Non-canonical inflammasome activation mediates the adjuvanticity of nanoparticles.

The non-canonical inflammasome sensor caspase-11 and gasdermin D (GSDMD) drive inflammation and pyroptosis, a type of immunogenic cell death that favors cell-mediated immunity (CMI) in cancer, infection, and autoimmunity. Here we show that caspase-11 and GSDMD are required for CD8+ and Th1 responses induced by nanoparticulate vaccine adjuvants. We demonstrate that nanoparticle-induced reactive oxygen species (ROS) are size dependent and essential for CMI, and we identify 50- to 60-nm nanoparticles as optimal inducers of ROS, GSDMD activation, and Th1 and CD8+ responses. We reveal a division of labor for IL-1 and IL-18, where IL-1 supports Th1 and IL-18 promotes CD8+ responses. Exploiting size as a key attribute, we demonstrate that biodegradable poly-lactic co-glycolic acid nanoparticles are potent CMI-inducing adjuvants. Our work implicates ROS and the non-canonical inflammasome in the mode of action of polymeric nanoparticulate adjuvants and establishes adjuvant size as a key design principle for vaccines against cancer and intracellular pathogens.

Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics.

In the recent of years, the use of lipid nanoparticles (LNPs) for RNA delivery has gained considerable attention, with a large number in the clinical pipeline as vaccine candidates or to treat a wide range of diseases. Microfluidics offers considerable advantages for their manufacture due to its scalability, reproducibility and fast preparation. Thus, in this study, we have evaluated operating and formulation parameters to be considered when developing LNPs. Among them, the flow rate ratio (FRR) and the total flow rate (TFR) have been shown to significantly influence the physicochemical characteristics of the produced particles. In particular, increasing the TFR or increasing the FRR decreased the particle size. The amino lipid choice (cationic-DOTAP and DDAB; ionisable-MC3), buffer choice (citrate buffer pH 6 or TRIS pH 7.4) and type of nucleic acid payload (PolyA, ssDNA or mRNA) have also been shown to have an impact on the characteristics of these LNPs. LNPs were shown to have a high (>90%) loading in all cases and were below 100 nm with a low polydispersity index (≤0.25). The results within this paper could be used as a guide for the development and scalable manufacture of LNP systems using microfluidics.

Scalable solvent-free production of liposomes.

OBJECTIVES: A major challenge faced with the manufacture of liposomes is the high volumes of organic solvents used during manufacturing. Therefore, we have implemented an organic solvent-free production method for drug-loaded liposomes and demonstrated its applicability with both aqueous core-loaded and bilayer-loaded drugs. METHODS: Liposomes were produced by high shear mixing dry powder lipids with an aqueous buffer, followed by down-sizing using a Microfluidizer processor. Liposomes were purified via tangential flow filtration and characterised in terms of size, polydispersity index, zeta potential and drug loading. KEY FINDINGS: Doxorubicin-loaded PEGylated liposomes can be manufactured using this solvent-free method with particle sizes of 100-110 nm, low polydispersity index (PDI) (<0.2) and high drug loading (97-98%). If required, liposomes can be further down-sized via microfluidic processing without impacting drug loading. Similar results were achieved with non-PEGylated liposomes. With bilayer-loaded amphotericin B liposomes, again liposomes can be prepared within a clinically appropriate size range (100-110 nm in size, low PDI) with high drug loading (98-100%). CONCLUSIONS: We apply a simple and scalable solvent-free method for the production of both aqueous core or bilayer drug-loaded liposomes.

Rapid scale-up and production of active-loaded PEGylated liposomes.

Manufacturing of liposomal nanomedicines (e.g. Doxil®/Caelyx®) is a challenging and slow process based on multiple-vessel and batch processing techniques. As a result, the translation of these nanomedicines from bench to bedside has been limited. Microfluidic-based manufacturing offers the opportunity to address this issue, and de-risk the wider adoption of nanomedicines. Here we demonstrate the applicability of microfluidics for continuous manufacturing of PEGylated liposomes encapsulating ammonium sulfate (250 mM). Doxorubicin was subsequently active-loaded into these pre-formed liposomes. Critical process parameters and material considerations demonstrated to influence the liposomal product attributes included solvent selection and lipid concentration, flow rate ratio, and temperature and duration used for drug loading. However, the total flow rate did not affect the liposome product characteristics, allowing high production speeds to be adopted. The final liposomal product comprised of 80-100 nm vesicles (PDI < 0.2) encapsulating ≥ 90% doxorubicin, with matching release profiles to the innovator product and is stable for at least 6 months. Additionally, vincristine and acridine orange were active-loaded into these PEGylated liposomes (≥ 90% and ~100 nm in size) using the same process. These results demonstrate the ability to produce active-loaded PEGylated liposomes with high encapsulation efficiencies and particle sizes which support tumour targeting.

Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP: A case study using protein-loaded liposomes.

Nanomedicines are well recognised for their ability to improve therapeutic outcomes. Yet, due to their complexity, nanomedicines are challenging and costly to produce using traditional manufacturing methods. For nanomedicines to be widely exploited, new manufacturing technologies must be adopted to reduce development costs and provide a consistent product. Within this study, we investigate microfluidic manufacture of nanomedicines. Using protein-loaded liposomes as a case study, we manufacture liposomes with tightly defined physico-chemical attributes (size, PDI, protein loading and release) from small-scale (1 mL) through to GMP volume production (200 mL/min). To achieve this, we investigate two different laminar flow microfluidic cartridge designs (based on a staggered herringbone design and a novel toroidal mixer design); for the first time we demonstrate the use of a new microfluidic cartridge design which delivers seamless scale-up production from bench-scale (12 mL/min) through GMP production requirements of over 20 L/h using the same standardised normal operating parameters. We also outline the application of tangential flow filtration for down-stream processing and high product yield. This work confirms that defined liposome products can be manufactured rapidly and reproducibly using a scale-independent production process, thereby de-risking the journey from bench to approved product.

Translating the fabrication of protein-loaded poly(lactic-co-glycolic acid) nanoparticles from bench to scale-independent production using microfluidics.

In the formulation of nanoparticles, poly(lactic-co-glycolic acid) (PLGA) is commonly employed due to its Food and Drug Administration and European Medicines Agency approval for human use, its ability to encapsulate a variety of moieties, its biocompatibility and biodegradability and its ability to offer a range of controlled release profiles. Common methods for the production of PLGA particles often adopt harsh solvents, surfactants/stabilisers and in general are multi-step and time-consuming processes. This limits the translation of these drug delivery systems from bench to bedside. To address this, we have applied microfluidic processes to develop a scale-independent platform for the manufacture, purification and monitoring of nanoparticles. Thereby, the influence of various microfluidic parameters on the physicochemical characteristics of the empty and the protein-loaded PLGA particles was evaluated in combination with the copolymer employed (PLGA 85:15, 75:25 or 50:50) and the type of protein loaded. Using this rapid production process, emulsifying/stabilising agents (such as polyvinyl alcohol) are not required. We also incorporate in-line purification systems and at-line particle size monitoring. Our results demonstrate the microfluidic control parameters that can be adopted to control particle size and the impact of PLGA copolymer type on the characteristics of the produced particles. With these nanoparticles, protein encapsulation efficiency varies from 8 to 50% and is controlled by the copolymer of choice and the production parameters employed; higher flow rates, combined with medium flow rate ratios (3:1), should be adopted to promote higher protein loading (% wt/wt). In conclusion, herein, we outline the process controls for the fabrication of PLGA polymeric nanoparticles incorporating proteins in a rapid and scalable manufacturing process. Scale-independent production of polymer nanoparticles.

The Impact of Solvent Selection: Strategies to Guide the Manufacturing of Liposomes Using Microfluidics.

The aim of this work was to assess the impact of solvent selection on the microfluidic production of liposomes. To achieve this, liposomes were manufactured using small-scale and bench-scale microfluidics systems using three aqueous miscible solvents (methanol, ethanol or isopropanol, alone or in combination). Liposomes composed of different lipid compositions were manufactured using these different solvents and characterised to investigate the influence of solvents on liposome attributes. Our studies demonstrate that solvent selection is a key consideration during the microfluidics manufacturing process, not only when considering lipid solubility but also with regard to the resultant liposome critical quality attributes. In general, reducing the polarity of the solvent (from methanol to isopropanol) increased the liposome particle size without impacting liposome short-term stability or release characteristics. Furthermore, solvent combinations such as methanol/isopropanol mixtures can be used to modify solvent polarity and the resultant liposome particle size. However, the impact of solvent choice on the liposome product is also influenced by the liposome formulation; liposomes containing charged lipids tended to show more sensitivity to solvent selection and formulations containing increased concentrations of cholesterol or pegylated-lipids were less influenced by the choice of solvent. Indeed, incorporation of 14 wt% or more of pegylated-lipid was shown to negate the impact of solvent selection.

