PMOZ strongly suggests that the former induces stronger CD8+ T-cell responses while the latter induces superior neutralizing titers. These findings augur well for the further development of PMOZ as a PEG replacement for LNP-based mRNA delivery approaches.

The delivery of therapeutics into the brain is highly limited by the blood-brain barrier (BBB). Although this is essential to protect the brain from potentially harmful material found in the blood, it poses a great challenge for the treatment of diseases affecting the central nervous system (CNS). Substances from the periphery that are required for the function of the brain must rely on active mechanisms of entry. One such physiological pathway is called receptor-mediated transcytosis (RMT). In this process, ligands bind to specific receptors expressed at the luminal membrane of endothelial cells composing the BBB leading to the internalization of the receptor-ligand complex into intracellular vesicles, their trafficking through various intracellular compartments and finally their fusion with the abluminal membrane to release the cargo into the brain. Targeting such RMT receptors for BBB crossing represents an emerging and clinically validated strategy to increase the brain permeability of biologicals. However, the choice of an appropriate receptor is critical to achieve the best selectivity and efficacy of the delivery method. Whereas the majority of work has been focused on transferrin (Tf) receptor (TfR), the search for novel receptors expressed in brain endothelial cells (BECs) that can deliver protein or viral vector cargos across the BBB has yielded several novel targets with diverse molecular/structural properties and biological functions, and mechanisms of transcytosis. In this review, we summarize well-studied RMT pathways, and explore mechanisms engaged in BBB transport by various RMT receptors. We then discuss key criteria that would be desired for an optimal RMT target, based on lessons-learned from studies on TfR and accumulating experimental evidence on emerging RMT receptors and their ligands.

Selective Laser Sintering (SLS) has the potential to offer a more accurate alternative to current-practice manipulation of oral dosage forms for pediatric, geriatric, and dysphagia-suffering patient groups. In order to create the best possible dosage forms for these patient groups, an in-depth look into how a dosage forms geometry impacts the overall properties is essential. In this study, the impact of geometry on SLS manufactured oral dosage forms on the tablet’s microstructure, actual-to-theoretical volume, mass deviation, disintegration, and dissolution was investigated. Three different shapes; cylinder, hollow cylinder, and conical frustum with similar surface area (SA), as well as three cylinders with different diameters, were investigated. The results indicate that the geometry has an impact on the mass uniformity, resultant volume, disintegration, and dissolution properties of the tablets. The mass uniformity analysis of the tablets provided the most variation between tablets of different sizes, with more uniformity for tablets with similar SA-to-volume ratio (SA/V). When examining the actual-to-theoretical volume of the tablets, a greater variance between the actual and theoretical volumes for shapes with higher overall SA was observed. The values found are approximately 1.05 for the three differently sized cylinders, 1.23 for the conical frustum, and 1.44 for the hollow cylinder, following this trend. Disintegration data supported a link between SA/V and average disintegration time, observed with the tablet of the highest SA/V disintegrating in 12 s and the tablet with the lowest SA/V disintegrating in 58 s. Dissolution results also indicated a strong dependence on SA/V. Hence, when novel ways to produce oral dosage form tablets become available by additive manufacturing, such as SLS, both geometry and SA/V must be taken into consideration in the tablet design process to ensure appropriate release kinetics and dosing standards.

Multi-RNA co-transfection is starting to be employed to stimulate immune responses to SARS-CoV-2 viral infection. While there are good reasons to utilize such an approach, there is little background on whether there are synergistic RNA-dependent cellular effects. To address this issue, we use transcriptome-induced phenotype remodeling (TIPeR) via phototransfection to assess whether mRNAs encoding the Spike and Nucleocapsid proteins of SARS-CoV-2 virus into single human astrocytes (an endogenous human cell host for the virus) and mouse 3T3 cells (often used in high-throughput therapeutic screens) synergistically impact host cell biologies. An RNA concentration-dependent expression was observed where an increase of RNA by less than 2-fold results in reduced expression of each individual RNAs. Further, a dominant inhibitory effect of Nucleocapsid RNA upon Spike RNA translation was detected that is distinct from codon-mediated epistasis. Knowledge of the cellular consequences of multi-RNA transfection will aid in selecting RNA concentrations that will maximize antigen presentation on host cell surface with the goal of eliciting a robust immune response. Further, application of this single cell stoichiometrically tunable RNA functional genomics approach to the study of SARS-CoV-2 biology promises to provide details of the cellular sequalae that arise upon infection in anticipation of providing novel targets for inhibition of viral replication and propagation for therapeutic intervention.