Current applications of Colloidal Liquid Aphrons: Predispersed solvent extraction, enzyme immobilization and drug delivery

Journal Pre-proof Current applications of Colloidal Liquid Aphrons: Predispersed solvent extraction, enzyme immobilization and drug delivery

Keeran Ward, Anasha Taylor, Akeem Mohammed, David C. Stuckey PII:

S0001-8686(19)30182-4

DOI:

https://doi.org/10.1016/j.cis.2019.102079

Reference:

CIS 102079

To appear in:

Advances in Colloid and Interface Science

Revised date:

30 October 2019

Please cite this article as: K. Ward, A. Taylor, A. Mohammed, et al., Current applications of Colloidal Liquid Aphrons: Predispersed solvent extraction, enzyme immobilization and drug delivery, Advances in Colloid and Interface Science(2019), https://doi.org/10.1016/ j.cis.2019.102079

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© 2019 Published by Elsevier.

Journal Pre-proof

Current Applications of Colloidal Liquid Aphrons: Predispersed Solvent Extraction, Enzyme Immobilization and Drug Delivery.

Keeran Warda , Anasha Taylora , Akeem Mohammeda , David C. Stuckeyb* a

Department of Chemical Engineering, University of the West Indies.

b

Department of Chemical Engineering, Imperial College London.

* Telephone: 44 207 5945591 email: [email protected]

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Abstract

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Colloidal Liquid Aphrons (CLAs) are micron sized discrete spherical solvent droplets formed by

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the dispersion of polyaphrons into a bulk aqueous phase at a low phase volume ratio where they can be kept homogenously suspended with only minimal agitation. CLAs have high stability due

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to the presence of a surfactant ‘shell’ surrounding the solvent core, and possess large surface areas per unit volume for mass transfer due to their small size. Therefore, CLAs are well suited

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for applications in pre-dispersed solvent extraction (PSE), enzyme immobilization, and have the potential to be used as a drug delivery system. Using PSE, CLAs have been used to remove

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metals such as Ni2+, Cu2+, Fe3+, Cr3+ and Mg2+ from dilute streams, separate organic dyes such as Yellow 1 from wastewater, extract succinic and lactic acid, reactively extract phenylalanine, and

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separate suspensions. CLAs have also been used to immobilize enzymes such as lipase,

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lysozyme and albumins with cases of superactivity being reported due to the influence of surfactant and solvent interactions with the enzyme. Furthermore, due to their similarity to current drug delivery systems such as microemulsions and hydrogels, and other advantages, CLA systems have the potential to be adapted for drug delivery systems also. This article provides a complete list of the current applications of Colloidal Liquid Aphrons (CLAs) in PSE and enzyme immobilization, and also presents insight into how CLAs can be utilized as a drug delivery method in the future. Finally, this review ends by summarizing potentially interesting research areas to pursue in this field.

Keywords: colloidal liquid aphron (CLA), pre-dispersed solvent extraction (PSE), enzyme immobilization, polyaphrons, drug delivery. 1

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Contents

1.

Introduction.............................................................................................................................. 3

2.

Polyaphrons and Colloidal Liquid Aphrons (CLAs) ............................................................... 4 Polyaphron Structure....................................................................................................... 4

2.2

Polyaphron Formulation and CLA Stability. .................................................................. 6

Colloidal Liquid Aphron Applications .................................................................................... 9 Predispersed Solvent Extraction (PSE)........................................................................... 9

3.2

Enzyme Immobilization and Characterisation .............................................................. 10

3.3

Possible Application of CLAs for Drug Delivery......................................................... 13

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3.1

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3.

2.1

Key Research Areas for Future Development ....................................................................... 15

5.

Conclusions............................................................................................................................ 18

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4.

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References ..................................................................................................................................... 20

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1. Introduction Colloidal Liquid Aphrons (CLAs) are a type of oil-in-water (O/W) macroemulsion, generally around 10-100 μm in droplet size [1]. Similar to CLAs, several other O/W systems have been characterized; nano-emulsions as well as microemulsions are translucent formulations ranging in size between 10-600 nm, are inherently stable, and are utilized in cosmetics, personal care as well as pharmaceutical applications and drug solubilization [2-6]. These O/W emulsion systems

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are generally formulated using oil (solvent), water and an emulsifying agent-usually a surfactant. Sebba [7] reported that the CLA structure consisted of an inner solvent core stabilised by a

ro

monolayer of solvent soluble surfactant which is surrounded by an outer shell containing a bilayer of aqueous soluble surfactant (Figure 1); the first phase of the bi-liquid foam could either

-p

be liquid or gas. He postulated that the structure consisted of a micron sized solvent droplet

re

encapsulated by a thin aqueous film of surfactant molecules termed a “soapy shell”. The core of the structure has the hydrophobic non-polar ends of the surfactants, while the second phase,

lP

which is usually aqueous, contains the hydrophilic ends with its hydrophobic counterparts being oriented into the third outer layer. This outer layer, which also prevents the coalescence of

the bulk fluid.

na

adjacent aphrons, consists of a layer of surfactant molecules with hydrophilic ends extending into

ur

Due to their size, CLAs possess large mass transfer surfaces, and hence are excellent for use in

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mass transfer applications such as enzyme immobilization and pre-dispersed solvent extraction (PSE). In addition to large interfaces, the solvent core of these droplets allow for extensive product partitioning as well as adsorption due to favourable hydrophobic interactions [3,8-12]. These properties also make CLAs interesting candidates for drug delivery. Successful formulations should have critical attributes such as ease of fabrication, being thermodynamically stable and enable high degrees of immobilization. The majority of past research has centred on the use of ionic surfactants, particularly anionic surfactants such as sodium dodecyl sulphate (SDS), for CLA formulation as they aid in greater enzyme retention due to the electrostatic interactions between the polar residues within the protein structure and polar surfactant head groups [1, 8]. Nevertheless, the use of anionic surfactants in CLA formulations are not considered ideal for drug delivery systems as they have

3

Journal Pre-proof been known to lead to protein denaturation, and there are concerns over their toxicology. In a recent study by Ward and Stuckey [9] using a non-ionic CLA formulation they found that nonionic surfactants (Tween 80/Tween 20), even in high concentrations, do not bind to proteins thereby allowing their active conformation to be preserved. Although this non-ionic system showed promise in enzyme retention, optimization of the formulation is still necessary if it is to be successfully used as a drug delivery system. However, despite their extremely useful properties, there has been considerable debate amongst surface chemists as to the exact structure of microemulsions and CLAs (see below). While we

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will mention highlights of these competing hypotheses, in this review we will focus more on the

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applied use of these CLAs, rather than discuss in depth issues about structure. In addition, while most large reviews have focused on the application and developments surrounding colloidal gas

-p

aphrons (CGAs) [10-14] this paper seeks to review the applications of CLAs and introduces the

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possibility of adapting CLAs to be effective alternatives for drug delivery.

