Membrane Emulsification in Pharmaceutics and Biotechnology

CHAPTER 7

Membrane Emulsification in Pharmaceutics and Biotechnology Goran T. Vladisavljevi
c Chemical Engineering Department, Loughborough University, Loughborough, United Kingdom

Abbreviations Chemicals and materials AOT APTES APTMS Brij 35 CA CATO CR-310 CR-500 CTAB DCM DOX EA EDA EDC GA Gelucire 44/14 HS-9 IPSO L-7D KPS Labrasol Lipoid E80 Lipoid S75 and Lipoid S100 MCE MCT ML-310 MCA NHS ODS PAA PAH PBS

docusate sodium salt (3-aminopropyl)-triethoxysilane (3-aminopropyl)-trimethoxysilane polyethylene glycol dodecyl ether cellulose acetate glyceryl behenate condensed ricinoleic acid tetraglycerin ester hexaglycerol polyricinoleate cetyltrimethylammonium bromide dichloromethane doxorubicin ethyl acetate ethylenediamine (1-ethyl-3(3-dimethylaminoprophyl)carbodiimide glytaraldehyde lauroyl polyoxylglycerides HS-11, stearic acid hexaglycerol esters iodinated poppy seed oil lauric acid decaglycerol ester potassium peroxydisulfate lauroyl polyoxylglycerides egg yolk lecithin manufactured by Lipoid GmbH soybean phospholipids manufactured by Lipoid GmbH mixed cellulose ester medium-chain triglycerides (Miglyol 812) tetraglycerol monolaurate mixed cellulose acetate N-hydroxysuccinimide octadecyldimethylchlorosilane poly(acrylic acid) poly(allylamine hydrochloride) phosphate-buffered saline

Current Trends and Future Developments on (Bio-) Membranes. https://doi.org/10.1016/B978-0-12-813606-5.00007-5 # 2019 Elsevier Inc. All rights reserved.

167

168 Chapter 7 PC PE PEG PES PGPR PG10-L PLA PLGA PLLA PNIPAM PO-500 Pol PrOH PS PSMA PST PTFE PVA PVDF Q10 QD SBO SDS SL Span 20 Span 80 SPG S-24D S-28D TEOS RIF TDC TGPR TM TMS Transcutol HP Tween 20 Tween 40 Tween 80 Tyl

polycarbonate polyester polyethylene glycol polyethersulfone polyglycerol polyricinoleate polyglyceryl-10-laurate polylactic acid or polylactide poly(lactic-co-glycolic acid) poly(L-lactic acid) poly(N-isopropylacrylamide) hexaglycerin penta ester poloxamer 188 (Pluronic F-68) 2-propanol polysulfone poly(styrene-co-maleic anhydride) polystyrene polytetrafluoroethylene polyvinyl alcohol polyvinylidene fluoride ubidecarenone quantum dot soybean oil sodium dodecyl sulfate sucrose laurate sorbitan monolaurate sorbitan monooleate Shirasu porous glass stearic acid decaglycerol esters tetraethylorthosilicate rifampicin terephthaloyl dichloride tetraglycerol polyricinoleate trimyristin trimethylchlorosilane highly purified diethylene glycol monoethyl ether polyoxyethylene (20) sorbitan monolaurate polyoxyethylene (20) sorbitan monopalmitate polyoxyethylene (20) sorbitan monooleate tyloxapol

Emulsions/dispersions O/W O/W/O S/O/W S/S W/O W/O/W

oil-in-water oil-in-water-in-oil solid-in-oil-in-water solid-in-solid water-in-oil water-in-oil-in-water

Membrane Emulsification in Pharmaceutics and Biotechnology 169 Acronyms DGU DME ME HAI HLB LAI ME NP PME SEM XMT XL

droplet generation unit direct membrane emulsification membrane emulsification hydrophilic active ingredient hydrophile-lipophile balance lipophilic active ingredient membrane emulsification nanoparticle premix membrane emulsification scanning electron microscopy X-ray microtomography cross-linker

Symbols Am A b Ca D Dcell D1 D2 d50 dd dpm dmi dp E f h J K0 K00 nb Q P Pcap Re rb rmo rp rtrans Tm U V W γ δ ε

cross sectional area of membrane, m2 amplitude of membrane oscillation, m height of stirrer blade, m capillary number, / stirrer diameter, m internal diameter of stirred cell, m hydraulic diameter of dispersed phase channel, m hydraulic diameter of continuous phase channel, m diameter of the drop that 50% of a sample’s volume is smaller than, m drop diameter, m drop diameter in premix, m inner diameter of membrane tube, m pore diameter, m energy input, J frequency of membrane oscillation, Hz height of cross-flow channel, m transmembrane flux, m3 m
2 s
1 proportionality constant between pore size and drop size in direct ME, / proportionality constant between pore size and drop size in premix ME, / number of stirrer blades, / volume flow rate, m3 s
1 pressure, Pa capillary pressure, Pa rotating Reynolds number of continuous phase, / inner radius of a cylinder in which the membrane is rotating outer radius of membrane tube pore radius, m radial distance from the axis of rotation at which the shear is maximal, m melting point, K fluid velocity, m s
1 volume, m3 width of cross-flow channel, m interfacial tension between water and oil phase, N m
1 membrane thickness, m membrane porosity, /

170 Chapter 7 η ξ ϕ θ τw ω

viscosity, Pa s pore tortuosity, / dispersed phase volume fraction, / contact angle, rad shear stress on surface, Pa angular velocity of membrane or stirrer, rad s
1

Subscripts c cr d e o w p

continuous phase critical dispersed phase emulsion oil phase water phase pore

1 Introduction In pharmaceutical industry, synthetic microporous membranes are mainly used in separation processes. In a typical membrane separation process such as ultrafiltration or microfiltration, the feed solution is split into retentate and permeate (Fig. 1A), due to selective passage of some feed stream components through the membrane (Mulder, 1996). Shear force is applied on the membrane surface to minimize concentration polymerization. In the last few decades, membrane dispersion processes have emerged, driven by rising interest in microfluidics and progress in microfabricaton technology. In a direct (single-step) membrane dispersion process, liquid I is extruded through the membrane into liquid II without any Membrane filtration

Feed

Direct membrane emulsification

Retentate P1

Continuous phase (II)

P2

Product emulsion P2

P1

Permeate

(A)

P2

Product emulsion

Premix membrane emulsification

Dispersed phase (I)

(B)

P1 Coarse emulsion

(C)

Fig. 1 Comparison between membrane filtration processes (ultrafiltration, microfiltration, and reverse osmosis) and membrane emulsification processes, where P1 > P2. In a membrane filtration process (A), feed stream (a dispersion or solution) is split into two product streams of different compositions (Mulder, 1996). In a direct membrane emulsification (B), two immiscible liquids are brought into contact by passing one liquid (I) through the membrane into another liquid (II) to form an emulsion. Premix membrane emulsification (C) involves passing a coarse emulsion through the membrane, which results in the droplet size reduction (Vladisavljevi
c, 2016b).

Membrane Emulsification in Pharmaceutics and Biotechnology 171 change in its composition. Two main examples of this process are: (i) mixing two miscible solvents, e.g., water and ethanol, usually coupled with nanoprecipitation or crystallization of solute(s) dissolved initially in liquid I, due to their poor solubility in liquid II (Chen et al., 2004), or (ii) dispersing liquid I in the form of drops into liquid II, which occurs if liquids I and II are immiscible. Direct membrane emulsification (direct ME, Fig. 1B) is the formation of drops directly from two separate liquids by passing one liquid (the dispersed or internal phase) through the membrane into another immiscible liquid (the continuous or external phase) (Nakashima et al., 2000; Nakashima et al., 1991). Premix ME (Fig. 1C) or membrane homogenization is the disruption of drops in a preexisting emulsion, achieved by passing the feed emulsion (a premix or pre-emulsion) through the membrane (Suzuki et al., 1996). Both processes have been widely used in pharmaceutics and biotechnology, mainly for encapsulation of active agents (drugs, quantum dots, cells, viruses, etc.).

2 Direct ME Versus Premix ME In direct ME, a pure dispersed phase is brought into contact directly with the continuous phase via membrane pores, which leads to the formation of drops due to mutual immiscibility of carrier liquids in the two phases. Shear force is applied to facilitate the detachment of drops from the membrane surface by providing drag force on the forming drops which overcomes the capillary force. Hydrophobic membranes are used to make water-in-oil (W/O) and oil-in-waterin-oil (O1/W/O2) emulsions (Cheng et al., 2008; Jing et al., 2006), while hydrophilic membranes are used to make oil-in-water (O/W) and water-in-oil-in-water (W1/O/W2) emulsions (Fig. 2A). If the driving pressure is below the critical pressure, the dispersed phase flow rate will be zero and the interface will be immobilized within the membrane pores. This mode of operation is used in membrane-assisted solvent extraction (Schlosser et al., 2005) and will not be discussed here. In premix ME, a pre-emulsion prepared by gentle mixing is pushed through the membrane (Suzuki et al., 1996) or a packed bed of small particles (van der Zwan et al., 2008; Yasuda et al., 2010; Laouini et al., 2014) and homogenized due to laminar shear and elongation stresses within the porous system. Hydrophobic and hydrophilic membranes are used for W/O and O/W emulsions, respectively (Fig. 2B). If the driving pressure is below the critical pressure, the drops will be rejected by the membrane, which will lead to the separation of the feed emulsion into pure continuous phase and concentrated emulsion (Koltuniewicz et al., 1995; Park et al., 2001). Droplet coalescence caused by wetting the membrane surface by the dispersed phase liquid can lead to demulsification, e.g., the break-up of the feed emulsion into two separate phases (Kukizaki and Goto, 2008) or phase inversion (Suzuki et al., 1999). In the phase inversion process, pre-emulsion is comprised of a mixture of high and low HLB surfactants dissolved in aqueous and oil phase, respectively. One example of such surfactants are ML-310 (HLB11) as a hydrophilic surfactant and CR-500 (HLB < 1) as a lipophilic surfactant (Suzuki et al., 1999). W/O emulsion can be inverted into an O/W emulsion by injecting through a hydrophilic

172 Chapter 7 Direct membrane emulsification (DME) W

O/W emulsion

Hydrophilic membrane O

W2

W1/O/W2 emulsion

Hydrophilic membrane W1/O emulsion

O

W/O emulsion

Hydrophobic membrane W

O2

O1 /W/O2 emulsion

Hydrophobic membrane O1/W emulsion

(A) Premix membrane emulsification (PME) without phase inversion O/W emulsion

W1/O/W2 emulsion

Hydrophilic membrane

Hydrophilic membrane

O/W premix

W1/O/W2 premix

W/O emulsion

O1/W/O2 emulsion

Hydrophobic membrane

Hydrophobic membrane

W/O premix

O1/W/O2 premix

(B) Fig. 2 Emulsion types that can be produced using membrane emulsification and the required wettability of the membrane.

membrane (Suzuki et al., 1999), whereas O/W or W/O/W emulsion can be inverted into a W/O emulsion by flowing through a hydrophobic membrane (Kawashima et al., 1991). Premix ME with phase inversion was used to prepare emulsions with densely packed drops in which the dispersed phase content, ϕ, was above 0.8 starting from pre-emulsions with low volume fractions of the dispersed phase (ϕ < 0.2). The highest ϕ value in phase-inverted emulsions achieved using hydrophilic and hydrophobic PTFE membrane was 0.90 and 0.84, for O/W and W/O emulsion, respectively (Suzuki et al., 1999). Direct ME suffers from low transmembrane flux, which means that the product emulsion must be recycled to achieve the desired dispersed phase concentration. For the same pore size, premix ME gives smaller drop size than direct ME, but the premix process leads to more significant membrane fouling and broader drop size distribution.

Membrane Emulsification in Pharmaceutics and Biotechnology 173

3 Comparison Between Membrane and Microfluidic Emulsification 3.1 Number of Drop Generation Units (DGUs) In microfluidic emulsification, drops are usually formed by injecting the inner fluid through a perpendicular channel into a cross-flowing stream of the outer fluid (T junction) (Thorsen et al., 2001) or forcing a coaxial jet of two immiscible liquids to flow through a narrow orifice, which leads to jet fragmentation (flow focusing device) (Anna et al., 2003). Although the drop generation rate in flow focusing devices can exceed 10,000 Hz (Yobas et al., 2006), the flow rate of inner fluid is typically 0.01–5 mL h
1, because there is only one drop generation unit (DGU). On the other hand, membranes have billions of pores per m2. SPG membrane has 109–1014 pores per m2, while silicon plates with straight-through microchannels can contain >108 microfabricated pores per m2(Kobayashi et al., 2012). Although only 1%–8% of the pores are active in direct ME with SPG membrane (Vladisavljevi
c et al., 2004a), the total number of active DGUs is still above 107 per m2.

3.2 Drop Generation Regimes In cross-flow ME, the ratio of the diameter of the cross-flow channel to the pore diameter, D2/D1, is 102–106, which means that droplets are unconfined during their growth on the membrane surface. In a microfluidic T junction, droplets are highly confined in the cross-flow channel (D2/D1 ¼1–10, Fig. 3). As a result, at low capillary numbers of the outer fluid (Cac < 0.015), drops are formed by the plugging-squeezing mechanism (Seemann et al., 2012; Vladisavljevi
c et al., 2012); they occupy almost the whole cross section of the main channel and the pinch-off occurs due to build-up of pressure upstream of the forming drop. In cross-flow ME, the forming drops occupy a small fraction of the total cross sectional area of the main channel and the pressure built up upstream of the forming drop is negligible. In a T junction at Cac > 0.3, the inner fluid forms a long jet in the main channel, prior to its break-up due to Plateau-Rayleigh instability. In direct ME, the main channel is so large that the shear force from the outer fluid is not high enough to extend the inner fluid into a long jet as it exits the pore. Instead, at high flow rates, the inner fluid flows continuously out of the pore due to high inertial forces, which is known as the continuous outflow regime (Kobayashi et al., 2003). In this regime, the drop retains its spherical shape while it grows on the pore mouth and detach after reaching a large size without being pulled into a jet (Vladisavljevi
c et al., 2007, 2011).

