Background/Objectives: The development of lipid nanoparticles (LNPs) as delivery platforms for nucleic acids has revolutionised possibilities for both therapeutic and vaccine applications. However, emerging studies highlight challenges in achieving reliable in vitro–in vivo correlation (IVIVC), which delays the translation of experimental findings into clinical applications. This study investigates these potential discrepancies by evaluating the physicochemical properties, in vitro efficacy (across three commonly used cell lines), and in vivo performance (mRNA expression and vaccine efficacy) of four LNP formulations. Methods: LNPs composed of DSPC, cholesterol, a PEGylated lipid, and one of four ionizable lipids (SM-102, ALC-0315, MC3, or C12-200) were manufactured using microfluidics. Results: All formulations exhibited comparable physicochemical properties, as expected (size 70–100 nm, low PDI, near-neutral zeta potential, and high mRNA encapsulation). In vitro studies demonstrated variable LNP-mediated mRNA expression in both immortalised and immune cells, with SM-102 inducing significantly higher protein expression (p < 0.05) than the other formulations in immortalised and immune cells. However, in vivo results revealed that ALC-0315 and SM-102-based LNPs achieved significantly (p < 0.05) higher protein expression without a significant difference between them, while MC3- and C12-200-based LNPs exhibited lower expression levels. As vaccine formulations, all LNPs elicited strong immune responses with no significant differences among them. Conclusions: These findings highlight the complexities of correlating in vitro and in vivo outcomes in LNP development and demonstrate the importance of holistic evaluation strategies to optimise their clinical translation.

Background/Objectives: Murine models play a key role in guiding formulation and immunogenicity studies across various vaccine platforms, including mRNA-based vaccines. Typically, a narrow age range (6 to 8 weeks) is used in these studies. Here, we investigated whether widening this age range could provide greater flexibility in experimental design without impacting pre-clinical outcomes. Methods: To achieve this, we evaluated two commonly used lipid nanoparticle (LNP) formulations (based on SM102 and ALC-0315 ionizable lipids) containing either firefly luciferase or ovalbumin mRNA in female BALB/c mice aged 4, 8, and 16 weeks. LNPs were prepared and purified via microfluidics, and their size, polydispersity, zeta potential, and encapsulation efficiency were measured. Mice were injected intramuscularly, and the in vivo bioluminescence and antibody titers were measured to evaluate mRNA expression profiles and immunogenicity across the three age groups. Results: Our findings show that the 4-week-old mice exhibited higher protein expression following mRNA administration compared to the older groups; however, no significant differences were observed between the 8- and 16-week-old mice. Despite the initial higher protein expression, the antibody responses after the prime dose were lower in the 4-week-old mice compared to the other two groups. However, following the booster dose, antibody levels were comparable across all three age groups. Conclusions: By identifying a broader age range window, we provide greater flexibility in study design, enhance data comparability across studies, and promote more efficient use of animal resources, all while maintaining reliable and representative results in these murine models.

Leishmaniasis is a protozoan disease responsible for significant morbidity and mortality. There is no recommended vaccine to protect against infection. In this study, transgenic Leishmania tarentolae expressing gamma glutamyl cysteine synthetase (γGCS) from three pathogenic species were produced and their ability to protect against infection determined using models of cutaneous and visceral leishmaniasis. The ability of IL-2-producing PODS^® to act as an adjuvant was also determined in L. donovani studies. Two doses of the live vaccine caused a significant reduction in L. major (p < 0.001) and L. donovani (p < 0.05) parasite burdens compared to their respective controls. In contrast, immunisation with wild type L. tarentolae, using the same immunisation protocol, had no effect on parasite burdens compared to infection controls. Joint treatment with IL-2-producing PODS^® enhanced the protective effect of the live vaccine in L. donovani studies. Protection was associated with a Th1 response in L. major and a mixed Th1/Th2 response in L. donovani, based on specific IgG1 and IgG2a antibody and cytokine production from in vitro proliferation assays using antigen-stimulated splenocytes. The results of this study provide further proof that γGCS should be considered a candidate vaccine for leishmaniasis.

Background/Objectives: Prostate cancer is the most commonly diagnosed cancer in men and a leading cause of cancer-related mortality, highlighting the need for delivery systems capable of efficiently transporting both chemotherapeutic drugs and therapeutic genes to tumor cells. Generation-3 diaminobutyric polypropylenimine (DAB) dendrimers display low toxicity, high drug loading capacity and efficient gene delivery, and can be engineered as camptothecin-bearing PEGylated carriers complexed with plasmid DNA. The aim of this study was to compare microfluidic processing with conventional hand mixing for the preparation of camptothecin-bearing PEGylated DAB dendriplexes and to evaluate the impact of formulation methods and microfluidic parameters on their physicochemical properties, cellular uptake and gene expression in prostate cancer cells. Methods: Camptothecin-bearing PEGylated DAB dendrimers were synthesized and complexed with plasmid DNA to form dendriplexes. Formulations were prepared either by microfluidics, using different total flow rates and aqueous: organic flow rate ratios, or by conventional hand mixing. The resulting dendriplexes were characterized for DNA condensation, particle size, polydispersity index and zeta potential. Morphology was assessed by transmission electron microscopy. Cellular uptake of fluorescein-labelled DNA and β-galactosidase reporter gene expression were evaluated in PC3-Luc and DU145 prostate cancer cells. Results: Both microfluidic and hand-mixed methods produced stable, nanosized, positively charged dendriplexes with efficient and sustained DNA condensation (more than 99% over 24 h). Microfluidic processing, particularly at an aqueous: organic flow rate ratio of 3:1, yielded dendriplexes with hydrodynamic diameters and zeta potentials comparable to or slightly improved over hand-mixed formulations. These microfluidic conditions significantly enhanced cellular uptake in both PC3-Luc and DU145 cells. In PC3-Luc cells, this translated into β-galactosidase expression levels comparable to hand-mixed dendriplexes and higher than naked DNA, whereas in DU145 cells, transfection efficiencies remained modest for all formulations despite increased uptake. Conclusions: Microfluidic processing enables the reproducible and scalable preparation of camptothecin-bearing PEGylated DAB dendriplexes with tunable physicochemical properties. Under selected conditions, in vitro cellular uptake and gene expression were comparable to conventional hand mixing, supporting microfluidics as a robust alternative platform for the manufacture of dendrimer-based systems for combined chemo–gene delivery in prostate cancer.

The authors would like to make the following corrections to the published paper