Polyaphron Structure

na

2.1

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2. Polyaphrons and Colloidal Liquid Aphrons (CLAs)

Polyaphrons are biliquid foams that are made up as the inner solvent phase is broken up into

ur

droplets encapsulated by an aqueous phase. The nature of the system is governed strongly by its

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close packing of the droplets within a set volume, allowing for distinct polyhedral type aphrons [1]. This closed-packed arrangement increases as the volume of oil used to create these droplets increases.

When polyaphrons are dispersed into a bulk continuous phase, the individual aphrons separate and are dispersed homogeneously, thus forming micron-sized discrete spherical droplets (5-20 µm) referred to as Colloidal Liquid Aphrons (CLAs). CLAs, due to their buoyant nature with most common solvents (less dense than water) form homogenous dispersions when mixed. Furthermore, the presence of a solvent core allows for product partitioning between the bulk aqueous and non-polar solvent phases. Sebba’s ‘soapy shell' accounts for the dispersibility of the polyaphrons, although they can freely rise to the surface when they are not mixed owing to differences in density between the aphrons and the bulk aqueous phase. Aphrons have been

4

Journal Pre-proof considered to be similar to High internal phase ratio emulsions (HIPREs). These HIPREs are microemulsions including both oil-in-water and water-in-oil systems in which the volume of the dispersed phase,

approaches or exceeds that of the close-packed-sphere packing,

[15]. Yan et al. [16] presented results that supported this opinion in an investigation geared towards providing insights into the microstructure of CLAs using a variety of different analytical methods such as freeze fracture transmission electron microscopy (FF-TEM) and small angle X-

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ray scattering (SAXS). They concluded from their data that the overall microstructure of the CLA was similar to the biphasic structure of HIPREs- consisting of a dispersed oil phase as a

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continuous phase, with an interfacial area thickness of 0.3-0.4 m. Additionally, the research highlighted supramolecular structures such as micelles or microemulsions within CLA oil and

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water regions unlike HIPREs where these structures are found within the water layer. Although

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these authors made conclusive findings on the thickness of the interface, as well as useful insights into CLA structure, more research is needed into the stability of these microemulsions

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based on formulation parameters such as solvent type, and surfactant type and concentration for the better “tuning” of CLAs to various systems.

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Other attempts are still being made to clarify the structure of the CLA utilising techniques such as differential scanning calorimetry (DSC), light scattering, as well as transmission electron

ur

microscopy (TEM) which can be used to determine mainly macroscopic features of the aphron

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structure. In a study by Lye and Stuckey [17], the macroscopic structure of aphrons was confirmed using cryo-transmission electron microscopy (Cryo-TEM) and other light scattering methods. It was reported that the thickness of the bilayer, approximately 0.03- 0.15 m, was in agreement with that of the model proposed by Sebba [7]. The authors however, did not report any findings on structural characteristics of the interface that can support CLA stability. Srivastava et al. [18] used a mixture of DSC and SAXS to provide direct evidence for the “soapy shell” of the CLA. Their study revealed that the interface had a unique arrangement similar to that of liquid-crystals composed of multiple layers. However, the order of these multiple layers are attributed to the surfactant properties and are most likely not constant over a broad range of formulation methodologies.

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Journal Pre-proof Although many studies have reported insights into the structure of the polyaphron model, with some evidence supporting the view of surfactant bilayers as well as monolayers, currently there is no solid evidence for the actual location and structure of the CLA interface. However, despite the lack of agreement in the literature on their basic structure, CLAs have many practical applications, and these are the areas we will focus on in this review.

Polyaphron Formulation and CLA Stability.

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2.2

Polyaphron properties depend on the formulation methods employed in their generation, while

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CLAs are mainly influenced by the nature of the continuous phase into which they are dispersed.

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For the general preparation of emulsions oil, water, surfactant and energy is needed; considerable energy is required to expand the surface to allow for droplet formulation. As the interfacial

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tension is very large due to the incompatibility of both fluids, a largely positive Gibbs free energy is created, and thus their production is non-spontaneous in nature [19]. Thus, high

lP

energies are required to allow for droplet deformation and expansion of the interface. In an effort to reduce the interfacial tension required for emulsion formation, surfactants are needed as they

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adsorb to the interface promoting mixing between phases. Generally, for CLAs, non-ionic surfactants are used to stabilized the solvent player while ionic/non-ionic surfactants are

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employed to stabilize the aqueous layer. Specifically, polyaphron production has been postulated

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to be limited by the quantity and solubility of surfactant required to stabilise the encapsulating film. However, the method for formulating polyaphrons is mostly trial and error as no specific methodology exists. In most cases, the Hydrophilic Lipophilic Balance (HLB) number is used as a guide. The HLB number gives the solubility of the surfactant in polar/non-polar solvents and thus, provides critical information. In a study by Matsushita et al. [20], anionic aqueous phase surfactants such as sodium dodecyl sulphate (SDS), and cationic surfactants-dodecyltrimethyl ammonium bromide (DTMAB) and cetyltrimethyl ammonium bromide (CTMAB) were used as well as a variety of oil phase alcohol ethoxylates (non-ionic surfactants). The results showed that stability was strongly driven by the solvent phase surfactant and its solubility within the polar organic phases. This allows for a decrease in interfacial tension and a greater encapsulation of the oil phase. However, for anionic aqueous phase surfactants, a decrease in concentration