3.3 Droplet Throughput The droplet throughputs in ME are much higher than in microfluidic devices. In premix ME, the flow rate of the inner fluid through SPG membrane with a cross sectional area of 4 cm2 and the pore size of 11 μm can exceed 104 mL h
1, resulting in the drop size of 10 μm

174 Chapter 7 Cross-flow ME (D2/D1»10)

T junction (D2/D1)

Dripping regime Dripping regime Continuous phase

Continuous phase

D2

D2 D1 D1

Dispersed phase Continuous outflow regime

Dispersed phase Jetting regime

Dispersed phase

(A)

(B) Fig. 3 Comparison of droplet generation in cross-flow membrane emulsification (ME) and microfluidic T junction. D1 and D2 are the hydraulic diameters of the channel for supplying the dispersed phase and continuous phase, respectively. In cross-flow ME, jetting is not possible due to low shear on the membrane surface. Instead of that, at high injection rates of the dispersed phase, the drops grow continuously on the membrane surface, until they finally detach.

(Vladisavljevi
c et al., 2004b). In industrial-scale modules with a membrane surface area of 10–100 m2, the flow rate of the inner fluid in premix ME can exceed 106 L h
1. The parallelization of microfluidic DGUs is difficult due to high-pressure drop in distribution channels and difficulties in providing uniform flow distribution in long channel networks.

3.4 Droplet Size Uniformity and Morphology In ME, the drop size variations are above 10% with a span ((d90–d10)/d50) of 0.3–0.6. In microfluidic systems, the drop size variations in the dripping regime are below 3% with a span of <0.05, which satisfies the definition of the monodisperse particle size distribution ( Jillavenkatesa et al., 2001). In addition, microfluidic devices can produce drops with complex internal structures (Vladisavljevi
c, 2016a) and manipulate (merge, split, sort, or move) individual drops with high accuracy after production. In ME, only one fluid can be injected through the membrane and all drops are collected together in a large channel. Therefore, the

Membrane Emulsification in Pharmaceutics and Biotechnology 175 drops cannot be individually tracked and manipulated, and multiple emulsions can only be formed via a two-step process where primary emulsification is achieved in a standard homogenizer and secondary emulsification in membrane device. In microfluidic devices, more than two fluids can be supplied simultaneously through different channels with high precision, enabling single-step on-chip generation of complex drops with multiple concentric layers (double, triple, quadruple, and quintuple drops), multiple inner drops (2–6) (Nabavi et al., 2017), distinct inner drops, and two or three distinct surface regions (Janus and ternary drops). Although multiphase flow microfluidics is a powerful tool for production of droplets and particles for drug delivery, this topic will not be covered in this chapter.

4 Membrane Versus Conventional Emulsification ME is associated with mild shear conditions, low energy consumption, isothermal operation at room temperature, and high encapsulation efficiency of active ingredients (Surh et al., 2007; Vladisavljevi
c and Williams, 2008; Dragosavac et al., 2012). Conventional homogenizers can damage shear- and heat-sensitive ingredients, such as proteins, vitamins, and pigments, due to high energy inputs and high shear forces used to disrupt drops (Karbstein and Schubert, 1995).

4.1 Shear Rate In direct ME, the shear rate at the membrane surface is (1–50)103 s
1, but micron-sized drops can be produced even at zero shear due to “push-off force” originating from steric hindrance effects (Kukizaki, 2009a; Kukizaki and Goto, 2009; Kosvintsev et al., 2008) or Laplace pressure gradient because of curvature imbalance along the interface between two immiscible fluids caused by an abrupt change in cross section of the channels (Kobayashi et al., 2011). In oscillating ME devices, shear is localized within the distance of 0.5 mm from the membrane surface and negligible outside this region (Silva et al., 2015). The shear rate in in-line mixers and colloid mills is (1–2)105 s
1 and can reach 107 s
1 in Microfluidizers. Furthermore, in conventional homogenizers, shear forces show high spatial fluctuations leading to nonuniform drops. On the other hand, in cross-flow and oscillating ME systems, shear rate is constant over the whole membrane surface.

4.2 Energy Input per Unit Volume (E/V) In premix ME, the pre-emulsion is injected through SPG membrane at the pressure of 1–50 bar, and thus, E/V is (1–50)  105 J m
3 per extrusion cycle. In direct ME, E/V ranges from 103 J m
3 for 30-μm drops and ϕ¼1 vol% to 106 J m
3 for 1-μm drops and ϕ¼50 vol% (Gijsbertsen-Abrahamse et al., 2004). In high-pressure valve homogenizers, E/V varies from 106 J m
3 for 1-μm drops and ϕ¼30 vol% to 108 J m
3 for 0.4-μm drops and ϕ¼30 vol%.

176 Chapter 7

5 Microporous Membranes for Emulsification The desirable properties of membranes used for emulsification are: (i) uniform pores with a wide range of pore sizes, so that drops with a narrow size distribution can be prepared over a wide range of sizes; (ii) low hydrodynamic resistance to prevent high transmembrane pressures; (iii) high mechanical durability; (iv) high thermal and chemical stability; (v) straightforward surface modification to easily change surface wettability from hydrophilic to hydrophobic or surface charge from negative to positive or vice versa; (vi) cheap fabrication process. In addition, the membranes used in pharmaceutical applications must be sterilizable and made of a biocompatible material. The most common emulsification membranes are Shirasu Porous Glass (SPG), polymeric, and microengineered membranes. SPG membrane is more porous than microengineered membranes, easier to modify due to high reactivity of surface silanol groups, has a wider range of available pore sizes, and is cheaper. On the other hand, microengineered membranes are thinner than SPG membrane and have a low hydrodynamic resistance and isoporous structure with regular spatial arrangement of the pores. Polymeric membranes are the cheapest and often used as disposable filters.

5.1 SPG Membrane Shirasu Porous Glass (SPG) membrane is widely used for emulsification. This membrane was fabricated from Shirasu (volcanic ash), CaCO3, and boric acid by spinodal decomposition of glass melt at 650–750°C (Nakashima and Kuroki, 1981; Nakashima and Shimizu, 1986; Kukizaki and Nakashima, 2004). The mean pore size is controlled by adjusting the duration and temperature of the phase separation process (Kukizaki, 2010). The duration of phase separation varies from several hours for submicron-sized pores to several tens of hours for the pores larger than 10 μm. SPG membrane has a uniform porous structure with highly branched cylindrical pores, as shown in Figs. 4A and B (Vladisavljevi
c et al., 2007). The membrane porosity is high (50%–60%) with a pore tortuosity of about 1.3 for all pore sizes. Due to large thickness (400–1000 μm), isotropic SPG membrane offers a high resistance to flow (108–1012 m
1), but it can be reduced by >10 times if the membrane has an anisotropic structure, shown in Fig. 4C (Kukizaki and Goto, 2007a). SPG is more mechanically robust than sintered alumina or zirconia (Nakashima et al., 1992) and more stable in alkaline solutions than Porous Vycor Glass, due to higher alumina content. Both glasses are unstable in highly alkaline solutions, but the stability of SPG at high pH values can be improved by adding zinc in the mother glass (Kukizaki, 2010).

Membrane Emulsification in Pharmaceutics and Biotechnology 177

Fig. 4 (A) SEM micrograph of the surface of isotropic SPG membrane polished with diamond paste to visualize droplet generation by metallurgical microscope; (B) XMT image of the cross section of isotropic SPG membrane. No difference between the internal structure and surface morphology was found (Vladisavljevi
c et al., 2007); (C) SEM micrograph of the cross section of anisotropic SPG membrane (Kukizaki and Goto, 2007b); (D, E) SEM image of track-etched polycarbonate (PC) membrane with a pore size of 670 nm manufactured by irradiating nonporous PC film with Cu3+ (Chen et al., 2008); (F) SEM image of hydrophilized polypropylene membrane filter with a pore size of 200 nm (He and Ulbricht, 2006); (G) optical micrograph of electroformed nickel microsieve with a pore size of 40 μm (Othman et al., 2016); (H) Optical micrograph of stainless steal membrane with 10-μm laser drilled pores (Othman et al., 2016); (I) SEM image of asymmetric silicon plate with cylindrical channels at the bottom side and microslots at the top side (Kobayashi et al., 2011).

SPG membrane is hydrophilic, but can be rendered hydrophobic by covalent treatment with organosilanes (Kukizaki and Wada, 2008) or coating the surface with hydrophobic silicone resin (Vladisavljevi
c et al., 2005), such as polymethylsilsesquioxane. Monochlorosilanes such as TMS and ODS are very suitable for hydrophobic treatment because they only react with the

178 Chapter 7 silanol groups on the SPG surface and not with each other (Kai et al., 2006). The membrane hydrophobicity can be tailored by adjusting the length of the hydrocarbon chain in the silane molecule (Kukizaki and Wada, 2008). Untreated SPG is negatively charged (Kukizaki, 2009b), but a positive charge can be induced with (3-aminopropyl)-trimethoxysilane (APTMS) and (3-aminopropyl)triethoxysilane (APTES).

5.2 Polymeric Membranes Track-etched polymeric membranes made from polycarbonate (PC), polyesters (PEs) such as polyethylene terephthalate (PET), and polyimide (PI) are the most common polymeric membranes used for emulsification. The track-etch method involves bombarding a nonporous polymer sheet with fission fragments or accelerated heavy ion beams from a nuclear reactor to break chemical bonds in the polymer backbone and create latent (sensitized) tracks, followed by chemical etching (typically using concentrated NaOH solution) to convert the latent tracks into hollow channels (Apel, 2001). Nuclepore™ track-etched PC membranes are available with a pore size ranging from 15 nm to 12 μm and the membrane diameter can range from 10 mm to 300 mm. The pore density is controlled by the irradiation time, while the pore shape and size depend on the etching time. Track-etched membranes are less sensitive to fouling than SPG membranes, because they are >10 times thinner and contain non-interconnected,straight-through pores (Fig. 4D and E). Both membranes are sterilizable, thus preventing cross-contamination during processing, but tracketched polymer membranes are cheaper than SPG membranes, and thus, can be disposed after use ( Joseph and Bunjes, 2012). A hydrophilicity of track-etched PC membrane can be enhanced by plasma treatment (Gehrmann and Bunjes, 2018). The most popular plasmas used for hydrophilic treatment are those obtained from Ar, He, O2, N2, CO2, and air. The thickness of track-etched polymeric membranes is <30 μm, but the porosity is relatively small (5%–30%), which limits the transmembrane flux. Disposable syringe filters made of polytetrafluoroethylene (PTFE), polyethersulfone (PES), cellulose acetate (CA), mixed cellulose ester (MCE) (cellulose nitrate with a small content of CA), nylon, polyesters (PEs), and polyvinylidene fluoride (PVDF) are widely used in premix ME (Gehrmann and Bunjes, 2016a). A porous structure of syringe filters is formed by phase inversion of a homogeneous polymer solution, which can solidify via: (i) thermally induced phase separation; (ii) controlled solvent evaporation from a mixture of polymer, solvent, and nonsolvent; (iii) precipitation from the vapor phase; (iv) immersion precipitation (Liu et al., 2011). Syringe filters have a thickness of 20–200 μm and highly porous sponge-like structure with a porosity of 60%–76% (Fig. 4F). Syringe filters are thicker than track-etched membrane and exhibit a broader pore size distribution (Gehrmann and Bunjes, 2018).

Membrane Emulsification in Pharmaceutics and Biotechnology 179

5.3 Microengineered or Microsieve Membranes Microengineered (microsieve) membranes have highly ordered uniform pores with welldefined geometry, manufactured by electroplating (nickel membrane, Fig. 4G) (Nazir et al., 2011; Schadler and Windhab, 2006; Egidi et al., 2008), reactive ion etching (RIE) (silicon nitride microsieves, van Rijn et al., 1997), laser ablation (stainless-steel membrane, shown in Fig. 4H) (Dowding et al., 2001; Vladisavljevi
c and Williams, 2006), deep reactive ion etching (DRIE) (straight-through silicon microchannel plate, Fig. 4I) (Kobayashi et al., 2003), and anodic oxidation of aluminium (Yanagishita et al., 2010; Lee and Mattia, 2013). In anodic oxidation process, formation of uniform pore arrays without defects and dislocations can be enhanced by imprinting aluminium substrate with a mold before anodization (Yanagishita et al., 2017). The pore size, interpore spacing, and membrane thickness can be controlled by the applied voltage, anodization time, electrolyte concentration, and temperature. The pore size and interpore spacing are proportional to the voltage and inversely proportional to the electrolyte concentration (Belwalkar et al., 2008). The membrane thickness increases with increasing acid concentration in the bath.

6 Equipment for ME Common batch ME devices are cross-flow modules with tubular membranes, about 10 mm in diameter and 100–500 mm in length, SPG micro kits with the same membrane diameter and a tube length of 7–20 mm, and membrane extruders. Recently, rotating, oscillating, and flow pulsing ME systems were introduced to achieve continuous production of emulsions.

