Development of a rapid in vitro pre-screen for distinguishing effective liposome-adjuvant delivery systems.

Liposomes are a strong supporting tool in vaccine technology, as they are a versatile system that not only act as antigen delivery systems but also adjuvants that can be highly effective at stimulating both innate and adaptive immune responses. Their ability to induce cell-mediated immunity makes their use in vaccines a useful tool in the development of novel, more effective vaccines against intracellular infections (e.g. HIV, malaria and tuberculosis). Currently, screening of novel liposome formulations uses murine in vivo models which generate data that often correlates poorly with human data. In addition, these models are both high cost and low throughput, making them prohibitive for large scale screening of formulation libraries. This study uses the cationic liposome formulation DDA:TDB (known as cationic adjuvant formulation 01 (CAF01)), as a lead formulation, along with other liposome formulations of known in vivo efficacy to develop an in vitro screening tool for liposome formulation development. THP-1-derived macrophages were the model antigen presenting cell used to assess the ability of the liposome formulations to attract, associate with and activate antigen presenting cells in vitro, crucial steps necessary for an effective immune response to antigen. By using a combination of in vitro functions, the study highlights the potential use of an in vitro screening tool, to predict the in vivo efficacy of novel liposome formulations. CAF01 was predicted as the most effective liposome formulation when assessing all in vitro functions and a measure of in vitro activation was able to predict 80% of the liposome correctly for their ability to induce an in vivo IFN-Ò¯ response.

Author Correction: Formation and purification of tailored liposomes for drug delivery using a module-based micro continuous-flow system.

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Formation and purification of tailored liposomes for drug delivery using a module-based micro continuous-flow system.

Liposomes are lipid based bilayer vesicles that can encapsulate, deliver and release low-soluble drugs and small molecules to a specific target site in the body. They are currently exploited in several nanomedicine formulations. However, their development and application is still limited by expensive and time-consuming process development and production methods. Therefore, to exploit these systems more effectively and support the rapid translation of new liposomal nanomedicines from bench to bedside, new cost-effective and scalable production methods are needed. We present a continuous process flow system for the preparation, modification and purification of liposomes which offers lab-on-chip scale production. The system was evaluated for a range of small vesicles (below 300 nm) varying in lipid composition, size and charge; it offers effective and rapid nanomedicine purification with high lipid recovery (> 98%) combined with effective removal of non-entrapped drug (propofol >95% reduction of non-entrapped drug present) or protein (ovalbumin >90% reduction of OVA present) and organic solvent (ethanol >95% reduction) in less than 4 minutes. The key advantages of using this bench-top, rapid, process development tool are the flexible operating conditions, interchangeable membranes and scalable high-throughput yields, thereby offering simultaneous manufacturing and purification of nanoparticles with tailored surface attributes.

Manufacturing Methods for Liposome Adjuvants.

A wide range of studies have shown that liposomes can act as suitable adjuvants for a range of vaccine antigens. Properties such as their amphiphilic character and biphasic nature allow them to incorporate antigens within the lipid bilayer, on the surface, or encapsulated within the inner core. However, appropriate methods for the manufacture of liposomes are limited and this has resulted in issues with cost, supply, and wider scale application of these systems. Within this chapter we explore manufacturing processes that can be used for the production of liposomal adjuvants, and we outline new manufacturing methods can that offer fast, scalable, and cost-effective production of liposomal adjuvants.

Microfluidics based manufacture of liposomes simultaneously entrapping hydrophilic and lipophilic drugs.