6

Journal Pre-proof directly decreases the size of the aphrons produced. This has also been reported with microemulsions where a reduction in concentration of the surfactant can cause the interface to retract, decreasing the overall size of the microemulsion, and is thought to be directly related to surfactant charge interactions [21]. Along with surfactant type and solubility, the nature of the solvent also has an effect on CLA formulation. Lee et al. [22] performed a study which shows that polar solvents decreased stability, and this was directly attributed to a higher organic phase solubility allowing greater destabilization of the CLA. Furthermore, the volume of oil available for droplet formulation

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increases the stability of the polyaphron as the phase volume ratio (PVR) increases. The PVR is

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the ratio of the volume of the dispersed phase to the aqueous phase and is considered a distinguishing feature of polyaphrons as it differentiates them from microemulsions which can

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destabilize at low PVRs. Generally, polyaphrons at low PVRs are Newtonian in behaviour and

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fairly spherical. However, as PVR increases the overall shape of the polyaphron changes to accommodate a growing population of droplets, increasing the overall viscosity to a Bingham

lP

type system. This further decreases the size of the individual aphron, with uniformity increasing with PVR [16, 23]. This decrease in size allows for greater adsorption capacity as a larger (Figure

2).

The system,

however,

can eventually appear

na

interfacial area is created

monodispersed as the effect of shearing ceases to affect the polyaphron size. In fact, as

ur

movement is restricted in this clustered polyaphron system, if shearing is continued the energy being inputted into the system destabilises the polyaphrons leading to phase inversion. Based on

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these findings it is apparent that CLA size has a clear influence on CLA stability, and although higher PVRs do support a more stable formulation, there is no ideal PVR that will guarantee stability for a desired formulation. Other than system parameters, environmental conditions can also affect the stability of the CLA. Lye et al. [1] proposed a collision-coalescence mechanism, suggesting that the temperature at which this process occurred would influence CLA stability. From their investigation, they confirmed that increased kinetic energy of the droplets at higher temperatures resulted in more frequent collisions, thereby increasing the probability of fusion occurring. As their results suggest, if break-up of CLAs is dependent on collisions between CLAs having sufficient energy to overcome the activation energy barrier, then a clear correlation between size and half-life

7

Journal Pre-proof should be expected. This correlation was evident in Scarpello and Stuckey [24] study on the stability of CLAs; they proposed a semi-empirical design equation which predicted the half-life of CLAs, and from this were able to show that with decreasing size the half-life of CLAs increased. They attributed their findings to a lower energy of collision of CLAs with decreasing size.

Lamb and Stuckey [25] also investigated the influence of process parameters including pH, temperature and ionic strength on CLA stability. They described CLA break-up as a first order

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chemical reaction which depends on collisions occurring with adequate energy to overcome the

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repulsive forces that stabilize the CLAs. Once the sum of the kinetic energies of the colliding CLAs was greater than the activation energy required for break-up, the CLAs break-up, or

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possibly coalesce. From their experimental results, they concluded that the rate of CLA break-up would rise with increasing kinetic energy, thereby increasing the average velocity of CLA

The authors also used CLAs as an immobilization

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charge repulsion led to lower break-up.

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collisions. They further elucidated that ionic strength was independent of CLA stability as the

support in a membrane reactor, and determined the effect of process parameters on the support

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stability to obtain half-life values for dispersed CLAs based on Equation 1: t1/2 = ln 2 /

(1)

Using the same analogy of a chemical reaction with first-order kinetics, the half-life values found

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for the dispersed CLAs were expressed in the order of minutes. Ultimately, they aimed to

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optimise the process with emphasis on immobilization, activity and support stability. For the polyaphron phase, no evident deterioration was observed over time, and quite a clear correlation between CLA concentration and half-life was reported. Due to the reliance of CLA stability on the collision energy, with increasing CLA concentrations the average number of collisions would be anticipated to increase, while the average collision energy would decrease. They suggested that an increase in CLA concentration restricts the necessary mean average velocity from being achieved, thereby leading to a reduction in the kinetic energy of CLAs and break-up.

Although several limitations are present within current methodologies, such as solvent addition rates, stirring rates and the possibility of excessive shearing during formulation, all of which can affect the nature of the results obtained, the general trend is clear that smaller CLAs are more

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Journal Pre-proof stable. Based on the influence of temperature and solvent type, it seems that these two parameters have optimums that can be used to produce very stable CLA formulations. While it is clear that higher PVRs result in more stable formulations, stability is still possible at lower PVRs and is directly related to the nature of the surfactants and solvents used. While limited research exists

to

confirm whether

there

is

an

optimum PVR

value

or

system conditions

(surfactant/solvent type), it can be presumed that the effect of these parameters is based on the intended purpose of the formulation. Until a more critical polyaphron formulation methodology is proposed, a trial and error approach will remain the only viable way to produce stable

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polyaphrons.

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3. Colloidal Liquid Aphron Applications

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3.1 Predispersed Solvent Extraction (PSE)

One of the major initial applications of CLAs was their use in Predispersed Solvent Extraction

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(PSE). Within the PSE process, CLAs allow for product partitioning (extraction) based on the presence of the oil (solvent) core as well as their large interfacial area. Thus, overall extraction

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kinetics are rapid (< 1 second) and often hard to measure. Furthermore, their natural buoyancy allows for easy separation from solution, and therefore mixing is usually unnecessary and

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unimportant.. Table 1 illustrates recent applications of CLAs for PSE.

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Several studies have utilized PSE for heavy metal ion separation such as Ni2+, Fe3+, Cu2+, Cr3+ and Mg2+ [26-30] with exceptional extraction efficiency (>90%). The use of CLAs in PSE overcomes most disadvantages of conventional solvent extraction, eg.; a mixing-settling stage, high solvent/aqueous ratios, and high initial metal concentrations. Within the separation mechanism, electrostatic interactions provide the basis for ion-exchange between the CLA phase and the bulk aqueous phase. Thus, for metal ions, charged surfaces are necessary for which the surfactant

choice

is

most

important.

Cationic

and

non-ionic

systems

such

as

hexadecyltrimethylammonium bromide (CTAB) and polyethylene glycol sorbitan monostearate (Tween 60), often do not promote the electrostatic charges needed to support high efficiencies within metal ion separation processes. Many formulations employ the use of anionic surfactants such sodium dodecyl benzene sulphate (SDBS), SDS, as it gives the best compatibility.