6.1 Batch Cross-Flow ME Systems In this system, a continuous phase/emulsion circulates in a closed loop between a holding tank and the membrane module (Fig. 5). The dispersed phase from the shell side of the module penetrates through the membrane into the continuous phase stream, driven by a compressed gas. The transmembrane pressure is 1.1–5 times greater than the capillary pressure (Vladisavljevi
c and Schubert, 2003). The most uniform drops are produced at low transmembrane pressures. The dispersed to continuous phase flow rate ratio (Qd/Qc) is very low, because the flux through the membrane must be kept at small values to obtain uniform drops and significant continuous phase flow rate is needed for shear generation at the membrane surface. Therefore, the product emulsion must be recycled, which can lead to secondary droplet disintegration. Cross-flow systems are easy to scale up and offer constant shear operation. To decrease the flow rate in cross-flow channel, while keeping a high shear at the membrane surface, turbulence promoters can be inserted inside the membrane tube (Koris et al., 2011) or cross-flow stream in the membrane tube can be brought to back-and-forth pulsations (Piacentini et al., 2013b, Holdich et al., 2013). The droplet uniformity in the product emulsion containing 30 vol% of dispersed phase was compared by running the process

180 Chapter 7

Vent 1

Membrane module

Continuous phase Compressed Dispersed phase gas

Fig. 5 Apparatus for cross-flow direct ME using tubular SPG membrane. During start-up, valve 1 is open to remove air from the shell side of the module (Nakashima et al., 1994). The space occupied by the dispersed phase is shown in dark gray, while the rig space occupied by the continuous phase/emulsion is shown in light gray.

for 8.5 h using steady or pulsed cross flow of continuous phase. In pulsed cross flow, the droplet size distribution remained constant over 8.5 h, while in steady cross flow, the product emulsion was polydispersed after 3 h due to higher recirculation rate (Piacentini et al., 2013b).

6.2 Batch SPG Micro Kits The total volume of the continuous phase in cross-flow systems is more than several hundred milliliters. SPG micro kits shown in Fig. 6 can operate with <50 mL of the continuous phase and the dead volume is very low, which is useful feature for pharmaceutical emulsions for clinical use (Higashi and Setoguchi, 2000) and formulations of new drugs entering early clinical development, because at that stage very limited amounts of drugs are available (Balbach and Korn, 2004). In the external pressure type micro kit for direct ME (Fig. 6A), the continuous phase is stirred with a magnetic bar or impeller at 300–1000 rpm and the membrane tube is immersed in the continuous phase. The drops are formed at the inner wall of the SPG tube, just like in crossflow systems. The membrane tube functions as a draft tube, resulting in more efficient mixing than in the internal pressure SPG kit. A water-jacketed pressure vessel is used to emulsify lipids

Membrane Emulsification in Pharmaceutics and Biotechnology 181 Dispersed phase

Vent

Premix

Membrane

Emulsion

(A)

(B)

CP20K

(C)

(D)

Fig. 6 Small-scale laboratory kits for membrane emulsification: (A) External pressure type micro kit for direct ME; (B) external pressure type micro kit for premix ME; (C) Internal pressure type micro kit for direct ME; (D) SPG filter kit. All kits are commercially available from SPG Technology Co., Ltd. (Sadowara, Japan). The kits are supplied with SPG membrane tube with a diameter of about 10 mm and an effective length of 10
15 mm.

which are solid at the room temperature and the beaker is placed on a hotplate ( Joseph and Bunjes, 2014). In the external pressure type micro kit for premix ME (Fig. 6B), the membrane tube is placed above the collection beaker and the premix is supplied from the pressure vessel under pressure, which is 1 bar for a 10-μm membrane, 10 bar for a 1-μm membrane, and 50 bar for a 0.1-μ m SPG membrane. The product emulsion flows under gravity from the membrane tube into the collection beaker. To decrease the droplet size, the product emulsion can be repeatedly extruded through the same membrane without cleaning between successive extrusions

182 Chapter 7 (Vladisavljevi
c et al., 2004b, 2006a, 2006b). Repeated membrane extrusion was developed by Olson et al. (1979) and used for homogenizing liposomal suspensions using track-etch polycarbonate filters. In the internal pressure type micro kit (Fig. 6C), the membrane tube is pressurized from the inner side, the maximum driving pressure that the module can withstand is 3 bar, while the external pressure type kit can withstand the pressures up to 5 bar. In a SPG filter kit (Fig. 6D), the dispersed phase is loaded into a stainless-steel syringe, which is attached to a syringe dispensing device driven by the controlled speed motor to control the flow rate through the membrane. The membrane is dipped in a glass vial filled with 6–10 mL of the continuous phase stirred with a magnetic bar.

6.3 Membrane Extruders Membrane extruders are used for extrusion of multilamellar liposomal suspensions and course emulsions. The Avestin LiposoFast-Basic extruder consists of two gas-tight syringes mounted on a stainless-steel holder that holds a PC membrane sandwiched between two nylon meshes ( Joseph and Bunjes, 2012). The extruder produces fine emulsions or unilamellar liposomes by extruding manually premixes or multilamellar liposome suspensions through the membrane. The sample is pushed back and forth multiple times between the two syringes until a desired size distribution is achieved in the product suspension. A disposable syringe filter can be used in the extruder instead of a PC membrane (Gehrmann and Bunjes, 2016a). The premix ME process in the LiposoFast-Basic involves two steps (Fig. 7A): (i) mixing the oil and aqueous phases together by passing the liquids repeatedly back and forth between the two syringes with no membrane in between, and (ii) homogenizing the premix by pushing it back and forth through the membrane mounted between the two syringes. The LiposoFast-Basic extruder can be placed in the LiposoFast-Stabilizer to assist in extrusion of highly concentrated samples, especially through the membranes with small pore sizes. The LiposoFast-Stabilizer can accommodate 0.5 mL and 1.0 mL syringes. For the preparation of nanoemulsions from molten lipids, the extruder is immersed in a water bath to maintain the extrusion temperature above the melting point of the lipid. The advantages of the LiposoFast-Basic are small sample volumes (0.2–1 mL), virtually no dead volume, and short production times (several minutes), making it convenient for preparation of large number of samples for screening purposes and handling of expensive, scarce, or highly potent drugs used in small quantities ( Joseph and Bunjes, 2012). The EmulsiFlex-C5 homogenizer can be used to process larger sample volumes (7–1000 mL) at the flow rate of 1–5 L/h, depending on the homogenizing pressure that can reach 207 MPa. It has an air driven pump and can be equipped with a standard homogenizing valve for high-pressure

Membrane Emulsification in Pharmaceutics and Biotechnology 183

Fig. 7 Preparation of nanoemulsions by membrane extrusion: (A) Manual extrusion involving: (i) Mixing together oil and aqueous phase by pushing pure liquids back and forth multiple times between two 20-ml syringes; (ii) Homogenization of the mixture by pushing the premix back and forth multiple times through a disposable syringe filter placed between the two 1-ml syringes (Gehrmann and Bunjes, 2016a); (B) Instrumented small-scale membrane extruder composed of a computercontrolled high-pressure syringe pump, pressure transducer, membrane holder, collection vial, and non-return valve for sample recycling (Gehrmann and Bunjes, 2016b).

homogenization or filter/extruder unit for premix ME (Gehrmann and Bunjes, 2016a). Unlike in the LiposoFast extruder, the sample passes through the membrane only in one direction. Gehrmann and Bunjes (2016b) developed an automated small-scale membrane extruder (Fig. 7B) composed of a stainless-steel syringe mounted on a high-pressure syringe pump and a

184 Chapter 7 membrane holder, which was the filter/extruder unit of the EmulsiFlex-C5 homogenizer. The membrane holder was connected to a syringe open to atmospheric pressure, which was connected to the outlet of the syringe pump via a non-return valve to allow sample recycling. The extrusion pressure and pump flow rate were recorded continuously every 0.1 s.

6.4 Rotating ME Systems In static ME systems, shear force on the membrane surface arises from mono-directional flow of the continuous phase or agitation over the membrane surface. In rotating membrane systems, shear on the membrane surface is provided by spinning the membrane tube in the continuous phase at 100–1500 rpm in laboratory rigs (Vladisavljevi
c and Williams, 2006; Aryanti et al., 2006) or up to 10,000 rpm in commercial MEGATRON devices (Graber, 2010). Rotating SPG ME systems can be operated batchwise (Pawlik and Norton, 2012, 2013) or continuously. In the continuous system, shear on the membrane surface is controlled by the membrane rotational speed rather than by the continuous phase flow rate, which has a low impact compared to the rotational speed. In dynamic membrane systems, concentrated emulsions can be produced continuously without product recycling, which can prevent damage of shear-sensitive active pharmaceutical ingredients.

6.5 Oscillating ME Systems In oscillating ME systems, membrane tube rotates within an otherwise static continuous phase periodically clockwise and counter-clockwise (Silva et al., 2015) or oscillates up and down (Holdich et al., 2010). Shear on the membrane surface is a sinusoidal function of time and is controlled by adjusting the frequency (10–90 Hz) and amplitude (0.1–7 mm) of membrane oscillations. Typical membranes used in oscillating ME systems are stainless-steel membranes with laser drilled pores (Silva et al., 2015), electroformed nickel membranes (Holdich et al., 2010), and stainless-steelmicro-screens (Zeng et al., 2013).

6.6 Stirred Cells Stirred cells are simple batch emulsification devices and consisted of a glass tube with a paddle stirrer suspended just above a flat-sheet membrane disc (Kosvintsev et al., 2005). They generate relatively uniform emulsion droplets with size distribution spans down to 0.3, despite variable shear on the membrane surface. The Dispersion Cell is the most common stirred cell for ME, available from Micropore Technologies Ltd. The glass body of the cell can have two oppositely placed tube connectors to achieve a continuous operation.

7 Prediction of Drop Size in Direct ME The droplet size in direct ME depends on the membrane properties (pore size and shape, pore spacing, wettability of the membrane surface, membrane charge and porosity), emulsion formulation (viscosity and density of the dispersed and continuous phase, concentration of

Membrane Emulsification in Pharmaceutics and Biotechnology 185 surfactants, and additives), and process parameters (shear on the membrane surface and flux through the membrane) ( Joscelyne and Tr€aga˚rdh, 2000).

7.1 Effects of Transmembrane Pressure and Flux In direct ME, the drops are formed by: (a) shear-controlled detachment caused by shear force on the membrane surface and (b) spontaneous (step) emulsification driven by the gradient in Laplace pressure due to curvature imbalance along the interface (Sugiura et al., 2002). Circular pores pinch off the dispersed phase due to shear force (Kosvintsev et al., 2005). Spontaneous detachment occurs at irregular pores of SPG membrane, terraced microchannels (Sugiura et al., 2002), and straight-through microchannels with high aspect ratio (Kobayashi et al., 2004). In the shear-controlled process, larger drops are formed at higher transmembrane fluxes, because the neck is not instantaneously pinched off, but after a finite necking time, during which an additional volume of fluid will flow into the forming droplet (van der Graaf et al., 2006). At high dispersed phase flow rates through the membrane, a push-off force, due to droplet-droplet squeezing on the membrane surface, facilitates the droplet detachment (Egidi et al., 2008). Droplet generation regime can be predicted from the capillary number of the dispersed phase: Cad ¼ Udηd/γ wo, where Ud is the velocity of the dispersed phase in a pore and μd is the viscosity of the dispersed phase. Dripping regime occurs at low Cad values (Cad < 0.05). In this regime, the interfacial tension force is much higher than the viscous force of the dispersed phase (Sugiura et al., 2002) and the drop size depends mainly on the pore size. At Cad > 0.05, the viscous force of the dispersed phase dominates over the interfacial tension and the drops are formed in the continuous outflow regime. In this regime, dd > 10dp and the drop size strongly depends on transmembrane flux (Kobayashi et al., 2003). The velocity of the dispersed phase at which the transition from a dripping to continuous outflow occurs is independent on the pore size (Kobayashi et al., 2011) and increases on decreasing the viscosity of the dispersed phase (Vladisavljevi
c et al., 2011). At ηd/ηc of 55, the critical velocity of the dispersed phase in the pores is 2–5 mm/s (Sugiura et al., 2002; Kobayashi et al., 2011). The transition from a dripping to continuous outflow does not occur simultaneously for all the pores, but depends on local hydrodynamic conditions, local velocity in a pore, and local membrane wettability. Also, local velocities in the pores are randomly distributed and can range significantly from pore to pore.

7.2 Effects of Pore Size and Shear Force In the dripping regime, the drop size is directly proportional to the pore size: dd ¼ K0 dp, where K0 ¼ 2.8–3.5 for SPG membrane (Kukizaki and Goto, 2009; Kukizaki and Goto, 2007b; Nakashima et al., 1991; Vladisavljevi
c et al., 2006a). The K0 value is higher at lower shear on the membrane surface, but even at zero shear K0 was 3.3 for SPG membrane and O/W emulsion produced at low transmembrane flux (Kukizaki and Goto, 2009).

186 Chapter 7 In shear-controlled ME process, a droplet grows on the pore mouth until the shear force exceeds the capillary force. According to a simplified shear model (Kosvintsev et al., 2005): rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 18τw rp + 2 81τ4w rp4 + 4rp2 τ2w γ 2 (1) dd ¼ 3τw where rp is the pore radius and τw is the shear stress on the membrane surface. According to Eq. (2), dd decreases on increasing τw and approaches 2rp as τw ! ∞. Table 1 gives equations for calculation of τw in different systems. The variations of τw with time and position along the membrane surface are shown in Fig. 8 (Laouini et al., 2013). In a pulsed cross-flow system (Holdich et al., 2013), flow pulsations are superimposed on cross flow, but the contribution of the cross flow to the overall shear stress is negligible (Piacentini et al., 2013a).