Despite the substantial body of research investigating the use of liposomes, niosomes and other bilayer vesicles for drug delivery, the translation of these systems into licensed products remains limited. Indeed, recent shortages in the supply of liposomal products demonstrate the need for new scalable production methods for liposomes. Therefore, the aim of our research has been to consider the application of microfluidics in the manufacture of liposomes containing either or both a water soluble and a lipid soluble drug to promote co-delivery of drugs. For the first time, we demonstrate the entrapment of a hydrophilic and a lipophilic drug (metformin and glipizide respectively) both individually, and in combination, using a scalable microfluidics manufacturing system. In terms of the operating parameters, the choice of solvents, lipid concentration and aqueous:solvent ratio all impact on liposome size with vesicle diameter ranging from ∼90 to 300nm. In terms of drug loading, microfluidics production promoted high loading within ∼100nm vesicles for both the water soluble drug (20-25% of initial amount added) and the bilayer embedded drug (40-42% of initial amount added) with co-loading of the drugs making no impact on entrapment efficacy. However, co-loading of glipizide and metformin within the same liposome formulation did impact on the drug release profiles; in both instances the presence of both drugs in the one formulation promoted faster (up to 2 fold) release compared to liposomes containing a single drug alone. Overall, these results demonstrate the application of microfluidics to prepare liposomal systems incorporating either or both an aqueous soluble drug and a bilayer loaded drug.

Rapid Quantification and Validation of Lipid Concentrations within Liposomes.

Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance, since this concentration impacts both efficacy and stability. In this paper, we outline a high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing, and microfluidics). The ELSD system was used to quantify four lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) bromide, and ᴅ-(+)-trehalose 6,6'-dibehenate (TDB). The developed method offers rapidity, high sensitivity, direct linearity, and a good consistency on the responses (R² > 0.993 for the four lipids tested). The corresponding limit of detection (LOD) and limit of quantification (LOQ) were 0.11 and 0.36 mg/mL (DMPC), 0.02 and 0.80 mg/mL (cholesterol), 0.06 and 0.20 mg/mL (DDA), and 0.05 and 0.16 mg/mL (TDB), respectively. HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.

Designing liposomal adjuvants for the next generation of vaccines.

Liposomes not only offer the ability to enhance drug delivery, but can effectively act as vaccine delivery systems and adjuvants. Their flexibility in size, charge, bilayer rigidity and composition allow for targeted antigen delivery via a range of administration routes. In the development of liposomal adjuvants, the type of immune response promoted has been linked to their physico-chemical characteristics, with the size and charge of the liposomal particles impacting on liposome biodistribution, exposure in the lymph nodes and recruitment of the innate immune system. The addition of immunostimulatory agents can further potentiate their immunogenic properties. Here, we outline the attributes that should be considered in the design and manufacture of liposomal adjuvants for the delivery of sub-unit and nucleic acid based vaccines.

Microfluidic-controlled manufacture of liposomes for the solubilisation of a poorly water soluble drug.

Besides their well-described use as delivery systems for water-soluble drugs, liposomes have the ability to act as a solubilizing agent for drugs with low aqueous solubility. However, a key limitation in exploiting liposome technology is the availability of scalable, low-cost production methods for the preparation of liposomes. Here we describe a new method, using microfluidics, to prepare liposomal solubilising systems which can incorporate low solubility drugs (in this case propofol). The setup, based on a chaotic advection micromixer, showed high drug loading (41 mol%) of propofol as well as the ability to manufacture vesicles with at prescribed sizes (between 50 and 450 nm) in a high-throughput setting. Our results demonstrate the ability of merging liposome manufacturing and drug encapsulation in a single process step, leading to an overall reduced process time. These studies emphasise the flexibility and ease of applying lab-on-a-chip microfluidics for the solubilisation of poorly water-soluble drugs.

Correlating liposomal adjuvant characteristics to in-vivo cell-mediated immunity using a novel Mycobacterium tuberculosis fusion protein: a multivariate analysis study.