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Other than surfactant type, an extractant is often employed that enables product partitioning. As with any ion-exchange process, pH becomes increasingly important as H+ ions moderate equilibria between metal ions and the extractant [28]. Furthermore, the PVR controls the population of available droplets required for product partitioning, and hence, also becomes an important parameter in promoting extraction efficiency.

PSE has also been utilized in the extraction of non-polar solutes and organic compounds such as

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erythromycin, phenylalanine, succinic acid, lactic acid and butanediol [24, 31-36] where the

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product is sparingly soluble in aqueous media. Within these systems, equilibria dominates extraction efficiency, although extraction efficiency can be enhanced through reactions with

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extractants. This reactivity allows for rapid mass transfer within the CLA phase, promoting

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higher efficiencies with reduced contact times (Figure 3).

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Although PSE has shown to be quite effective in product separation, several drawbacks are affiliated with product yield and purity as well as CLA separation. After extraction of the product using PSE, CLAs need to be stripped for complete recovery. In some cases, stripping is not

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completely successful and complexes can form between the product and the surfactants that results in slow mass transfer kinetics. Furthermore, selectivity within the extraction process can

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be problematic if compounds or impurities present have similar electrostatic interactions. These

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drawbacks, however, can be minimized using reactive extractants with a higher removal (partitioning) efficiency and selectivity, and membrane filtration which can result in faster separation, increased mass transfer kinetics and the possibility of reusing the filtered CLAs after stripping of the solute [37]. In an effort to understand the use of PSE on a large scale more research is needed to examine the problems mentioned above, and the ideal characteristics suited to individual applications.

3.2

Enzyme Immobilization and Characterisation

Immobilization of enzymes has been an effective way of maintaining enzyme activity in harsh environments through encapsulation, cross-linking, adsorption onto solid supports, or covalent

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Journal Pre-proof attachment and entrapment in polymeric gels [41]. CLAs create an environment that can both stabilise and preserve enzyme conformation, and interactions between an enzyme and the oilwater interface allow attachment of the enzyme, thereby immobilising it from the bulk environmental

effects.

Table

2

illustrates

recent

applications

of

CLAs

for

enzyme

immobilization.

One of the first studies utilising CLAs as a support for enzyme immobilization was carried out by Lye et al. [1] where Candida cylindracea lipase was immobilised using SDS. Immobilization

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was governed predominantly by electrostatic interactions between SDS and lipase, with low pH

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allowing for higher enzyme retention. The existence of these electrostatic interactions allowed for greater mass transfer, and thus produced multilayers of adsorbed protein. The authors found

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that, upon successive dispersions of the enzyme loaded CLA formulation, enzyme layers furthest away from the soapy shell were stripped away allowing for monolayer coverage (Figure 4).

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However, the amount of protein immobilized increased as the enzyme loading increased,

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showcasing the effect of hydrophobic interactions in increasing the net coverage of adsorbed protein.

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In an effort to understand the effects of hydrophobic and electrostatic interactions, Lamb and Stuckey [8] investigated the use of anionic SDS, cationic DTMAB and CTMAB and non-ionic

- amylase and

-galactosidase. They found that pH had a significant effect only

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lysozyme,

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Synperonic A20 and Atlas G1300, on the immobilization of lipase, trypsin, ribonuclease-A,

when the protein was predominantly positively charged (pH
interactions

supporting

immobilization.

However,

for

globular

and

largely

hydrophobic proteins such as - amylase, -galactosidase and lipase, immobilization was found to be higher due to hydrophobic interactions allowing for greater protein compressibility. Furthermore, upon analysis of enzyme activity, SDS interactions led to gross denaturation with the exception of

- amylase, -galactosidase and ribonuclease-A attributed to favourable

surfactant- mediated active site excitation.

In a separate case, Lamb and Stuckey [38] generated CLAs from both ionic (SDS, DTMAB, CTMAB, AOT80) and non-ionic surfactants (Synperonic A20, Atlas G1300, Brij 78) as well as

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Journal Pre-proof various solvent phases shown in Table 3, for the immobilization of -galactosidase. Results showed that immobilization was dominated by hydrophobic interactions, however, electrostatic interactions were needed to initiate immobilization- this was apparent from the increased immobilization observed when polar solvents were used with ionic surfactants. Immobilization also changed the pH profile and increased the activity of the enzyme due to electrostatic interactions between surfactant monomers and the protein surface.

While past studies mainly focused on ionic/polar CLA formulations, recent work by Ward and

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Stuckey [9] examined the ability of non-ionic (Tween 80, Tween 20)/non-polar (Mineral oil)

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CLAs to preserve enzyme functionality upon immobilization and release. Investigations using the circular dichroism (CD) of desorbed proteins such as Bovine Serum Albumin (BSA-Figure

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5) showed the preservation of structural integrity, with no significant alterations before and after adsorption as non-ionic interactions were minimal; the study concluded that CLA formulation

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has a significant role in the promotion of immobilization through adsorption. Their results also

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showed no changes in optimum conditions, such as pH and temperature, after immobilization as the microenvironment of the protein was unaffected. Furthermore, analysis of activation energy

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for the immobilized enzyme showed an increase in activity at higher temperatures. For both systems a decrease in the Gibbs free energy,

G, followed by a decrease in enthalpy,

H, was

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observed. This suggests that activity was thermodynamically favourable at higher temperatures. Also, results supported hydration effects accounted for a decrease in

G, illustrating that non-

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ionic CLAs can provide the hydration level required for an active enzyme conformation. Based on these findings the ability of non-ionic CLAs to preserve catalytic activity conditions and enzyme structural integrity upon desorption, shows its potential as a support for immobilization.

Although anionic surfactants have proven to be effective in increasing enzyme retention and allowing for superactivity among CLA immobilized enzymes, their toxicological and denaturing properties raise a number of concerns in their use in future applications such as drug delivery. In contrast, non-ionic systems have shown considerable promise in their ability to preserve enzyme conformation, however, their weak interactions do not allow for effective enzyme retention. Furthermore, limitations in the proposed CLA model poses major challenges for enzyme immobilization such as identifying exactly where enzymes sit within the CLA structure, and how

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Journal Pre-proof the structure can influence retention as well as enzyme conformation due to the presence of micellar structures or microemulsions. Hence, in order to develop an optimal CLA formulation for enzyme immobilization, critical understanding of the effects of these micellar structures as well as the steric orientation of the enzyme within the soapy shell is needed.