7.3 Effect of Surfactant The rate of adsorption of surfactants at the drop surface affects the final drop size significantly (Schr€ oder et al., 1998; van der Graaf et al., 2004; Rayner et al., 2005). The surfactants molecules should adsorb fast to the newly created interface, but they should not bind to the membrane surface. It means that nonionic or anionic surfactants must be used with negatively charged SPG membrane (Nakashima et al., 1993). Zwitterionic surfactants must be avoided (Surh et al., 2008), even if their overall charge is negative. To prepare droplets stabilized with cationic surfactants, SPG membrane must be positively charged or anionic surfactant must be displaced with a cationic surfactant after ME (Vladisavljevi
c and McClements, 2010).

8 Factors Affecting Droplet Size in Premix ME In premix ME, interfacial tension effects (Rayleigh and Laplace instabilities), snap-off effects due to localized shear forces, and steric hindrance effects cause the drop break-up, as they are carried through the pores with the continuous phase (van der Zwan et al., 2006). The final drop size depends on the pore size, the shear stress at the pore walls caused by flow Table 1 Shear stress on the membrane surface in direct ME for different membrane shapes and shear generation techniques (Vladisavljevi
c, 2016b) Shear Generation Technique

Membrane Shape

Shear Stress on Membrane Surface

Cross flow

Cylindrical

τw ¼ 8ηdcmiUc

Cross flow Rotating

Flat Cylindrical

Paddle stirrer

Flat

c ωrtrans pffiffiffiffiffiffiffiffiffiffiffiffi ffia τw , max ¼ 0:825η

Oscillating

Any

τw, max ¼ (2πAf)3/2(ηcρc)1/2

3Qc ηc τw ¼ 2h 2W

c rmo ω τw ¼ 2η r 2
r 2 2

b

a

  b 0:036 rtrans ¼ 1:23 D2 0:57 + 0:35 DDcell nb 0:116 Re=ð1000 + 1:43ReÞ. T

mo

ηc =ðρc ωÞ

Membrane Emulsification in Pharmaceutics and Biotechnology 187 Stirred cell

Cross flow

w

h or dmi

Qaq Dispersed phase

t

rtrans

r

Dispersed phase

t

tmax

t = const

Membrane area

r

0

(A)

(B)

Oscillating membrane without cross flow Pulsed cross flow

Dispersed phase

Qaq Dispersed phase

a, f t t max

t max

t max

0

t max

t CF

0 Time

Time

–t max

–t max Contribution from cross-flow

(C)

(D) Fig. 8 Spatial or temporal variations of shear stress at the membrane surface in different direct ME devices: (A) Stirred cell; (B) Cross flow; (C) Oscillating membrane; (D) Pulsed cross-flow. The variation of the shear stress with time for pulsed cross-flow (D) is the same as in the oscillation membrane system (C), except that temporal shear variations are superimposed on constant shear stress (τCF) generated as a result of cross flow (Laouini et al., 2013).

of the premix through pores, the number of extrusion cycles applied, the viscosity of both phases, and the interfacial tension (Nazir et al., 2010). The mean drop size decreases on increasing shear stress at the walls of the pores:   (2) τw, p ¼ 8ηe Jξ= εdp

188 Chapter 7 where ηe is the viscosity of emulsion inside the pores. Therefore, smaller droplets are generated at higher transmembrane fluxes. The mean drop size is a power function of the pore size: dd ¼ K00 (dp)n, where n < 1. 1 < dd/dp < 1.5 for SPG membranes with dp ¼ 5–20 μm at τw. c et al., 2006a). The mean drop size is reduced after each extrusion and p ¼ 200 Pa (Vladisavljevi
reaches a constant steady-state value after completing large number of extrusions (Vladisavljevi
c et al., 2004b; Laouini et al., 2014).

9 Production of Pharmaceutical Microemulsions and Nanoemulsions Using ME 9.1 Microemulsions Vs. Nanoemulsions An O/W microemulsion is a thermodynamically stable dispersed system consisting of oil droplets dispersed within an aqueous phase (McClements, 2012). An O/W nanoemulsion is a thermodynamically unstable (but kinetically stable) dispersed system consisting of oil droplets dispersed in aqueous phase. In both dispersions, the mean droplet diameter is below 200 nm (McClements, 2012), but the fabrication process is significantly different. For a microemulsion, the free energy of the dispersed system is lower than the free energy of two separate phases. Microemulsions can be formed by bringing the two phases together and supplying very limited amount of external energy, because there is a kinetic energy barrier (the activation energy, ΔG*) that must be overcome. In direct ME, the external energy is supplied by driving the dispersed phase through the membrane at certain pressure and providing a gentle agitation or cross flow of the continuous phase (Oh et al., 2011). The drop size distribution depends on the hydrodynamic conditions in the continuous phase and emulsion formulation, but very slightly on the membrane pore size. The role of membrane in formation of microemulsion is primarily to inject the dispersed phase into the continuous phase at controlled rate and provide uniform shear conditions during emulsification. In the preparation of nanoemulsions, the drop size is directly proportional to the pore size and high amount of energy must be provided, usually in the form of high transmembrane pressure, to force the dispersed phase or premix through the membrane nanopores. Microemulsions can only be formed for certain mass ratios of the main system constituents (water, oil, and surfactants). Fig. 9 shows the pseudo-ternary phase diagram for the system comprised of water, DCM and Tween 20/Tween 80 1:1 vol/vol surfactant blend. A O/W microemulsion can only be formed within the gray-shaded area, i.e., for relatively high surfactant concentrations (low interfacial tensions) and low oil contents. The minimum concentration of surfactant required for the formation of microemulsion can be determined by preparing a series of emulsions with increasing concentration of surfactant and measuring the drop size at each surfactant concentration. This method was used by Adamczak et al. (2014) to determine the critical concentration of docusate sodium

Membrane Emulsification in Pharmaceutics and Biotechnology 189 Tween 20:Tween 80=1:1 70

30

80

Nanoemulsion or Macroemulsion

90

20 Microemulsion

10

DCM 100 0

10

20

30

40

50

60

70

80

90

0 100 Water

Fig. 9 The pseudo-ternary phase diagram for the dispersed system composed of dichloromethane (DCM), Tween 80/Tween 20 1:1 vol/vol surfactant blend, and water (Oh et al., 2011). Within the gray microemulsion region, the drop size in direct ME is much smaller than the membrane pore size and independent on the pore size. In the nanoemulsion or macroemulsion region, the drop size in direct ME is larger than the pore size.

Fig. 10 The effect of the concentration of AOT (docusate sodium) in the dispersed phase on the average size of squalene drops formed by direct ME in a stirred cell using ceramic membrane with a pore size of 0.4 μm (Adamczak et al., 2014). In the macroemulsion region, the average drop size was several times larger than the membrane pore size, while in the microemulsion region, the average drop size was 2–5 times smaller than the membrane pore size. The dispersed phase was AOT solution in squalene and the continuous phase was 100 ppm PAH in 0.015 M NaCl solution.

(AOT) in the dispersed phase for preparation of squalene O/W microemulsions (Fig. 10). Squalene is a linear triterpene that is extensively used as a principal component of parenteral emulsions for drug and vaccine delivery (Fox, 2009). At low surfactant concentrations in the dispersed phase, the average drop size in direct ME was in the micron size range (2–5 μm) and several times larger than the pore size of 0.4 μm. In addition, the drop size was in

190 Chapter 7 good agreement with an existing force balance model (the dashed line in Fig. 10), indicating that a macroemulsion was formed. A decrease of the average drop size with an increase in surfactant concentration in this region is a consequence of the decrease in interfacial tension, which facilitates detachment of the droplets from the membrane surface under agitation. At the surfactant concentration above 10
2 M, the average droplet size was 2–5 times smaller than the pore size, indicating the formation of a microemulsion. An abrupt decrease in drop size at the critical surfactant concentration of 10
2 M indicates the change in drop generation mechanism from membrane-assisted drop generation to self-emulsification. In Tables 2 and 3, the term nanoemulsion will be used for all emulsions with a mean droplet size <1 μm.

Table 2 Examples of nanoemulsions prepared using direct membrane emulsification (dd—average droplet size, DP—dispersed phase; CP—continuous phase) Process

Product

Formulation

Authors

O/W nanoemulsions for delivery of poorly water-soluble drugs O/W nanoemulsions (A) Internal pressure type loaded with itraconazole micro kit, 2.5-μm SPG (dd ¼ 410–550 nm) membrane

(B) Internal pressure type micro kit, 2.5-μm SPG membrane

O/W nanoemulsion loaded with flurbiprofen (dd ¼ 360 nm)

(C) Internal pressure type micro kit, 1.1-μm SPG membrane

O/W nanoemulsion loaded with fenofibrate (dd ¼ 157 nm)

(D) Cross-flow SPG membrane, 0.9-μm pore size

O/W nanoemulsion loaded with vitamin E (dd ¼ 106 nm, span ¼ 0.3, ϕ ¼0.18)

DP: 40 wt% itraconazole in DCM CP: 5% (w/v) surfactant mixture (Transcutol HP/ Span 20 at 1:1 mass ratio) + 2.5% (w/v) dextran in water DP: 20 wt% flurbiprofen in DCM CP: 5% (w/v) surfactant mixture (Tween 20/ Tween 80 at 1:1 mass ratio) + 1% (w/v) PVA in water DP: 0.2 wt% flurbiprofen +0.3 wt% Labrafil M 1944 CS + 0.2 wt% Capryol PGMC in ethanol CP: 0.075 wt% Labrasol in water DP: 5 wt% vitamin E in MCT CP: 2.7% (w/v) Tween 80 + 2.7% (w/v) Brij 35 in water

Choi et al. (2012)

Oh et al. (2011)

Pradhan et al. (2013)

Laouini et al. (2012)

Table 2

Examples of nanoemulsions prepared using direct membrane emulsification (dd—average droplet size, DP—dispersed phase; CP—continuous phase)—cont’d Process

Product

(E) Stirred cell, ceramic 0.45-μm ceramic membrane

Squalene O/W nanoemulsions (dd ¼ 80–180 nm, ϕ ¼ 0.001)

(F) External pressure type micro kit with jacketed pressure vessel, 0.1-μm SPG membrane

O/W nanoemulsions (ϕ ¼ 0.01–0.05, dd ¼ 400–900 nm)

Formulation

Authors

Parenteral O/W nanoemulsions DP: 0.01–0.5 M AOT in Adamczak et al. (2014) squalene CP: 100 ppm PAH in 0.015 M NaCl aqueous solution DP: MCT, SBO, 0.5 wt% Joseph and Bunjes (2014) Span 80 in SBO CP: 5 wt% SDS or 1–2 wt% Tween 20

O/W/O Nanoemulsions for delivery of water-soluble drugs W1: 1.0% (w/v) calcein in 100 mM PBS at pH 7.4 O: 28.6 wt% CR-310 in soybean oil W2: 1% (w/v) HS-11, S-24D, L-7D, Gelucire 44/14, or Labrasol +1% (w/v) PVA + 5% (w/v) glucose in water (H) Internal pressure type W1/O/W2 nanoemulsion W1: 0.5% (w/v) DOX in loaded with doxorubicin water micro kit, 1.4-μm SPG (dd ¼ 440 nm) O: 5% (w/v) PGPR in membrane soybean oil W2: 3% (w/v) Tween 20 in water (G) SPG filter kit, 0.1-μm SPG membrane

W1/O/W2 nanoemulsions loaded with calcein (dd ¼ 117–788 nm)

Koga et al. (2010)

Pradhan et al. (2014)

Table 3 Examples of nanoemulsions prepared using premix membrane emulsification Process

Product

(A) Repeated premix ME, LiposoFast-basic extruder, 50-nm,100-nm, and 200-nm PC membrane

O/W nanoemulsions (dd ¼ 100–200 nm, ϕ ¼0.05–0.2)

(B) External pressure type micro kit with jacketed pressure vessel, 0.1, 0.2, 0.3, 0.5, and 1.1-μm SPG membrane (C) Syringe filters, 0.2-μm + 0.1-μm PES, MCA, CE, and nylon membrane

O/W nanoemulsions (dd ¼ 100–200 nm, ϕ ¼ 0.1)

O/W nanoemulsions (dd < 500 nm)

Formulation

Authors

DP: MCT or SBO Joseph and Bunjes (2012) CP: Aqueous solution containing 18.75 wt% SDS or 18.75 wt% Pol or 18.75 wt% SL or 18.75 wt % PG10-L Joseph and Bunjes (2014) DP: MCT CP: Aqueous solution containing 7.5 wt% SDS, 7.5 wt% Pol, or 7.5 wt% Tween 20 Gehrmann and Bunjes DP: MCT (2016a) CP: Aqueous solution containing 2.5 wt% Lipoid S100 or 2.5 wt% Lipoid S75 or 5 wt% Tween 80 or 5 wt% SL (Continued)

192 Chapter 7 Table 3

Examples of nanoemulsions prepared using premix membrane emulsification—cont’d Process

Product

Formulation

Authors

(D) Syringe filters, 0.2-μm pore-sized PC, PE, PES, PS, PVDF, nylon, and CA membrane (E) Anodisc aluminium oxide membrane filter, 0.2-μm pore size

O/W nanoemulsions (dd ¼ 0.08–11 μm, ϕ ¼ 0.2)

DP: MCT or peanut oil CP: 6.25 wt% aqueous solution of SDS, Pol, Tween 80, Tyl, or SL DP: MCT or peanut oil CP: 6.25 wt% aqueous solution of SDS, Pol, Tween 80, Tyl, or SL DP: MCT CP: 18.75 wt% aqueous solution of SDS

Gehrmann and Bunjes (2018)