Scale-Independent Microfluidic Production of Cationic Liposomal Adjuvants and Development of Enhanced Lymphatic Targeting Strategies.

Cationic liposomes prepared from dimethyldioctadecylammonium bromide (DDAB) and trehalose 6,6'-dibehenate (TDB) are strong liposomal adjuvants. As with many liposome formulations, within the laboratory DDAB:TDB is commonly prepared by the thin-film method, which is difficult to scale-up and gives high batch-to-batch variability. In contrast, controllable technologies such as microfluidics offer robust, continuous, and scale-independent production. Therefore, within this study, we have developed a microfluidic production method for cationic liposomal adjuvants that is scale-independent and produces liposomal adjuvants with analogous biodistribution and immunogenicity compared to those produced by the small-scale lipid hydration method. Subsequently, we further developed the DDAB:TDB adjuvant system to include a lymphatic targeting strategy using microfluidics. By exploiting a biotin-avidin complexation strategy, we were able to manipulate the pharmacokinetic profile and enhance targeting and retention of DDAB:TDB and antigen within the lymph nodes. Interestingly, redirecting these cationic liposomal adjuvants did not translate into notably improved vaccine efficacy.

Formulation and manufacturing of lymphatic targeting liposomes using microfluidics.

The lymphatics are a target for a range of therapeutic purposes, including cancer therapy and vaccination, and both vesicle size and charge have been considered as factors controlling lymphatic targeting. Within this work, a range of liposomal formulations were investigated to develop a liposomal lymphatic targeting system. Initial screening of formulations considered the effect of charge, with neutral, cationic and anionic liposomes being investigated. Biodistribution studies demonstrated that after intramuscular injection, anionic liposomes offered the most rapid clearance to the draining lymphatics with cationic liposomes forming a depot at the injection site. Anionic liposomes containing phosphatidylserine showed higher clearance to the lymphatics and this may result form preferential uptake by macrophages. In terms of vesicle size, smaller unilamellar vesicles gave high lymphatic targeting and a 10-fold increase in concentration was achieved in dose escalation studies. Given that effective trafficking to the lymphatics was achieved, the next step was to enhance retention of the liposomes within the lymphatics, therefore the liposome formulation was combined with an avidin/biotin complex mechanism. The affinity of avidin for biotin allows biotinylated liposomes to complex in the presence of avidin. By pre-dosing with avidin, the biotin-avidin complex can be exploited to promote longer retention of the liposomes at the draining lymphatics. To load these small, biotinylated liposomes with protein, microfluidics manufacturing was used. Using microfluidics, protein could easily be incorporated in these small (~90nm) biotinylated liposomes. Both liposome and protein retention at the local draining lymph nodes was demonstrated with the liposome-biotin-avidin system. These results demonstrate that microfluidics can be used to prepare protein-loaded liposomes that offer enhanced lymphatic targeting and retention of both the liposomes and entrapped antigen.

Investigating Prime-Pull Vaccination through a Combination of Parenteral Vaccination and Intranasal Boosting.

Formulation of inhalable delivery systems containing tuberculosis (TB) antigens to target the site of infection (lungs) have been considered for the development of subunit vaccines. Inert delivery systems such as poly (lactic-co-glycolic acid) (PLGA) are an interesting approach due to its approval for human use. However, PLGA suffers hydrolytic degradation when stored in a liquid environment for prolonged time. Therefore, in this study, nano- and microparticles composed of different PLGA copolymers (50:50, 75:25 and 85:15), sucrose (10% w/v) and L-leucine (1% w/v) encapsulating H56 TB vaccine candidate were produced as dried powders. In vitro studies in three macrophage cell lines (MH-S, RAW264.7 and THP-1) showed the ability of these cells to take up the formulated PLGA:H56 particles and process the antigen. An in vivo prime-pull immunisation approach consisting of priming with CAF01:H56 (2 × subcutaneous (s.c.) injection) followed by a mucosal boost with PLGA:H56 (intranasal (i.n.) administration) demonstrated the retention of the immunogenicity of the antigen encapsulated within the lyophilised PLGA delivery system, although no enhancing effect could be observed compared to the administration of antigen alone as a boost. The work here could provide the foundations for the scale independent manufacture of polymer delivery systems encapsulating antigens for inhalation/aerolisation to the lungs.