3.3

Possible Application of CLAs for Drug Delivery

The advent of protein immobilization has helped in the development of various methods for the

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coupling of proteins to solid supports for a variety of applications; these include protein

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digestion, protein separation, molecular delivery, and biocatalysis [39]. Conventionally, solid support systems used for enzymes include microparticles, silica gel, hydrogels, and nanoporous

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inorganic materials [40], while drug encapsulation utilizing liposomes, reverse micelles and

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microemulsions has made the oral delivery of essential drugs more attractive. Cubosomes and hexosomes are colloidal nanoparticles (microemulsions) with confined internal

lP

reversed structures of a 3D well-ordered bi-continuous cubic (V2 ), and 2D columnar hexagonal (H2 ) phase, respectively [41]. These are formed by means of steric or electrostatic processes

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controlled by the self-assembly of surfactant-like lipids that form nonlamellar phases in excess water in the presence of a stabilizer [41, 42]. The cubosome is a complex structure comprised of

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a curved lipid bilayer which subdivides the 3D space into an intertwined but non-penetrating network of hydrophilic nanochannels [43], while the hexosome is a 2D structure consisting of

The

structure

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water filled cylindrical rods embedded in a continuous hydrophobic medium. of

colloidal

nanoparticles

enables

biologically

active

molecules

to

be

accommodated within the aqueous domains, or be directly coupled to the hydrophobic lipid region, and as such has the potential to be utilized as an alternative drug delivery system with desired properties. These nanostructured liquid crystalline particles cause the efficient loading of various hydrophilic and hydrophobic drugs, and can allow for nano-engineered carriers for selective tissues and for enhancing cell penetration [4, 44, 45]. One of the most useful properties of cubosomes and hexosomes is the; efficient solubilization of poorly water soluble drugs, increased bioavailability, sustained drug release, and enhanced skin penetration in oral, topical and parenteral routes [46-50]. However, the internal nanostructures of

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Journal Pre-proof these dispersions are sensitive to guest materials which may affect the internal nanostructures and drug delivery and release mechanisms [4]. The structure of CLAs are similar to those of cubosomes and hexosomes where there exists both an oil and aqueous phase oriented similarly with an oil core and aqueous shell. Thus, it can be postulated that similarities in these colloidal particles to the CLAs further prove that CLAs can be used as an efficient alternative drug delivery vehicle. Matricardi et al. [51] investigated hydrogels which are environmentally sensitive, as ‘smart’ drug delivery systems that have the ability to release, at the right time and site of action, encapsulated

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drugs in response to specific physiological triggers. Hydrogels can be produced from virtually

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any water-soluble polymer which encompasses an extensive range of chemical compositions and bulk physical properties, and can be made in a range of physical forms which include slabs,

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microparticles, nanoparticles, coatings, and films [52]. Their high water content (70-99%)

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provides a physical similarity to tissues as they have a soft consistency and low interfacial tension with aqueous media [53], and gives rise to high biocompatibility and the ability to

lP

encapsulate hydrophilic drugs [54]. Furthermore, the danger of drug denaturation and aggregation upon contact with organic solvents is diminished as these hydrogels are usually

na

formed in aqueous solutions. These hydrogels have to ability to safeguard the drugs from hostile environments such as the presence of enzymes and low pH in the stomach, and can control drug

ur

release by altering the structure of the gel in response to changes in the environment [55].

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Thus, these properties of hydrogels have sparked interest in their application as drug delivery systems for tissue engineering [56], diagnostics [57], immobilization of cells [58], separation of biomolecules [59] and barrier materials to regulate biological adhesions [60]. The hydrogels’ structure is very porous and can easily be manipulated to control the density of the gel’s crosslinks within its matrix in addition to the hydrogel’s affinity for the aqueous environment in which they become swollen. The porosity of the hydrogel permits loading of drugs into the gel matrix and subsequent drug release giving rise to largely pharmacokinetic benefits [52]. As stated previously, the soapy shell of the CLA maintains the structural integrity of the enzyme; this finding highlights the most compatible part of the CLA as a potential drug delivery system while utilising non-ionic surfactants. Non-ionic systems not only resolve the toxicity issue due to ionic surfactants, but are also less denaturing, two properties that are essential for CLA

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Journal Pre-proof application in drug delivery. The primary goals in the growth of drug delivery systems are to protect an active therapeutic molecule from premature degradation, enhance drug efficacy, and minimize side effects. With these goals in mind, the CLA in comparison to liposomes, microemulsions and hydrogels possess several similarities; CLAs have a solvent core to incorporate hydrophobic drugs similarly to liposomes which have an aqueous core that stabilizes hydrophilic drugs. Furthermore, the CLA bilayer supports adsorption as well as encapsulation increasing the drug loading similarly to hydrogels. Like microemulsions, CLAs do not agglomerate and have a characteristic buoyancy which enables them to disperse easily in

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solution. Finally, the CLAs are composed of similar constituents as microemulsions with low

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particle sizes allowing for high mass transfer making them potential drug delivery systems. In a first study of its kind, CLAs were formulated for the immobilization of insulin through the

-p

use of charge neutralization with sodium alginate. Ward et al. [61] formulated a system

re

consisting of a non-ionic surfactant, a non-polar solvent phase and sodium alginate as a possible drug delivery formulation. The CLAs showed marked stability with a PVR of 4, and were able to

lP

adsorb almost 100% of insulin at a pH of 3.7. The study revealed that by using sodium alginate insulin can be charge neutralized, resulting in a hydrophobic complex with a high binding

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affinity for the non-ionic/non –polar CLAs. By utilizing the high surface area of these droplets, insulin was able to bind successfully to the CLA. Furthermore, by tuning the pH of the system

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insulin can be released successfully without any change in structural conformation. Similar to earlier findings for hydrogels, microemulsions and liposomes, the use of CLAs to bind and

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release valuable polypeptides such as insulin shows the potential application of these systems for drug delivery. While CLAs can fulfil the basic criteria for drug delivery, there are still key aspects of its structure that has to be understood before this application can be optimized for various types of proteins/drugs.