(F) Instrumented smallscale extruder, 0.2-μm pore-sized PC, PE, PES, PS, and PVDF membrane

O/W nanoemulsions (dd ¼ 77–250 nm, ϕ ¼ 0.2) O/W nanoemulsions (dd ¼ 100–200 nm, ϕ ¼ 0.2)

Gehrmann and Bunjes (2018)

Gehrmann and Bunjes (2016b)

9.2 Preparation of Pharmaceutical Micro/Nano-Emulsions Using Direct ME O/W nanoemulsions can be used as delivery systems for oral, parenteral, and transdermal administration of poorly water-soluble drugs such as itraconazole, flurbiprofen, fenofibrate, and vitamin E (Table 2A–D). An O/W microemulsion composed of itraconazole (a triazole antifungal agent for fungal infections), dichloromethane (DCM), water, Transcutol HP/Span 20 1:1 (wt/wt) surfactants blend, and dextran in the weight ratio of 2/5/100/5/2.5 was prepared using an internal pressure type micro kit with a 2.5-μm SPG membrane at an agitator speed of 150 rpm, a feed pressure of 15 kPa, and a temperature of 25°C (Choi et al., 2012). Transcutol HP was added as a good solubilizing agent for itraconazole and Span 20 was used as a cosurfactant to expand the microemulsion region in the phase diagram (Fig. 11). The prepared microemulsion was spray-dried to produce solid particles composed of surfactant-coated drug nanoparticles (NPs) embedded within a hydrophilic dextran matrix. The release rate of itraconazole from the solid particles was significantly higher than that from pure itraconazole powder due to formation of a microemulsion after reconstitution which enabled higher dissolution rates, leading to the higher drug bioavailability. A pharmacokinetic study in rats showed that the total plasma concentration of itraconazole after oral administration of solid emulsion was significantly higher than that of pure itraconazole powder with the same drug dose. The z-average diameters of the liquid emulsion drops and reconstituted solid emulsion drops were 410 nm and 453 nm, respectively. The differential scanning calorimetry (DSC) results showed that itraconazole was present in the solid emulsion as a molecular dispersion or in drug-rich amorphous domains. Laouini et al. (2012) prepared O/W nanoemulsions loaded with vitamin E for pulmonary delivery of vitamin E using cross-flow SPG membrane (Table 2D). The combined use of two

Span 20 0 10

100 90

20

80

30

70

40

60

50

50

60

40

70

30

80

20

90

10

100 0 Water

10

20

30

40

50

60

70

80

0 100 DCM

90

(A) Trancutol HP: Span 20 (1:1) 0 10

100 90

20

80

30

70

40

60

50

50

60

40

70

30

80

20

90

10

100 0 Water

10

20

30

40

50

60

70

80

90

0 100 DCM

(B) Fig. 11 The pseudo-ternary phase diagram for: (A) Span 20, DCM, and water; (B) Span 20:Transcutol HP 1:1 surfactants blend, DCM, and water. The black regions are stable microemulsion regions and point 1 represents the composition of the system used to prepare a microemulsion by direct ME in SPG micro kit, Table 3A (Choi et al., 2012).

194 Chapter 7 surfactants (Tween 80 and Brij 35) resulted in the largest microemulsion region and the smallest drop size as compared with the systems composed of individual surfactants. Under optimal conditions, a nanoemulsion composed of water, medium chain triglyceride (MCT), and Tween 80/Brij 35 blend in the weight ratio of 80/17.75/2.25 had an average drop size of 78 nm and a span of 0.25. Emulsions shown in Table 2A–E are microemulsions, which can be seen by the fact that the mean drop size was 5–8 times smaller than the mean pore size and the operating pressure was below 100 kPa. Joseph and Bunjes (2014) prepared O/W nanoemulsions using external pressure type SPG micro kit with a pore size of 0.1–1.1 μm (Table 2F). The ratio of the median drop size to the membrane pore size ranged from 2.8:1 to 8.2:1 when nonpolar oils such as soybean oil or MCT oil were dispersed at 200–800 kPa into gently stirred aqueous phase containing 5 wt% SDS or 1–2 wt% Tween 20. Submicron drops were consistently produced only with a 0.1 μm pore-sized SPG membrane. However, injecting more polar Gelucire 44/14 lipid melt at 55°C and 10 kPa through a 1.1-μm SPG membrane into a heated aqueous solution containing 2 wt% Tween 20 resulted in an emulsion with a median drop size of 70 nm indicating that a microemulsion was formed. Self-emulsifying properties of Gelucire 44/14 were confirmed by preparing NPs by gently mixing the molten lipid at 55°C with 2% Tween 20 solution without using any membrane. After cooling the mixture to 5°C, the particle size was the same as after ME. It shows that emulsification of Gelucire 44/14 melt was not controlled by the membrane. W/O/W emulsions prepared by ME can be used as colloidal carriers for highly water-soluble drugs (Table 2G–H). Koga et al. (2010) prepared W1/O/W2 nanoemulsions for enhanced intestinal absorption of BCS class III drugs using SPG filter kit. BCS class III drugs are highly hydrophilic drugs with a low permeability across the lipophilic intestinal membrane, which can be encapsulated in a W1/O/W2 or S/O/W emulsion to enhance their bioavailability. W1/O/W2 nanoemulsions with calcein (model drug) entrapped in the inner water phase were prepared using a 28.6 wt% solution of CR-310 in soybean oil as the middle phase and aqueous solutions of different hydrophilic surfactants with and without PVA as the outer aqueous phase. A primary W1/O emulsion prepared using a Polytron high-speed rotor/stator homogenizer at 16,000 rpm was extruded at 0.1 mL/h through a 0.1-μm pore-sized SPG membrane into a glass vial containing 9 mL of the stirred outer phase. The mean size of W1/O drops, calcein entrapment efficiency, and intestinal absorption of calcein by rat intestine were significantly affected by the choice of the hydrophilic surfactant. In the absence of any hydrophilic surfactant, the drop size was 322 nm and not affected by the presence of PVA. The smallest drop size of 105 nm was achieved in the presence of 1 wt% HS-11 in the outer aqueous phase. On the other hand, the mean drop size was above 1 μm in the presence of HS-9 and S-28D surfactants. After intraduodenal administration of 1.0 mg/kg of calcein entrapped in the inner phase of the prepared nanoemulsion into rats, the maximum concentration of calcein in the blood serum reached 11847 ng/mL. After administration of the same dose of calcein in the form of calcein

Membrane Emulsification in Pharmaceutics and Biotechnology 195 solution, the maximum concentration of calcein in the serum was only 13.75.4 ng/mL. Pradhan et al. (2014) prepared W1/O/W2 microemulsions loaded with doxorubicin using SPG membrane with a pore size of 1.4 μm. The oil phase was 5% (w/v) PGPR in soybean oil and the external aqueous phase was 3% (w/v) Tween 20 solution (Table 2H).

9.2.1 Factors affecting the mean drop size of micro/Nano-emulsions using direct ME Temperature: Temperature affects the viscosity of the dispersed and continuous phases and also the HLB value of the surfactant and its solubility. Higher temperatures could lead to coarsening of the emulsion via Ostwald ripening and/or coalescence. Choi et al. (2012) prepared O/W microemulsions at 25°C and 35°C and found that smaller drop sizes with narrower size distributions were obtained at 25°C and the same trend was observed by Laouini et al. (2012). Hydrodynamic conditions: Choi et al. (2012) investigated the effect of stirring rate over a range of 150–700 rpm on the average size of microemulsion drops formed in internal pressure type SPG micro kit. They found that the z-average and polydispersity index (PDI) increased as the agitator speed increased to 700 rpm, probably because of the increased probability of collisions between droplets, which caused their coalescence and increase in mean droplet size. However, Laouini et al. (2012) obtained microemulsions with smaller drops and narrower particle size distributions at higher cross-flow velocities. Pressure difference: The pressure difference used in direct ME depends on the type of emulsion formed. In the case of microemulsions, the applied pressure difference is often <100 kPa and the membrane pore size is typically above 1 μm. As an example, Oh et al. (2011) prepared microemulsions with the z-average between 50 and 100 nm at the pressure difference of 15–80 kPa using 2.5 μm pore-sized SPG membrane. They reported a decrease in droplet size with an increase in pressure difference. However, using the same membrane, Choi et al. (2012) observed an increase in the z-average value from 410 to 550 nm when the pressure difference increased from 15 kPa to 40 kPa. The driving pressure below15 kPa was not enough to force the dispersed phase through the membrane. In most cases, an increase in driving pressure led to higher PDI values (Choi et al., 2012; Laouini et al., 2012). If a nanoemulsion is formed, the applied pressure difference is typically much above 100 kPa, because the required membrane pore size is well below 1 μm. For example, to make O/W nanoemulsions comprised of soybean oil and MCT oil using an SPG membrane with a pore size of 0.1 μm, a pressure difference of 800 kPa was needed ( Joseph and Bunjes, 2014). Surfactant concentration: Laouini et al. (2012) investigated the effect of surfactant concentration on the formation of O/W microemulsion in cross-flow emulsification with SPG membrane. The presence of surfactant in the aqueous phase lowers the interfacial tension between the oil and aqueous phase and facilitates droplets disruption given that the interfacial tension is a drop holding force during the process. In addition, surfactants

196 Chapter 7 stabilize the formed droplets against coalescence and aggregation. For the system composed of MCT oil, water, and Tween 80/Brij 35 1:1 w/w mixture at the mass ratio of 14/80/5/6, two peaks were observed in particle size distribution curve; the main peak at 125 nm corresponded to oil droplets and another peak at 11 nm was due to micelles formation. When the total surfactant content was reduced from 6 to 2.25 wt%, the peak corresponding to micelles decreased progressively and disappeared at 2.25 wt%, which was accompanied by a decrease in the size distribution span from 0.52 to 0.25. Further decrease in the total surfactant content from 2.25 to 2 wt% led to broadening a size distribution curve and the main peak occurred at 290 nm. Therefore, there is an optimum surfactant concentration for the preparation of microemulsions with monomodal size distribution and small average droplet size. Drug loading: The effect of loading vitamin E in MCT oil in the range of 5–10 wt% on the particle size distribution in cross-flow ME was investigated by Laouini et al. (2012). Increasing vitamin E loading led to an increase in the average droplet size and broadening of the particle size distribution curve, due to the higher dispersed phase viscosity and possible effects of vitamin E on the interfacial tension. Oh et al. (2011) found that the z-average of O/W microemulsions increased from 98 nm to 360 nm when DCM drops were loaded with 20 wt% of flurbiprofen (Oh et al., 2011). Joseph and Bunjes (2014) prepared trimyristin (TM) nanoemulsions loaded with ubidecarenone using external pressure type micro kit with jacketed pressure vessel. The emulsification temperature was 10°C higher than the melting point of TM. The particle size was independent of the drug loading in the range between 0 and 5 wt%. Membrane pore size: The ratio between the drop size and the pore size depends on whether a microemulsion or nanoemulsion is formed. The mean drop size of a microemulsion formed by direct ME is significantly smaller than the mean pore size. Oh et al. (2011) prepared an O/W microemulsion with a z-average of 98.5 nm using 2.5-μm SPG membrane in the presence of 5% (w/v) surfactant blend (Tween 20/Tween 80) and 1% (w/v) PVA in the aqueous phase. Therefore, the mean droplet size was 25 times smaller than the mean pore size, which means that the oil phase was self-emulsified and the drop size was not controlled by the pore size. If a nanoemulsion was formed with the same membrane using smaller amounts of surfactant, the mean drop size would be about 8 μm. Laouini et al. (2012) prepared O/W microemulsions using SPG membrane with three different pore sizes. The average particle size was very similar using 0.4-μm and 0.9-μm pore sized membranes, but the injection rate was very low with 0.4-μm membrane. When 10.2-μm membrane was used, the injection rate was very high, but the resultant particle size distribution was very broad with a large average particle size. It clearly shows that the pore size of SPG membrane for preparation of microemulsions should be above 0.4 μm, but well below 10 μm. In most cases, microemulsions were made using SPG membrane with a pore size between 0.45 μm and 2.5 μm (Table 2A–D). On the other hand, O/W nanoemulsions can be formed by direct ME if the pore size of SPG membrane is 0.1 or 0.2 μm ( Joseph and Bunjes, 2014).