OBJECTIVE: In this study, we have used a chemometrics-based method to correlate key liposomal adjuvant attributes with in-vivo immune responses based on multivariate analysis. METHODS: The liposomal adjuvant composed of the cationic lipid dimethyldioctadecylammonium bromide (DDA) and trehalose 6,6-dibehenate (TDB) was modified with 1,2-distearoyl-sn-glycero-3-phosphocholine at a range of mol% ratios, and the main liposomal characteristics (liposome size and zeta potential) was measured along with their immunological performance as an adjuvant for the novel, postexposure fusion tuberculosis vaccine, Ag85B-ESAT-6-Rv2660c (H56 vaccine). Partial least square regression analysis was applied to correlate and cluster liposomal adjuvants particle characteristics with in-vivo derived immunological performances (IgG, IgG1, IgG2b, spleen proliferation, IL-2, IL-5, IL-6, IL-10, IFN-γ). KEY FINDINGS: While a range of factors varied in the formulations, decreasing the 1,2-distearoyl-sn-glycero-3-phosphocholine content (and subsequent zeta potential) together built the strongest variables in the model. Enhanced DDA and TDB content (and subsequent zeta potential) stimulated a response skewed towards a cell mediated immunity, with the model identifying correlations with IFN-γ, IL-2 and IL-6. CONCLUSION: This study demonstrates the application of chemometrics-based correlations and clustering, which can inform liposomal adjuvant design.

High-throughput manufacturing of size-tuned liposomes by a new microfluidics method using enhanced statistical tools for characterization.

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows for reproducible mixing in miliseconds on the nanoliter scale. Here we investigate microfluidics-based manufacturing of liposomes. The aim of these studies was to assess the parameters in a microfluidic process by varying the total flow rate (TFR) and the flow rate ratio (FRR) of the solvent and aqueous phases. Design of experiment and multivariate data analysis were used for increased process understanding and development of predictive and correlative models. High FRR lead to the bottom-up synthesis of liposomes, with a strong correlation with vesicle size, demonstrating the ability to in-process control liposomes size; the resulting liposome size correlated with the FRR in the microfluidics process, with liposomes of 50 nm being reproducibly manufactured. Furthermore, we demonstrate the potential of a high throughput manufacturing of liposomes using microfluidics with a four-fold increase in the volumetric flow rate, maintaining liposome characteristics. The efficacy of these liposomes was demonstrated in transfection studies and was modelled using predictive modeling. Mathematical modelling identified FRR as the key variable in the microfluidic process, with the highest impact on liposome size, polydispersity and transfection efficiency. This study demonstrates microfluidics as a robust and high-throughput method for the scalable and highly reproducible manufacture of size-controlled liposomes. Furthermore, the application of statistically based process control increases understanding and allows for the generation of a design-space for controlled particle characteristics.

A case-study investigating the physicochemical characteristics that dictate the function of a liposomal adjuvant.

A range of particulate delivery systems have been considered as vaccine adjuvants. Of these systems, liposomes offer a range of advantages including versatility and flexibility in design format and their ability to incorporate a range of immunomodulators and antigens. Here we briefly outline research, from within our laboratories, which focused on the systematic evaluation of cationic liposomes as vaccines adjuvants. Our aim was to identify physicochemical characteristics that correlate with vaccine efficacy, with particular consideration of the interlink between depot-forming action and immune responses. A variety of parameters were investigated and over a range of studies we have confirmed that cationic liposomes, based on dimethyldioctadecylammonium bromide and trehalose 6,6'-dibehenate formed a depot at the injection site, which stimulates recruitment of antigen presenting cells to the injection site and promotes strong humoral and cell-mediated immune responses. Physicochemical factors which promote a strong vaccine depot include the combination of a high cationic charge and electrostatic binding of the antigen to the liposome system and the use of lipids with high transition temperatures, which form rigid bilayer vesicles. Reduction in vesicle size of cationic vesicles did not promote enhanced drainage from the injection site. However, reducing the cationic nature through substitution of the cationic lipid for a neutral lipid, or by masking of the charge using PEGylation, resulted in a reduced depot formation and reduced Th1-type immune responses, while Th2-type responses were less influenced. These studies confirm that the physicochemical characteristics of particulate-based adjuvants play a key role in the modulation of immune responses.