4. Key Research Areas for Future Development 1) Although CLAs have been utilized successfully in areas such as PSE and Enzyme immobilization, and show promise in the area of drug delivery, several areas for development exist in an effort to fully understand and optimize their formulation. One of the major areas that still needs work is the aphron structure. Postulations made by past researchers have only resulted

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Journal Pre-proof in little insight into the existence of a bilayer leading to the stability of the aphron itself as well as the its natural characteristics. Efforts into determining the formation of the bilayer, and the critical role of the surfactant as well as solvent effects in its stabilization need to be addressed in order to be able to improve its formulation methodologies. 2) The stability of the aphron is dependent upon several parameters such as temperature, droplet size, solvent and surfactant type and bulk solution in which they are applied. Very little has been done in developing heuristic rules dictating which factor(s) is/are the most important in contributing to stability. Past studies have explored the process of determining a stable

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formulation based simply on a trial and error method. In most cases, the HLB number can be

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used as a guide to surfactant solubility. There also lies a threshold whereby the PVR is able to create a monodispersed droplet system dependent on the concentration of surfactant used;

-p

furthermore, the solvent compatibility with the aqueous phase is also important in creating a

re

stable formulation.

3) It is clear from past studies that there is no formulation that suits all applications. The

lP

chemical characteristics such as solvent and surfactant type and the physical characteristics such as PVR, temperature and mixing phenomena should all be examined based on the CLA

na

application; there are no set guidelines for examining these characteristics. For the solvent type, there is a strong dependence on hydrophobic interactions that govern protein immobilization and

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its structural preservation. However, protein adsorption also depends on charge interactions that

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are able to mediate in the transfer of the protein from bulk solution to the CLA interface. Surfactants are known to be very important in this process facilitating charge interactions due to their polar head groups. Hence, it is important to examine the level of immobilization needed, and the integrity of the protein immobilized. This is particularly important for drug delivery where the nature of the surfactant, the solvent, and the charge mediator, need to be nondenaturing in an effort to protect the protein/drug conformation and its efficacy. 4) For PSE, the nature of both the solvent and surfactant and their influence on extraction efficiency can also be governed by similar effects. Polar/non-polar interactions can effectively increase extraction depending on the extractant used and its affinity for the solute, while the mass transfer rate can be increased by decreasing the droplet size and increasing the PVR and removing the CLAs rapidly from solution allowing for enhanced removal. For PSE, physical

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Journal Pre-proof characteristics seem to be most important, but still a compromise needs to be established between the solubility of the surfactant and the type of solvent used to generate stable aphrons. Hence, more work needs to be done to fully understand and develop guidelines in aphron

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formulation suited to each CLA application.

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5. Conclusions Colloidal Liquid Aphrons (CLAs), are formed from discrete micron-sized solvent droplets upon dispersion into a bulk continuous aqueous phase. Their buoyant nature enables them to form homogeneous dispersions when mixed, while the presence of a solvent core allows for product partitioning between non-polar and polar environments. With respect to CLA stabilization, surfactant interactions are more important than solvent choice, however, there seems to be no standard approach in selecting surfactants for CLA formulation. In addition to the nature of the

of

surfactant used, PVR also plays a crucial role in CLA stability. From past research it is evident

ro

that higher PVRs yield more stable formulations, however, it was noted that destabilisation can

-p

occur even at very high PVR values.

The large interfacial area supported by CLAs have made them increasingly attractive, with

re

applications including PSE, enzyme immobilization and biocatalysis. PSE utilizes CLAs for

lP

separating by the extraction of the desired compound into the oil (solvent) core. Although the non-selectivity of the PSE process can be a potential drawback, the use of reactive extractants for

na

polar solutes can aid in enhanced selective extraction. However, more research is needed with

ur

respect to product recovery to allow for an efficient extraction process.

CLAs have also been successfully applied as a support for enzyme immobilization as it has been

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shown to provide an environment which achieves stabilisation by the preservation of the enzyme conformation, and in some cases it enhances the enzyme’s reaction rate. The hydration effect required to preserve conformation during adsorption can be attributed to the possibility of micellar structures existing within the CLA structure . However, as most of the literature focuses on the applications of aphrons and little on the actual aphron structure, it is difficult to prove that micellar structures support CLA stability and immobilization characteristics.

Advances in the understanding of the mechanisms relating to enzyme-CLA immobilization have simplified the process of biocatalysis. The use of ionic surfactants for CLA formulations have been

extensively

studied,

with most research directed

towards a greater degree of

immobilization. Studies show that ionic surfactant systems usually result in conformational

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Journal Pre-proof changes as interactions between surfactants and proteins occur, however, there are toxicological concerns associated with such systems. There exists limited research into the use of non-ionic systems, however, all the existing literature proves that these systems not only reduce CLA denaturing capacity, but they also minimize the toxicity of the system. As these are both requirements for drug delivery systems, further research into enzyme-CLA formulation utilizing non-ionic surfactants could lead to advances for future applications.

This review reinforces the need for more research into the proposed CLA model by Sebba. For

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optimizing CLA formulations for any of these applications examined in this review, it is evident

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that more advances can be made once the structure has been more clearly understood.