Membrane Emulsification in Pharmaceutics and Biotechnology 197

9.3 Preparation of Pharmaceutical Nanoemulsions Using Premix ME Joseph and Bunjes (2012) prepared pharmaceutical O/W nanoemulsions by repeated premix ME through PC membranes with a pore size of 50, 100, or 200 nm using LiposoFast-basic extruder or EmulsiFlex-C5 homogenizer equipped with a high-pressure filter/extruder. The dispersed phase was MCT oil or soybean oil and the continuous phase was an aqueous solution of SDS, poloxamer 188 (Pol), sucrose laurate (SL), or polyglyceryl-10-laurate (PG10-L). The oil was predispersed in the aqueous surfactant solution with an Ultra-Turrax mixer and the resulting premix containing 20 wt% oil, 15 wt% surfactant, and 65% water was extruded through the membrane 11 or 21 times. For extrusion through a 50-nm membrane, the premixes were preextruded 11 times using a 200-nm membrane, and then 11 or 21 times using a 50-nm membrane. The nanodispersions had a mean particle size of 100–200 nm after 21 extrusion cycles, depending on the pore size, surfactant type, and the extrusion device used. The smallest particle size was obtained with Pol and the largest particle size with PG10-L; the most uniform particle size distribution was observed with SDS and the broadest with PG10-L. The minimum amount of hydrophilic surfactant depends on the pore size. For the pore size of 200 nm or 100 nm and nanoemulsions comprised of 20 wt% MCT oil, the concentration of SDS could be reduced from 15% to 4% without significant effect on the mean drop size. For smaller pore sizes, SDS concentration of at least 8% was needed to achieve monomodal particle size distributions after 21 cycles. Gehrmann and Bunjes (2016a) prepared O/W nanoemulsions stabilized with phospholipids (Lipoid S75 and S100), Tween 80 and SL by extruding premix through disposable syringe filters with polyethersulfone (PES), cellulose acetate (CA), mixed cellulose ester (MCE), or nylon membranes (Table 3C). The premix was prepared by injecting the liquids back and forth between the two syringes starting from two separate phases in two different syringes (Fig. 7A (i)). The premix with a median particle size of 10–24 μm was pre-extruded through a 1-μm glass fiber filter to reduce the particle size to 1–6 μm, followed by 21 extrusion cycles through a 200-nm membrane and one subsequent cycle through a 100-nm membrane. In most cases, the last cycle was only possible after a 1:5 dilution of the nanoemulsion with the continuous aqueous phase. The final median drop size achieved with PES membrane was 400 nm or below irrespective of the surfactant. In all cases, the most substantial decrease in drop size was achieved after first extrusion. When oil drops were stabilized with Lipoid S100 and SL, the median drop size was below 1 μm already after first cycle, whereas the drop sizes for nanoemulsions stabilized with Lipoid S75 and Tween 80 were still in the micrometer range. The most uniform size distributions (span  0.5) were achieved with Tween 80 and SL and the largest median particle size after 21 cycles was obtained using CA and nylon membranes. Gehrmann and Bunjes (2018) extruded premixed MCT emulsions comprised of five different pharmaceutically relevant water-soluble surfactants through seven different hydrophilic polymeric membranes with a pore size of 200 nm. The particle size distribution of emulsions

198 Chapter 7 obtained after 21 cycles differed significantly with the median drop size ranging between 150 nm and 11 μm. A key factor in determining the median drop size was the contact angle between a 6.25 wt% emulsifier solution and the membrane surface, critically dependent on the combination of surfactant and membrane material used. The microstructure and thickness of the membrane and the type of dispersed phase were less important as the similar results were obtained with peanut oil and MCT oil with widely different viscosities (60 and 30 mPa s, respectively). Based on their ability to create O/W nanoemulsions containing 20 wt% of MCT oil with a d50 value smaller than 500 nm after 21 extrusion cycles at the flow rate of 25 mL/s, Gehrmann and Bunjes (2018) divided hydrophilic polymeric membranes into three groups. The first group (Nylon and CA) are the membranes that produced emulsions with d50 < 500 nm and low span values with all pharmaceutically relevant surfactants (SDS, Pol, Tween 80, Tyl, and SL) when present in the aqueous phase at 6.25 wt%. The second group (PC, PS, and PVDF) yielded d50 < 500 nm only with SDS. The third group (PES and PE) showed the performance between the two groups. Depending on the membrane and surfactant, the contact angle between the 6.25% surfactant solution and the membrane surface varied between 17° and 77° (Fig. 12). The contact angle of <49° led to nanoemulsions with d50 < 500 nm in all cases, whereas emulsions with d50 > 3 μm were obtained for all contact angles higher than 55°. To prove the notion that the final drop size of an emulsion after repeated extrusion through a polymeric membrane critically depends on membrane hydrophilicity, PC and PE membranes were treated with nitrogen and oxygen plasma at 25–75 W for 5–10 min. After this hydrophilic treatment, smaller d50 values were achieved with both membranes. Furthermore, after 21 extrusion cycles through a highly hydrophilic aluminium oxide

Fig. 12 Median droplet sizes of emulsions obtained by extrusion through different syringe filters with a pore size of 200 nm (21 cycles, transmembrane flow rate: 0.25 mL/s, dispersed phase: MCT oil; continuous phase: 6.25 wt% surfactant solution) as a function of the contact angle between the surfactant solution and membrane surface. Each point is a mean of three repeated experiments (Gehrmann and Bunjes, 2018).

Membrane Emulsification in Pharmaceutics and Biotechnology 199 Anopore™ membrane with a pore size of 200 nm, d50-values below 250 nm were achieved with all surfactants, with the minimum d50-value of 77 4 nm reached for SDS (Gehrmann and Bunjes, 2018). Even smaller particle sizes were achieved by using an alumina membrane with a pore size of 100 nm (Gehrmann and Bunjes, 2017). 9.3.1 Factors affecting the mean drop size of micro/Nano-emulsions using premix ME Pore size distribution and contact angle: For repeated extrusion, a narrow pore size distribution of the membrane is not critically important for the drop size uniformity, because the probability that all droplets will be disrupted by the smallest pores increases with every extrusion cycle (Gehrmann and Bunjes, 2018). This explains why polymeric membranes are more often used in premix ME, while SPG membranes are predominantly used in direct ME. A low contact angle between the membrane and the surfactant solution is crucial for the generation of oil droplets in the nano-range. An incomplete wetting of the membrane with the continuous phase resulted in polydisperse emulsions with larger droplets irrespective of the formulation (Gehrmann and Bunjes, 2018). Membrane pore size: Smaller droplets can be achieved using membranes with smaller pore sizes, but the effect of pore size on the drop size is less pronounced in premix ME than in direct ME ( Joseph and Bunjes, 2012, 2014). The d50 value for SDS-stabilized MCT oil drops in the prepared nanoemulsions was smaller or equal to the pore size after 21 cycles through a tracketched PC membrane with a pore size of 200 nm (Gehrmann and Bunjes, 2016b). The ratio of d50 to the membrane pore size for a 200-nm track-etched PC membrane ranged from 1:1 to 0.6:1 at the transmembrane flux of 0.27 and 3.8 m3 m
2 h
1, respectively. After one pass through SPG membrane with a pore size of 100–300 nm at 900 kPa, the ratio of d50 to the membrane pore size was in the range from 0.4:1 to 1.1:1 for nanoemulsions comprised of 2.5–10 wt% MCT oil stabilized with SDS. The prepared nanoemulsions were monomodal with d50 < 200 nm. Membrane structure: The use of track-etched PC or PE membranes with straight-through pores led to smaller d50 values than the use of sponge-like PES, PS, and nylon membranes (Gehrmann and Bunjes, 2016b). On the other hand, track-etched PC membranes were found to be less efficient in reducing the drop size than SPG membranes due to their non-tortuous, rectilinear pores and smaller thickness (<20 nm as compared to 700–900 nm for SPG membranes). For example, one extrusion cycle through SPG membrane with a pore size of 100–300 nm led to smaller drops and narrower size distributions than 21 extrusion cycles through tracked-etched PC membranes with the same pore size ( Joseph and Bunjes, 2014). Zhou et al. (2009) also observed that a higher thickness of a membrane with branched pores increased the level of droplet disruption and led to smaller droplet sizes. Number of extrusion cycles and premix quality: The drop size and transmembrane pressure decrease on increasing number of extrusion cycles at constant transmembrane flux, because

200 Chapter 7 smaller droplets cause less friction with the pore walls. After about 20 cycles at constant flux through the membrane, d50 and transmembrane pressure were stabilized and reached constant values. Track-etched membranes with well-defined pore structure required a smaller number of cycles to reach a steady-state droplet size than polymeric membranes with highly branched pores (Gehrmann and Bunjes, 2018). After only two extrusions through the track-etched PE membrane with a pore size of 200 nm, a monomodal particle size distribution of SDS-stabilized oil droplets with a d50 value of 320 nm was obtained (Gehrmann and Bunjes, 2016b). The greatest reduction in drop size and transmembrane pressure were observed during the first three cycles. The quality of the premix had no influence on the quality of the nanoemulsion after 5 cycles (Gehrmann and Bunjes, 2017). Transmembrane flux and extrusion pressure: At constant transmembrane flux, the extrusion pressure has the highest value during first extrusion cycle and then declines to reach a plateau value after about 20 cycles (Gehrmann and Bunjes, 2016b). This trend is caused by the reduction in droplet size leading to reduced resistance to flow until a steady-state is established. The extrusion pressure increased with increasing flow rate through the membrane due to higher hydrodynamic resistance (Gehrmann and Bunjes, 2016b). At constant extrusion pressure, a higher transmembrane flux was observed at higher extrusion pressures and for the higher number of cycles (Vladisavljevi
c et al., 2004b). In most cases, smaller droplets and narrower particle size distributions were achieved at higher extrusion pressures (fluxes). The extrusion pressure of 3 bar was sufficient to produce SDS-stabilized oil droplets with a median size of just above 200 nm after 21 cycles through a track-etched PC membrane with a pore size of 200 nm (Gehrmann and Bunjes, 2016b).

10 Production of NPs Loaded with Pharmaceutical Active Agents Using ME Nanodroplets prepared by ME can be solidified into NPs. Depending on the formulation, solidification reaction can include ionic cross-linking, solvent evaporation, or cooling crystallization.

10.1 Hydrogel NPs Hydrogel NPs are nanospheres composed of cross-linked hydrophilic polymers, which can entrap water in their interstitial spaces (Hoare and Kohane, 2008). Hydrogel NPs are one of the most promising nanoparticulate drug delivery systems owing to their ability to release their contents in response to external triggers (Hamidi et al., 2008). Hydrogel NPs can be produced by emulsifying an aqueous solution of hydrophilic polymer (chitosan, sodium alginate, etc.) in an organic solution of oil-soluble surfactant via hydrophobic membrane and subsequent ionic or covalent cross-linking of the polymer within the formed nanodroplets.

Membrane Emulsification in Pharmaceutics and Biotechnology 201 Table 4 Hydrogel nanoparticles (NPs) produced from nanodroplets prepared by membrane emulsification (DP—dispersed phase, CP—continuous phase, XL—cross-linking agent) Process

Product

Emulsion Formulation

Authors

External gelation (A) Repeated premix ME, 1.4 μm SPG membrane, 5 cycles

Chitosan NPs (dNP ¼ 370 nm)

(B) Direct ME, external pressure type micro kit, 0.5 μm SPG membrane

W/O nanoemulsion (dd ¼ 780 nm, ϕ ¼0.09) Chitosan NPs (dNP ¼ 390 nm)

Lv et al. (2009); Ma et al. DP: 0.2 wt% chitosan (2010) + 1 wt% acetic acid in water CP: 4 wt% PO-500 in liquid paraffin/petroleum ether (1:2, v/v) XL: Toluene saturated by glutaraldehyde Wang et al. (2005) DP: 1.5 wt% chitosan +1 wt% acetic acid +0.9 wt% NaCl in water CP: 4 wt% PO-500 in liquid paraffin/petroleum ether (7:5, v/v) XL: toluene saturated with glutaraldehyde

Gelation triggered by droplet merging (C) Premix ME, 1.4 and 2.8 μm SPG membrane

Alginate NPs (dNP ¼ 370 and 700 nm, respectively)

DP1: 1 wt% alginate in water DP2: 5 mol L
1 CaCl2 in water CP: 4 wt% PO-500 in liquid paraffin/petroleum ether (1/2, v/v)

Nan et al. (2014)

Chitosan NPs were produced by repeated premix ME using a 1.4-μm SPG membrane (Table 4A and Fig. 13A) or direct ME using a 0.5-μm SPG membrane (Table 4B and Fig. 13B). In both cases, the continuous phase was a mixture of liquid paraffin and petroleum ether containing 4 wt% of hexaglycerin penta ester (PO-500). Chemical (covalent) cross-linking of chitosan or sodium alginate can be achieved by dripping toluene saturated with glytaraldehyde in the prepared W/O nanoemulsion. In physical (ionic) cross-linking, charged functional groups along the polymer chains interact with oppositely charged divalent or polyvalent ions to form ionic bridges that cross-link the polymer. Alginate NPs with an average size of 370 nm were prepared by mixing together two W/O nanoemulsions: nanoemulsion 1 containing droplets of 1 wt% alginate solution and mini-emulsion 2 containing droplets of 5 M CaCl2 (Nan et al., 2014). The cross-linking was achieved by merging colliding drops of different emulsions upon mechanical agitation, which triggered the cross-linking reaction between negatively charged alginate chains and Ca2+

202 Chapter 7 Premix ME and external gelation

Aqueous biopolymer solution

Direct ME and external gelation

Oil + lipophilic surfactant

Aqueous biopolymer solution

Oil + lipophilic surfactant

Stirring

Direct ME

W/O preemulsion

W/O emulsion

``

water oil

Biopolymer

Crosslinker

Crosslinker

Crosslinking

Premix ME

Crosslinker

Hydrogel NPs W/O emulsion Crosslinking

(B) Crosslinker

Hydrogel NPs

(A) Premix ME and gelation by coalescing biopolymer and crosslinker drops

Aqueous alginate solution

1

Paraffin/petroleum ether + PO-500

Naalginate

Stirring W/O preemulsion

Aqueous CaCl2 solution

–

Oil

– –

– – – – – – – – –

Paraffin/petroleum ether + PO-500

– –

–

Ultrasonication

Premix ME

– –

Ca2+ Ca2+Ca2+ Ca2+ – Ca2+ Ca2+

W/O emulsion 2

W/O emulsion 1

(C)

–

– –

2

Ca2+ Ca2+ Ca2+ Ca2+ 2+ Ca Ca2+

Emulsion mixing

Ca2+ Ca2+ Ca2+ 2+ Ca Ca2+

Alginate NPs

Ca2+

Fig. 13 Preparation of hydrogel NPs from W/O nanoemulsions: (A) Premix ME and subsequent external cross-linking (Lv et al., 2009); (B) Direct ME and external cross-linking (Wang et al., 2005); (C) Premix ME and subsequent cross-linking triggered by coalescence of biopolymer and cross-linker water-in-oil emulsion droplets (Nan et al., 2014).

ions within fused droplets (Fig. 13C). Emulsion 1 was prepared by premix ME at the transmembrane pressure of 10 bar using 1.4-μm SPG membrane and mini-emulsion 2 was prepared by ultrasonication. Hybrid chitosan/alginate nanospheres with alginate core and chitosan shell were prepared by dispersing the produced alginate NPs into 0.7 wt% chitosan solution.

