RESUMEN
Immune stimulating complexes (ISCOMs) are safe and effective saponin-based adjuvants formed by the self-assembly of saponin, cholesterol, and phospholipids in water to form cage-like 30-40 nm diameter particles. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) in ISCOM particles yields a promising next-generation adjuvant termed Saponin-MPLA NanoParticles (SMNP). In this work, we detail protocols to produce ISCOMs or SMNP via a tangential flow filtration (TFF) process suitable for scalable synthesis and Good Manufacturing Practice (GMP) production of clinical-grade adjuvants. SMNP or ISCOM components were solubilized in micelles of the surfactant MEGA-10, then diluted below the critical micelle concentration (CMC) of the surfactant to drive ISCOM self-assembly. Assembly of ISCOM/SMNP particles using the purified saponin QS-21 used in clinical-grade saponin adjuvants was found to require controlled stepwise dilution of the initial micellar solution, to prevent formation of undesirable kinetically-trapped aggregate species. An optimized protocol gave yields of ~77% based on the initial feed of QS-21 and the final SMNP particle composition mirrored the feed ratios of the components. Further, samples were highly homogeneous with comparable quality to that of material prepared at lab scale by dialysis and purified via size-exclusion chromatography. This protocol may be useful for clinical preparation of ISCOM-based vaccine adjuvants and therapeutics.
RESUMEN
The recent clinical success of multiple mRNA-based SARS-CoV-2 vaccines has proven the potential of RNA formulated in lipid nanoparticles (LNPs) in humans, and products based on base-modified RNA, sequence-optimized RNA, and self-replicating RNAs formulated in LNPs are all in various stages of clinical development. However, much remains to be learned about critical parameters governing the manufacturing and use of LNP-RNA formulations. One important issue that has received limited attention in the literature to date is the identification of optimal storage conditions for LNP-RNA that preserve long-term activity of the formulations. Here, we analyzed the physical structure, in vivo expression characteristics, and functional activity of alphavirus-derived self-replicating RNA (repRNA)-loaded LNPs encoding HIV vaccine antigens following storage in varying temperatures, buffers, and in the presence or absence of cryoprotectants. We found that for lipid nanoparticles with compositions similar to clinically-used LNPs, storage in RNAse-free PBS containing 10% (w/v) sucrose at -20 °C was able to maintain vaccine stability and in vivo potency at a level equivalent to freshly prepared vaccines following 30 days of storage. LNPs loaded with repRNA could also be lyophilized with retention of bioactivity.
Asunto(s)
COVID-19 , Nanopartículas , Vacunas , Humanos , Vacunas contra la COVID-19 , SARS-CoV-2 , ARN , Nanopartículas/química , ARN Interferente Pequeño/químicaRESUMEN
The majority of in vitro studies assessing cancer treatments are performed in two-dimensional (2D) monolayers and are subsequently validated in in vivo animal models. However, 2D models fail to accurately model the tumour microenvironment. Furthermore, animal models are not directly applicable to mimic the human scenario. Three-dimensional (3D) culture models may help to address the discrepancies of 2D and animal models. When cancer cells escape the primary tumour, they can invade at distant organs building secondary tumours, called metastasis. The development of metastasis leads to a dramatic decrease in the life expectancy of patients. Therefore, 3D systems to model the microenvironment of metastasis have also been developed. Several studies have demonstrated changes in cell behaviour and gene expression when cells are cultured in 3D compared to 2D and concluded a better comparability to cells in vivo. Of special importance is the effect seen in response to anti-cancer treatments as models are built primarily to serve as drug-testing platforms. This review highlights these changes between cancer cells grown in 2D and 3D models for some of the most common cancers including lung, breast and prostate tumours. In addition to models aiming to mimic the primary tumour site, the effects of 3D cell culturing in bone metastasis models are also described. STATEMENT OF SIGNIFICANCE: Most in vitro studies in cancer research are performed in 2D and are subsequently validated in in vivo animal models. However, both models possess numerous limitations: 2D models fail to accurately model the tumour microenvironment while animal models are expensive, time-consuming and can differ considerably from humans. It is accepted that the cancer microenvironment plays a critical role in the disease, thus, 3D models have been proposed as a potential solution to address the discrepancies of 2D and animal models. This review highlights changes in cell behaviour, including proliferation, gene expression and chemosensitivity, between cancer cells grown in 2D and 3D models for some of the most common cancers including lung, breast and prostate cancer as well as bone metastasis.