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References

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[1] Lye GJ, Pavlou OP, Rosjidi M, Stuckey DC. Immobilization of Candida cylindracea lipase on colloidal liquid aphrons (CLAs) and development of a continuous CLA -membrane reactor. Biotechnology and Bioengineering. 1996;51:69-78. [2] H N, S T, M O. First Emulsion. Paris. France1993. [3] Bouchemal K, Briançon S, Perrier E, Fessi H. Nano-emulsion formulation using spontaneous emulsification: solvent, oil and surfactant optimisation. International Journal of Pharmaceutics. 2004;280:241-51. [4] Azmi I, Moghimi S, Yaghmur A. Cubosomes and hexosomes as versatile platforms for drug delivery. Therapeutic delivery. 2015;6:1347-64. [5] Klier J, Tucker CJ, Kalantar TH, Green DP. Properties and Applications of Microemu lsions. Advanced Materials. 2000;12:1751-7. [6] McClements D. Nanoemulsions versus microemulsions: Terminology, differences, and similarities. Soft Matter. 2012;8:1719-29. [7] Sebba F. Foams and biliquid foams, aphrons. Chichester [West Sussex]; New York: Wiley; 1987. [8] Lamb SB, Stuckey D. Enzyme immobilisation on colloidal liquid aphrons (CLAs): The influence of protein properties. Enzyme and Microbial Technology. 1999;24:541-8. [9] Ward K, Stuckey DC. Refractive index matching to develop transparent pol yaphrons: Characterization of immobilized proteins. Colloids Surf B Biointerfaces. 2016;142:159-64. [10] Molaei A, Waters KE. Aphron applications — A review of recent and current research. Advances in Colloid and Interface Science. 2015;216:36-54. [11] Pugh RJ. Foaming, foam films, antifoaming and defoaming. Advances in Colloid and Interface Science. 1996;64:67-142. [12] Jauregi P, Varley J. Colloidal gas aphrons: potential applications in biotechnology. Trends in Biotechnology. 1999;17:389-95. [13] Hashim MA, Mukhopadhyay S, Gupta BS, Sahu JN. Application of colloidal gas aphrons for pollution remediation. Journal of Chemical Technology & Biotechnology. 2012;87:305-24. [14] Ivan CD, Quintana JL, Blake LD. Aphron-Base Drilling Fluid: Evolving Technologies for Lost Circulation Control. SPE Annual Technical Conference and Exhibition. New Orleans, Louisiana: Society of Petroleum Engineers; 2001. p. 6. [15] Princen HM. On "an unusual gel without a gelling agent". Langmuir. 1988;4:486-7. [16] Yan YL, Zhang NS, Qu CT, Liu L. Microstructure of colloidal liquid aphrons (CLAs) by freeze fracture transmission electron microscopy (FF-TEM). Colloid Surface A. 2005;264:139-46. [17] Lye GJ, Stuckey DC. Structure and stability of colloidal liquid aphrons. Colloids and Surfa ces A: Physicochemical and Engineering Aspects. 1998;131:119-36. [18] Srivastava P, Hahr O, Buchholz R, Worden RM. Enhancement of mass transfer using colloidal liquid aphrons: Measurement of mass transfer coefficients in liquid–liquid extraction. Biotechnology and Bioengineering. 2000;70:525-32. [19] Tadros T, Izquierdo P, Esquena J, Solans C. Formation and stability of nano-emulsions. Advances in Colloid and Interface Science. 2004;108-109:303-18. [20] Matsushita K, Mollah AH, Stuckey DC, del Cerro C, Bailey AI. Predispersed solvent extraction of dilute products using colloidal gas aphrons and colloidal liquid aphrons: Aphron preparation, stability and size. Colloids and Surfaces. 1992;69:65-72. [21] Gelbart W, Ben-Shaul A, Roux D. Micelles, Membranes, Microemulsions, and Monolayers. New York, NY: Springer; 1994. [22] Lee DW, Hong WH, Lee T-y, Lee CH. Influence of continuous aqueous phase on the preparation and stability of colloidal liquid aphrons. Separation Science and Technology. 2002;37:1897-909.

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[23] Ward K, Xi J, Stuckey DC. Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Surface and enzyme effects. Colloids Surf B Biointerfaces. 2015;136:424-30. [24] Scarpello JT, Stuckey D. The reactive extraction of phenylalanine with Aliq uat 336: Buffer coextraction equilibrium and mass transfer kinetics2000. [25] Lamb SB, Stuckey DC. Factors influencing the stability of a novel enzyme immobilisation support colloidal liquid aphrons (CLAs). Journal of Chemical Technology & Biotechnology . 2000;75:681-8. [26] Molaei A, Kökkılıç O, Waters KE. An investigation into predispersed solvent extraction of nickel (II) ions from dilute aqueous solutions. Separation and Purification Technology. 2017;174:396-407. [27] Luo JH, Li J, Duan XX, Jin Y. Extraction of Fe3+ from Sodium Dihydrogen Phosphate with Colloidal Liquid Aphrons. Industrial & Engineering Chemistry Research. 2013;52:4306-11. [28] Molaei A, Waters K. Copper ion removal from dilute solutions using colloidal liquid aphrons. Separation and Purification Technology. 2015;152:115-22. [29] Luo JH, Li J, Qi YB, Cao YQ. Study on the removal of chromium(III) by solvent extraction. Desalination and Water Treatment. 2013;51:2130-4. [30] Luo J, Li J, Jin Y, Zhang Y, Zheng D. Study on Mg2+ Removal from Ammonium Dihydrogen Phosphate Solution by Predispersed Solvent Extraction. Industrial & Engineering Chemistry Research. 2009;48:2056-60. [31] Lye GJ, Stuckey DC. Extraction of erythromycin‐A using colloidal liquid aphrons: I. Equilibrium partitioning. Journal of Chemical Technology & Biotechnology. 2000;75:339-47. [32] Kim BS, Hong YK, Hong WH. Effect of ph on the extraction characteristics of succinic acid and the stability of colloidal liquid aphrons. Korean Journal of Chemical Engineering. 2002;19:669-72. [33] Kim BS, Hong YK, Hong WH. Effect of salts on the extraction characteristics of succinic acid by predispersed solvent extraction. Biotechnology and Bioprocess Engineering. 2004;9:207-11. [34] Lee DW, Hong WH, Hwang KY. Removal of an Organic Dye from Water Using a Predispersed Solvent Extraction. Separation Science and Technology. 2000;35:1951-62. [35] Hong YK, Lee DW, Cheon Lee P, Hong WH, Chang HN. Extraction of lactic acid with colloidal liquid aphrons and comparison of their toxicities with solvents without surfactant on the viability of Lactobacillus rhamnosus. Biotechnology Letters. 2001;23:983-8. [36] Birajdar SD, Rajagopalan S, Sawant JS, Padmanabhan S. Continuous predispersed solvent extraction process for the downstream separation of 2,3-butanediol from fermentation broth. Separation and Purification Technology. 2015;151:115-23. [37] Rosjidi M, Stuckey DC. Parameters influencing the separation of colloidal liquid aphrons(CLAs) using inorganic crossflow microfiltration.: Institution of Chemical Engineers; 1994. p. 127-9. [38] Lamb SB, Stuckey DC. Enzyme immobilization on colloidal liquid aphrons (CLAs): the influence of system parameters on activity. Enzyme and Microbial Technology. 2000;26:574-81. [39] Sheldon AR. Enzyme Immobilization: The Quest for Optimum Performance. Advanced Synthesis & Catalysis. 2007;349:1289-307. [40] Shieh F-K, Wang S-C, Yen C-I, Wu C-C, Dutta S, Chou L-Y, et al. Imparting Functionality to Biocatalysts via Embedding Enzymes into Nanoporous Materials by a de Novo Approach: Size-Selective Sheltering of Catalase in Metal–Organic Framework Microcrystals. Journal of the American Chemical Society. 2015;137:4276-9. [41] Kaasgaard T, Drummond CJ. Ordered 2-D and 3-D nanostructured amphiphile self-assembly materials stable in excess solvent. Physical Chemistry Chemical Physics. 2006;8:4957-75. [42] Hyde S, Ninham B, Andersson S, Larsson K, Landh T, Blum Z, et al. Lipid Self -Assembly and Function In Biological Systems. 1997. p. 199-235. [43] Mariani P, Luzzati V, Delacroix H. Cubic phases of lipid-containing systems: Structure analysis and biological implications. Journal of Molecular Biology. 1988;204:165-89.

