pharmaceutics Article The Impact of Solvent Selection: Strategies to Guide the Manufacturing of Liposomes Using Microfluidics 1, 1, 1,2 1 Cameron Webb y, Swapnil Khadke y, Signe Tandrup Schmidt , Carla B. Roces , Neil Forbes 1, Gillian Berrie 1 and Yvonne Perrie 1,* 1 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK; [email protected] (C.W.); [email protected] (S.K.); [email protected] (S.T.S.); [email protected] (C.B.R.); [email protected] (N.F.); [email protected] (G.B.) 2 Department of Infectious Disease Immunology, Center for Vaccine Research, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark * Correspondence: [email protected]; Tel.: +0141-548-2244 These authors contributed equally to this work. y Received: 26 October 2019; Accepted: 30 November 2019; Published: 4 December 2019 Abstract: 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. Keywords: liposomes; microfluidics; solvents; formulation; particle size; pegylation; alcohol 1. Introduction Liposomes are a versatile group of nanoparticles, which are approved for use in the clinic to improve the delivery of cancer agents, antifungal agents and vaccine adjuvants [1,2]. Currently, most approved liposome products contain already-approved drugs (e.g., amphotericin B, doxorubicin, bupivacaine, verteporfin) [3]. Recently, there has been a rise in follow-on ‘nanosimilars’ or generic liposome products being approved for use. Indeed, this has resulted in new recommendations for the naming of liposomal medicines such that the qualifier ‘liposomal’ or ‘pegylated liposomal’ should be added to the name. This recommendation, made jointly by the EMA’s human medicines committee and the Coordination Group for the mutual Recognition and Decentralised Procedures, aims to provide clearer distinctions between liposomal and non-liposomal formulations and hence avoid medication errors [4]. Pharmaceutics 2019, 11, 653; doi:10.3390/pharmaceutics11120653 www.mdpi.com/journal/pharmaceutics PharmaceuticsPharmaceutics 20192019,, 1111,, 653653 2 of 1615 However, despite the increase in their use, the manufacturing processes used in the production of liposomesHowever, remains despite therelatively increase unchanged, in their use, with the manufacturingtime-consuming, processes multi-stage used batch in the production ofprocedures liposomes being remains common. relatively Furthermore, unchanged, in many with time-consuming,liposome research multi-stage studies, small-scale batch production methods proceduresare still used being that common. do not offer Furthermore, realistic line-of-sight in many liposome to a manufacturing research studies, process. small-scale This methodsreduces our are stillability used to thattranslate do not liposome offer realistic technology line-of-sight from bench to a manufacturing to patient. 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[10,11], Using thisnucleic method, acids we[12] have and demonstratedproteins [13,14] the within ability liposomes. to entrap small We have molecules also demonstrated [10,11], nucleic the acids scale-independent [12] and proteins production [13,14] within of liposomes.liposomal adjuvants We have also using demonstrated this method the [15–18]. scale-independent production of liposomal adjuvants using this methodWhen adopting [15–18]. microfluidics as a production method for liposomes, the lipids are dissolved in an organicWhen adoptingsolvent (normally microfluidics alcohol), as a productionwhich is then method mixed for with liposomes, the aqueous the lipids phase are to dissolved promote innanoprecipitation an organic solvent and (normally liposome alcohol),production. which Therefor is thene, the mixed solvent with selected the aqueous must phasedissolve to promotethe lipid nanoprecipitationcomponent and be and water liposome soluble. production. The water Therefore, miscibility the of solvent organic selected solvents must is directly dissolve related the lipid to componentcarbon chain and length be water and soluble.surface tension The water as miscibilityshown in Figure of organic 1 (adapted solvents from is directly [19]). relatedAs the tolength carbon of chainthe carbon length chain and surface increases, tension the as polar shown OH in Figuregroup1 (adaptedbecomes froma smaller [ 19]). component As the length of of the the solvent carbon chainmolecule. increases, The solubility the polar and OH the group polarity becomes of alcohol a smaller decreases component correspondingly. of the solvent The molecule. solubility The of solubilityorganic solvents and the in polarity water ofis alcoholalso driven decreases by hydrogen-bonding correspondingly. Theinteractions solubility between of organic water solvents and insolvent. water For is also example, driven water by hydrogen-bonding and short-chain solvents interactions such betweenas ethanol water are completely and solvent. miscible For example, due to waterthe ability and short-chainof the ethanol solvents molecules such as to ethanol form hydrogen are completely bonds miscible with water due tomolecules the ability as of well the ethanolas with moleculeseach other to[20]. form However, hydrogen when bonds considering with water solvent molecules selection as wellfor use as in with microfluidics, each other [lipid20]. However,solubility whenand water considering miscibility solvent are not selection the only for factors. use in microfluidics, Whilst in the lipidpurification solubility process and water solvent miscibility is removed, are notworking the only with factors. solvents Whilst that have in the low purification toxicity potential process and solvent defined is removed, as Class working3 in the ICH with Q3C solvents (R6) that[21] (e.g., have ethanol low toxicity and isopropanol potential and (IPA)) defined is preferable as Class 3 followed in the ICH by Q3Cthose (R6) in Class [21] (e.g.,2 (e.g., ethanol methanol) and isopropanol(Figure 1). (IPA)) is preferable followed by those in Class 2 (e.g., methanol) (Figure1). Figure 1.1. The relationship between aqueous solubility andand the number of carbon atoms inin alcoholsalcohols determined by by surface surface tension tension (adapted (adapted from from [19]) [19 and]) and physical physical properties properties of methanol, of methanol, ethanol, ethanol, and andisopropanol. isopropanol. Typically, IPAIPA hashas beenbeen usedused asas thethe lipidlipid solventsolvent inin microfluidicmicrofluidic processesprocesses [[8,22–24],8,22–24], withwith fewer studies exploring other solvents such as ethanol as a less toxic alternative for medicinal applications, whichwhich wouldwould alsoalso comply with routine industrial processesprocesses [[25].25]. However, thethe choice of solvent used during microfluidics may have an impact; Zook and Vreeland hypothesised that liposomes formed Pharmaceutics 2019, 11, 653 3 of 15 duePharmaceutics to the alcohol2019, 11 ,and 653 aqueous phase mixing, increasing the polarity of the solvent, which3 of 16caused the lipids to become progressively less soluble and self-assemble into planar