Membrane Emulsification in Pharmaceutics and Biotechnology 203

10.2 Solid Lipid NPs Solid lipid NPs are comprised of saturated fatty acids, triesters of glycerol and saturated fatty acids, and mono-,di-, and triesters of glycerol, and polyethylene glycol. The melting point, Tm, of these lipids is typically between 40°C and 80°C and ME must be performed at the temperature which is at least 5°C above Tm to prevent pore clogging by fat particles. The resulting nanodroplets crystallize into solid NPs by rapid cooling to 5°C in an ice bath. D’oria et al. (2009) produced Compritol 888 and Gelucire 44/14 NPs with a mean size between 50 nm and 750 nm by injecting the lipid melt through an SPG membrane with a pore size of 0.2–1 μm at 65–80°C and 0.84 m3 m
2 h
1 into cross-flowing aqueous solution of a hydrophilic surfactant (Table 5A). Joseph and Bunjes (2012) prepared TM NPs by extruding TM melt at 65° C through a PC track-etched membrane with a pore size of 200 nm using EmulsiFlex-C5 homogenizer equipped with a high-pressure filter/extruder (Table 5B). The continuous phase was 18.75 wt% aqueous solution of SDS, Pol, SL, or PG10-L. The mean particle size ranged between 100 and 200 nm after 11–21 extrusion cycles at the driving pressure of 25–180 bar. Joseph and Bunjes (2014) prepared glyceryl behenate (CATO) NPs by direct ME at 60 kPa using a 0.2-μm SPG membrane. CATO NPs stabilized with 5 wt% SDS were much smaller (d50 ¼ 80 nm) than those stabilized with 5 wt% Pol (d50 ¼ 950 nm). On the other hand, SL-stabilized TM NPs were smaller (d50 ¼ 72 nm) than SDS-stabilized TM NPs (d50 ¼ 360 nm) when prepared with a 0.1 μm SPG membrane at 800 kPa.

Table 5 Solid lipid NPs fabricated by solidification of melted lipid nanodroplets prepared by membrane emulsification Process

Product

(A) Direct ME, cross-flow system, 0.2, 0.4, and 1 μm SPG membrane

Gelucire 44/14 NPs (Tm ¼ 44°C, dNP ¼ 50–130 nm) and Compritol 888 NPs (Tm ¼ 69–74°C, dNP ¼ 560–760 nm) TM NPs (Tm ¼ 55–58°C, dNP ¼ 100–200 nm)

Emulsion Formulation

Authors

DP: Gelucire 44/14 melt D’oria et al. (2009) at 65°C or Compritol 888 melt at 80°C CP: Aqueous 0.125 wt% Tween 20 or 1.26 wt% Pluronic F68 solution DP: TM melt at 65°C Joseph and Bunjes (2012) (B) Repeated premix ME, CP: Aqueous solution EmulsiFlex-C5 containing 18.75 wt% homogenizer with filter/ SDS or 18.75 wt% Pol or extruder unit, 0.2-μm PC 18.75 wt% SL or 18.75 wt membrane % PG10-L TM NPs, CATO NPs, (C) Direct ME, External DP: 0–5 wt% Q10 in TM Joseph and Bunjes (2014) Gelucire 44/14 NPs, and melt at 66°C, CATO melt pressure type micro kit Q10-loaded TM NPs at 80°C or Gelucire 44/14 with jacketed pressure melt at 54°C vessel, 0.1, 0.2 and (dNP ¼ 70–400 nm) CP: 5 wt% SDS or 15 wt% 0.3 μm SPG membrane SL in water

204 Chapter 7

10.3 Synthetic Biodegradable Polymer NPs Synthetic biodegradable polymer NPs loaded with a lipophilic active ingredient (LAI) were prepared by dissolving a mixture of LAI and polymer (PLGA or PLA) in a volatile organic solvent, such as DCM, ethyl acetate, and isopropyl acetate. ME was used to disperse the organic phase in an aqueous surfactant solution to prepare an O/W emulsion. Finally, the organic solvent was evaporated from the droplets to form solid polymer matrix with embedded LAI. Doan et al. (2011) prepared PLGA NPs with a diameter of 640 nm loaded with antibiotic rifampicin by extruding the premix three times through the 5.9-μm SPG membrane at 100 kPa (Fig. 14A). Synthetic biodegradable polymer NPs can also be used for encapsulation of hydrophilic active ingredients (HAIs). HAI can be entrapped within inner water droplets or adsorbed onto the outer surface of the particle. Zhang et al. (2014a) prepared PLA NPs with a diameter of 820 nm that have been tested as immunologic adjuvants. The process involved premix ME using five extrusion cycles through a 2.8-μm SPG membrane, solvent evaporation, and incubation of freeze-dried particles in an antigen solution. The adsorption of antigen onto the particle surface was confirmed by mice immunization and Confocal Laser Scanning Microscopy (CLSM) using particles with fluorescently labeled antigen. Synthetic biodegradable polymer NPs loaded with HAIs are usually prepared by double emulsion-solvent evaporation method (Zhang et al., 2014b). The first step is ultrasonication or rotor-stator homogenization leading to W1/O emulsion with HAI (i.e., a protein) dissolved in the inner water phase (W1). Ultrasonication produced smaller droplets, more uniformly distributed in a polymer matrix after solvent evaporation (Qi et al., 2014). The second step is the formation of W1/O/W2 emulsion using direct or premix ME, followed by solvent evaporation. Synthetic biodegradable polymer NPs loaded with both HAI and LAI can be prepared using ME (Ma et al., 2014b). In that case, HAI is trapped in the inner aqueous phase (W1) and LAI is dissolved in the oil phase (Fig. 14B). In Fig. 14B, Lipid A was used as a LAI, while HAI was a model antigen (ovalbumin). Due to its amphiphilic character, Lipid A was embedded within the interfacial phospholipid layer with its diglucosamine part orientated towards the water environment and acyl chains pointing to the interior of the particle. When used as lipophilic surfactants in double emulsion-solvent evaporation method, phospholipids were found to form efficient protective barriers at W1/O and O/W2 interfaces against the release of encapsulated proteins from the polymer matrix (Ma et al., 2014a).

10.4 Inorganic NPs Kong et al. (2010, 2012, 2013) prepared porous silica nanocapsules loaded with magnetite NPs and drug camptothecin using mini-emulsion polymerization, ME, and sol-gel processing. Nanodroplets comprising Fe3O4 NPs dispersed in octane prepared by sonication were mixed

Membrane Emulsification in Pharmaceutics and Biotechnology 205

Fig. 14 Preparation of synthetic biodegradable polymeric nanoparticles by membrane emulsification/solvent evaporation: (A) encapsulation of hydrophilic active ingredient (HAI) (Doan et al., 2011); (B) combined encapsulation of lipophilic active ingredient (LAI) and HAI (Ma et al., 2014a, 2014b, 2014c). LAI is lipid A and HAI is ovalbumin.

206 Chapter 7 Organic solvent + magnetite

Aqueous surfactant solution

Sonication Magnetite nanodroplets

Monomer + solvent

Aqueous surfactant solution

Initiator

Direct ME

Mini-emulsion polymerisation

Monomer droplets

Magnetic polymer nanoparticles Silica coating Silica-coated magnetic polymer nanoparticles Polymer removal Magnetic silica nanoshells Drug loading Magnetic silica nanoshells loaded with drug

Drug

Fe 3O4

Polymer

Silica

Fig. 15 Porous silica nanoshells loaded with magnetite nanoparticles and hydrophobic drug prepared by membrane emulsification, miniemulsion polymerization, and sol-gel processing (Kong et al., 2012).

with 4-μm styrene droplets prepared by direct ME using 1.2-μm SPG membrane (Fig. 15). After mixing, styrene spontaneously diffused into octane nanodroplets until an equilibrium was established. The polymerization of styrene was initiated by potassium peroxydisulfate (KPS), a water-soluble initiator added to the aqueous phase, and involved three steps: (i) Thermal 2
% decomposition of KPS: S2O2
8 ! 2SO4 ; (ii) Formation of oligoradicals in the aqueous phase 2
% % by reaction between the monomer (M) units and KPS free radicals: nM + SO2
4 ! MnSO4 ; (iii) Transfer of oligoradicals into monomer-swollen nanodroplets, once their chain becomes sufficiently hydrophobic. The probability for oligoradicals to diffuse into styrene droplets is very low, because their interfacial area is small compared to that of the nanodroplets. The role of ME is to create micron-sized styrene droplets with negligible amount of nanodroplets, to minimize entry of reactive oligoradicals into pure styrene droplets and formation of magnetitefree polymer particles. Here, styrene droplets act only as monomer depots and

Membrane Emulsification in Pharmaceutics and Biotechnology 207 polymerization occurs within monomer-swollen nanodroplets. It clearly differs from emulsion polymerization, where polymerization occurs within monomer-swollen micelles (Schork et al., 2005). In mini-emulsion polymerization, droplets are smaller than 500 nm and not only compete effectively for radicals with micelles, but also cause the surfactant molecules from micelles to dissociate and occupy their large surface area. Magnetic NPs were coated with silica by ammonia-catalyzed hydrolysis of TEOS, according to the St€ ober method. Polystyrene cores of the coated NPs were removed by dissolving or burning the polymer. Hollow silica nanocapsules were then loaded with camptothecin, a hydrophobic anticancer drug, which was trapped and retained inside the capsules due to surface repulsion from the hydrophilic shell (Fig. 15). The drug release from the capsules was triggered by applying magnetic field which led to heating and increased drug diffusivity (Kong et al., 2013).

11 Production of Multiple Emulsions Loaded with Pharmaceutical Active Agents Using ME Multiple W1/O/W2 emulsions can be prepared by ME using a two-step process: (a) Preparation of W1/O emulsion with submicron-sized droplets from two separate immiscible liquids using a Microfluidizer or sonication (Mine et al., 1996); (b) Dispersion of W1/O emulsion in the outer aqueous phase (W2) using the membrane with a pore size at least two times larger than the size of inner drops. The concentration of inner drops in the oil phase is typically 30–50 vol% (Mine et al., 1996; Vladisavljevi
c et al., 2006a). The inner drops contain a hydrophilic active ingredient (HAI), which could be small-molecule drugs or macromolecular biological actives, such as enzymes and polyphenols. The lipophilic surfactants used in these emulsions are PGPR and TGPR in the concentrations between 5 wt% and 10 wt%. A release of entrapped HAI from inner drops into the outer aqueous medium can be minimized by solidifying or coating the middle phase, which can be done by spray drying (Berendsen et al., 2015), evaporating volatile organic solvent from the middle phase (Ma et al., 2014a), or melt cooling (Kukizaki and Goto, 2007b). Inner drops can be also dehydrated and transformed into surfactant-coated nanocrystals of HAI (Kukizaki, 2009c) or inner drops can be gelified by heat treatment (Surh et al., 2007). If inner drops of a W/O/W emulsion are solidified, the resulting structure is known as the solid-in-oil-in-water emulsion (S/O/W). Higashi and Setoguchi (2000) and Higashi et al. (1995) prepared W1/O/W2 emulsions for hepatic arterial infusion pump chemotherapy using internal pressure type SPG micro kit (Fig. 16). A W1/O emulsion comprised of an aqueous solution of epirubicin (anticancer drug) and glucose (osmotic modifier) dispersed in a mixture of iodized poppy seed oil (Lipiodol) and polyoxyethylene (40) hydrogenated castor oil was prepared by sonication. This W1/O emulsion

208 Chapter 7

Fig. 16 (A) W1/O/W2 emulsion prepared by conventional agitation (Kanematsu et al., 1984); (B) W1/O/W2 emulsion loaded with eprubicin for transcatheter arterial injection chemotherapy of hepatocellular carcinoma prepared by internal pressure type SPG micro kit. W1: water +5.8 wt% glucose +1.2 wt% epirubicin hydrochloride, oil: IPSO +9 wt% PGPR, W2: physiological saline +1% polyoxyethylene 60 stearate (Higashi et al., 1995); (C) Particle size distribution of W1/O droplets immediately after preparation and after 40 days (Higashi et al., 1995).

Membrane Emulsification in Pharmaceutics and Biotechnology 209 was further emulsified using a hydrophilic SPG membrane with a mean pore size of 20 μm to prepare a W1/O/W2 emulsion. Clinical trials showed that the prepared multiple emulsion was effective in treating liver cancer, when administered into the hepatic artery. ME enabled to prepare multiple emulsion drops with more uniform size distribution and higher drug encapsulation efficiency compared to other traditional methods (Fig. 16C).

12 Production of Microparticles Loaded with Pharmaceutical Active Agents Using ME 12.1 Solid Lipid Microparticles Common microcarriers for lipophilic active ingredients (LAIs) are oil droplets, biodegradable synthetic polymer microparticles, and solid lipid microparticles. The ME-O/W melt dispersion method was used to encapsulate vitamin E (Laouini et al., 2014) and bisphenols, such as hydroxytyrosol (Bazzarelli et al., 2017). In this method, LAI was dissolved in a hot melted lipid; the solution was mixed with a hot aqueous surfactant solution and the premix was extruded through the membrane (Fig. 17). Alternatively, the lipid solution can be directly extruded through the membrane into the aqueous surfactant solution (D’oria et al., 2009). The prepared O/W emulsion was cooled to room temperature to solidify the melted lipid, washed to remove the hydrophilic surfactant, and collected by filtration or centrifugation. The ME-W1/O/W2 melt dispersion method (Fig. 18) can be used to encapsulate a hydrophilic active ingredient (HAI), such as vitamin B12. Kukizaki and Goto (2007b) prepared a W1/O/W2 Aqueous phase

LAI + melted lipid phase Stirring

Melted lipid

LAI

O/W premix T > Tm Premix ME O/W emulsion Cooling

Cooling T < Tm

Filtration

Solid lipid

S/S carrier for LAI

Fig. 17 Preparation of solid lipid microparticles for entrapment of a lypophilic active ingredient (LAI) using premix membrane emulsification (PME) above the melting point of the lipid, Tm, and subsequent lipid solidification at T < Tm. ① Oil droplet composed of LAI dispersed or dissolved in a melted lipid; ② Solid lipid particle composed of LAI dispersed within the solidifed lipid matrix (Vladisavljevi
c, 2015).

210 Chapter 7

Fig. 18 (A) Preparation of solid lipid microparticles for entrapment of a hydrophilic active ingredient (LAI) by two-stage direct membrane emulsification (DME) at T > Tm. ① W1/O emulsion composed of an aqueous solution of HAI dispersed in a melted lipid; ② W1/O/W2 emulsion droplet; ③ W1/S carrier comprised of aqueous droplets of HAI solution embedded within a solid lipid matrix (Vladisavljevi
c, 2015); (B) The SEM micrograph of W/S carrier with a diameter of 17.7 μm composed of aqueous droplets of 1 wt% B12 solution embedded in a tripalmitin solid matrix (Kukizaki and Goto, 2007b).

emulsion by the two-step direct ME process using SPG membranes with a pore size of 0.3 μm and 1–9.9 μm, respectively. The mean droplet size was 3.3–3.4 larger than the mean pore size in both emulsification steps. The prepared W1/O/W2 emulsion was cooled down below Tm to solidifiy the droplets and form a W1/S/W2 dispersion. Finally, W1/S microparticles were washed, filtered, and dried at 293 K. Fig. 18B shows a tripalmitin particle with a diameter of 17.7 μm with embedded droplets containing 1 wt% aqueous B12 solution. The particle has a pleat-like surface, suggesting that tripalmitin was in a crystalline form. The encapsulation efficiency of B12 was 94.6% and the pore size of the SPG membrane used in the second emulsification step (DME 2) was 5.2 μm. Using the same method, Shimizu et al. (2002) prepared tripalmitin particles containing embedded aqueous droplets of anticancer drug irinotecan (CPT-11) with the encapsulation efficiency of >90%. W1/S microparticles contain 40 wt% of water embedded within solid lipid matrix. To improve microbiological quality of the particles, inner water can be evaporated from the W1/O emulsion before second emulsification step. It can be done by vacuum evaporation at 60°C (Kukizaki, 2009c) or freeze drying (Toorisaka et al., 2003). The resulting nanodispersion is composed of surfactant-coated nanocrystals of HAI dispersed in the melted lipid with a water content below 0.7 wt% (Fig. 19). PGPR added to the lipid phase inhibited coalescence of inner droplets and agglomeration of nanocrystals during drying, so the final size of PGPR-coated nanocrystals of B12 in a glycerol trimyristate melt was 130 nm. This S/O

Membrane Emulsification in Pharmaceutics and Biotechnology 211

Fig. 19 (A) Preparation of solid-in-solid (S/S) lipid carrier by premix membrane emulsification (PME) at T > Tm using dehydrated W1/O emulsion containing surfactant-coated nanocrystals of HAI suspended in the lipid melt. ① W1/O emulsion composed of an aqueous solution of HAI dispersed in a melted lipid; ② S/O nanodispersion; ③ W1/O/W2 emulsion droplet; ④ S/S carrier containing surfactant-coated nanocrystals of HAI embedded in a solid lipid matrix (Vladisavljevi
c, 2015); (B) The SEM micrograph of a S/S carrier with a diameter of 15.5 μm comprised of PGPR-coated B12 nanocrystals embedded in a glycerol trimyristate matrix (Kukizaki, 2009c).

nanodispersion was then gently mixed with a hot 1% Tween 40 aqueous solution to prepare a S/O/W2 premix, which was extruded through SPG membrane. The mean particle size was 1–1.5 larger than the mean pore size (Kukizaki, 2009c). The S/O/W emulsion was cooled down to room temperature to obtain solid lipid microparticles with embedded nanocrystals of vitamin B12. Fig. 19B is an SEM image of the S/S carrier with a diameter of 15.5 μm prepared using a 14.8-μm SPG membrane at the transmembrane flux of 11.8 m3 m
2 h
1. The encapsulation efficiency of B12 was 93.5%.

12.2 Thermoresponsive Core-Shell Microparticles Chu et al. (2002) prepared thermoresponsive core-shell polymeric microspheres by ME and interfacial polymerization (Fig. 20). Porous polyamide membrane was created at the droplet surface by polycondensation of two monomers of different solubility. A hydrophobic

212 Chapter 7 Monomer II (EDA)

Monomer I (TDC) + Organic solvents

Interfacial polycondensation

Porous polymer (PA) Freeze drying

Graft polymerisation

Organic solvents

(A) Drug loading

40°C

Loaded capsule Gates closing

25°C

25°C

40°C

(B)

(C)

(D)

Fig. 20 (A) Preparation of thermoresponsive liquid-core-polymer-shell microparticles by membrane emulsification, interfacial polycondenzation, and graft polymerization (Chu et al., 2002). A drug (vitamin B12) was loaded and released at 40°C and kept in the capsules at 25°C; (B) Outer surface of the capsule with a diameter of 4 μm prepared using SPG membrane with a pore size of 1.2 μm at 40 kPa (Chu et al., 2003); (C) Magnified view of the shell surface (Chu et al., 2003); (D) Cross sectioned capsules prepared using SPG membrane with a pore size of 2.5 μm (Chu et al., 2003).

monomer, terephthaloyl dichloride (TDC), was dissolved in the dispersed phase (benzene/ xylene mixture) before ME, while a hydrophilic monomer, ethylenediamine (EDA), was added in the prepared emulsion. Polymerization proceeded at the organic/aqueous interface. After polymerization, PNIPAM chains were grafted on the inner pore surface of the polyamide

Membrane Emulsification in Pharmaceutics and Biotechnology 213 membrane to serve as thermoresponsive gates. The release rate of NaCl and vitamin B12 from the capsules was slow at 25°C compared to 40°C, since the extended PNIPAM chains obstructed the diffusion at 25°C, while the collapsed chains opened the pores at 40°C. A positive thermoresponsive behavior was attributed to the small number of grafted chains per unit surface area of the pore walls. Fig. 20D shows the cross section of the capsules prepared using an SPG membrane with a pore size of 2.5 μm. The capsule thickness was very small compared to the diameter, due to small monomer concentration in the organic phase. To make the capsule wall more robust, the TDC concentration in the organic phase should be at least 1.5 mol/L (Chu et al., 2003).

12.3 Quantum Dot Loaded Polymeric Microparticles Han et al. (2015) prepared quantum dot (QD)-embedded polystyrene (PST) microbeads using SPG membrane with a pore size of 15 μm (Fig. 21). PST and QDs were dissolved in DCM at 10 w/v% and 1 mg/mL, respectively, and the resultant solution was injected through the membrane into a 1 wt% SDS aqueous solution. The beads were formed after DCM evaporation and functionalized by exposing them sequentially to PAH and PAA. The beads were first incubated in a PAH solution to adsorb PAH polycations onto the negatively charged bead surface. The excess PAH was removed by washing and the PAH-layered PST beads were then resuspended in a PAA solution. After deposition of PAA, antibodies could be covalently attached onto the beads via an EDC/NHS-mediated amidation reaction. Fluorescence images in Fig. 21B show that blue and green QDs were successfully embedded within PST beads. The surface of PST beads was smooth on SEM images, indicating that QDs were embedded inside the beads (Fig. 21C). The bead sizes were between 20 and 27 μm with an average size of 24 μm (Fig. 21D). PSMA microbeads embedded with QDs were made by injecting a mixture of QDs, PSMA, and toluene through SPG membrane into an aqueous surfactant solution (Wang et al., 2013). Toluene was evaporated from the droplets to create solid particles and the surface of the particles was then functionalized to introduce carboxy groups, which are suitable for covalent attachment of antibodies (ABs). A library consisting of 12 barcodes has been created from only one type of QDs by combining four fluorescence intensities and three particle sizes. Different intensity levels were achieved using various concentrations of QDs in toluene, while different particle sizes were obtained by changing the membrane pore size.

13 Conclusions and Future Trends Microporous membranes are increasingly used for preparation of pharmaceutical microand nanoemulsions and solid nanodispersions. ME enables preparation of dispersions with a narrow particle size distribution and controlled particle size using low energy consumption

214 Chapter 7 Evaporation of DCM

SDS PAA

PAH

AB

QD PS + DCM

(A)

(B) 0.30

Fraction of microbeads

0.25 0.20 0.15

0.10 0.05 0.00

(C)

(D)

18

20

22

24

26

28

30

Diameter (um)

Fig. 21 (A) Preparation of quantum dot (QD)-embedded polystyrene (PS) microbeads using membrane emulsification, solvent evaporation, and layer-by-layer (LbL) deposition technique (Han et al., 2015); (B) Fluorescence images of PS microbeads with a diameter of 24 μm embedding green (left) and blue (right) QDs. The microbeads are prepared using an SPG membrane with a pore size of 15 μm; (C) SEM image of QD-embedded PS microbeads; (D) Size distribution of QD-embedded microbeads with a mean diameter of 24 μm (Han et al., 2015).

and mild hydrodynamic conditions. In direct ME, a pure dispersed phase is injected through the membrane at controlled injection rate and under controlled shear on the membrane surface. In premix ME, a preemulsified mixture of the dispersed and continuous phase is extruded (often repeatedly) through the membrane. Microemulsions are usually prepared via

Membrane Emulsification in Pharmaceutics and Biotechnology 215 direct ME using membranes with a pore size in the 0.45–2.5 μm range and the average drop size is significantly smaller than the pore size. Nanoemulsions can be prepared with direct or premix ME using nanoporous membranes and the average drop size is greater than the pore size. The most common emulsification membranes are SPG membrane, polymeric membranes, and microsieve membranes. Nanoemulsions comprising soybean oil or MCT oil dispersed in aqueous solutions of SDS or Tween 20 can be prepared by direct ME using SPG membranes with a maximum pore size of 0.2 μm and the ratio of the median drop size to the mean pore size ranges from 2.8:1 to 8.2:1. Due to higher ability to generate nanodrops, premix ME is more convenient process for preparation of nanoemulsions than direct process. The median drop size can be reduced by decreasing the pore size and increasing the number of extrusion cycles and transmembrane pressure. Track-etched polycarbonate membranes are less efficient in reducing the drop size in premix ME than SPG membranes, due to their rectilinear pores and smaller thickness (<20 nm) as compared to SPG membrane (700–900 nm). One extrusion of premix through SPG membrane with a pore size of 100–300 nm led to smaller drops than 11 or 21 extrusion cycles through tracked-etched PC membranes with the same pore size. The median drop size in premix ME using polymeric membrane primarily depended on the contact angle between the continuous aqueous phase and the membrane surface. The contact angle between the surfactant solution and the membrane surface must be <49° to create nanoemulsions with a d50 value smaller than 500 nm after 21 extrusion cycles at the flow rate of 25 mL/s through a polymeric membrane filter. Droplets prepared by ME processes can be solidified by ionic or covalent cross-linking of the polymer dissolved within the drops, solvent evaporation, cooling crystallization, spray drying, and free-radical polymerization. The main advantages of using ME in the pharmaceutical industry and biotechnology are: (1) controlled droplet /particle size, which allows controlled drug release rate and predictable drug biodistribution and biodegradation rate of the particles; (2) narrow particle size distribution; (3) high encapsulation efficiency of encapsulated active agents due to low shear during the process; (4) low energy input per unit volume, which leads to constant-temperature operation with minimal thermal degradation of the products and economical operation; (5) small consumption of chemicals per single batch due to small size of ME equipment. Due to new cost-effective shear manipulation techniques in ME devices and new membrane discoveries, ME will be increasingly used in pharmaceutical industry and biotechnology. The process evolved from production of traditional macroemulsions to the preparation of micro- and nanoemulsions and functional micro/nanoparticles.

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Further Reading Kotta, S., Khan, A.W., Pramod, K., Ansari, S.H., Sharma, R.K., Ali, J., 2012. Exploring oral nanoemulsions for bioavailability enhancement of poorly water-soluble drugs. Expert Opin. Drug Deliv. 9, 585–598. Loxley, A., Vincent, B., 1998. Preparation of poly(methylmethacrylate) microcapsule with liquid cores. J. Colloid Interface Sci. 208, 49–62. Nakashima, T., 2002. In: Porous glass material and its recent applications.Paper presented at 38th International SPG Forum on Membrane and Particle Science and Technology in Food and Medical Care in Sadowara, Japan. Sawalha, H., Fan, Y., Schroe¨n, K., Boom, R., 2008. Preparation of hollow polylactide microcapsules through premix membrane emulsification—effects of nonsolvent properties. J. Membr. Sci. 325, 665–671. Zhao, C.X., 2013. Multiphase flow microfluidics for the production of single or multiple emulsions for drug delivery. Adv. Drug Deliv. Rev. 65, 1420–1446.