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[44] Patel RTJ, Patel TN. Liquid Crystals and Their Application in the Field of Drug Delivery. Colloids in Drug Delivery. Vol. 150: CRC Press; 2010. p. 311-36. [45] Rumiana K, Boris T. Recent Patents on Nonlamellar Liquid Crystalline Lipid Phases in Drug Delivery. Recent Patents on Drug Delivery & Formulation. 2013;7:165-73. [46] Esposito E, Ravani L, Mariani P, Contado C, Drechsler M, Puglia C, et al. Curcumin containing monoolein aqueous dispersions: A preformulative study. Materials Science and Engineering C. 2013;33:4923-34. [47] Puglia C, Cardile V, Panico AM, Crascì L, Offerta A, Caggia S, et al. Evaluation of Monooleine Aqueous Dispersions as Tools for Topical Administration of Curcumin: Characterization, In Vitro and Ex Vivo Studies. Journal of Pharmaceutical Sciences. 2013;102:2349-61. [48] Murgia S, Falchi AM, Meli V, Schillén K, Lippolis V, Monduzzi M, et al. Cubosome formulations stabilized by a dansyl-conjugated block copolymer for possible nanomedicine applications. Colloids and Surfaces B: Biointerfaces. 2015;129:87-94. [49] Linkevičiūtė A, Misiūnas A, Naujalis E, Barauskas J. Preparation and characterization of que rcetinloaded lipid liquid crystalline systems. Colloids and Surfaces B: Biointerfaces. 2015;128:296-303. [50] Bonifácio BV, Silva PBd, Ramos MADS, Negri KMS, Bauab TM, Chorilli M. Nanotechnology -based drug delivery systems and herbal medicines: a review. International journal of nanomedicine. 2014;9:115. [51] Matricardi P, Di Meo C, Coviello T, Hennink WE, Alhaique F. Interpenetrating Polymer Networks polysaccharide hydrogels for drug delivery and tissue engineering. Advanced Drug Delivery Reviews. 2013;65:1172-87. [52] Hoare T, Kohane D. Hoare TR, Kohane DS. Hydrogels in drug delivery: progress and challenges. Polymer 49: 1993-20072008. [53] Sharpe LA, Daily AM, Horava SD, Peppas NA. Therapeutic applications of hydrogels in oral drug delivery. Expert Opinion on Drug Delivery. 2014;11:901-15. [54] Li J, Mooney DJ. Designing hydrogels for controlled drug delivery. Nature Reviews Materials. 2016;1:16071. [55] Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Advanced Drug Delivery Reviews. 2001;53:321-39. [56] Lee KY, Mooney DJ. Hydrogels for Tissue Engineering. Chemical Reviews. 2001;101:1869-80. [57] van der Linden HJ, Herber S, Olthuis W, Bergveld P. Stimulus-sensitive hydrogels and their applications in chemical (micro)analysis. Analyst. 2003;128:325-31. [58] Jen AC, Wake MC, Mikos AG. Review: Hydrogels for cell immobilization. Biotechnology and Bioengineering. 1996;50:357-64. [59] Wang KL, Burban JH, Cussler EL. Hydrogels as separation agents. In: Dušek K, (editor). Responsive Gels: Volume Transitions II. Berlin, Heidelberg: Springer Berlin Heidelberg; 1993. p. 67-79. [60] Bennett SL, Melanson DA, Torchiana DF, Wiseman D, M., Sawhney AS. Next‐Generation HydroGel Films as Tissue Sealants and Adhesion Barriers. Journal of Cardiac Surgery. 2003;18:494-9. [61] Ward K, Cortes JGC, Stuckey D. Alginate as a support ligand for enhanced colloidal liquid aphron immobilization of proteins and drug delivery. Biotechnol Bioeng. 2019;0. [62] Molaei A, Waters KE. Copper ion removal from dilute solutions usi ng colloidal liquid aphrons. Separation and Purification Technology. 2015;152:115-22. [63] Hahm HC, Lee DW, Hong WH, Lee T-y, Lee CH. Predispersed solvent extraction of negatively complexed copper from water using colloidal liquid aphron containing a quate rnary ammonium salt. Korean Journal of Chemical Engineering. 2003;20:716-23. [64] Ward K, Xi J, Stuckey DC. Immobilization of enzymes using non‐ionic colloidal liquid aphrons (CLAs): Activity kinetics, conformation, and energetics. Biotechnology and Bioengineering. 2015;113:970-8.

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List of Tables Table 1: Recent CLA Predispersed Solvent Extraction (PSE) studies.

Anionic/Cationic/Non-ionic

Anionic

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Cationic

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Anionic Anionic

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Anionic

Anionic

Anionic/Non-ionic Anionic/Non-ionic

[62]

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Anionic/Non-ionic

Anionic/Cationic

Ref [26]

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Anionic

PSE Application Ni2+ Extraction from dilute systems Cu2+ Removal from dilute systems Fe3+ removal from NaH2 PO4 Cr3+ extraction from wastewater Erythromycin extraction from broth Phenylalanine Extraction Succinic acid extraction Succinic acid extraction Organic dye removal from wastewater Lactic acid extraction Extraction of negatively complexed copper from water Separation of 2,3butanediol from fermentation broth Mg2+ removal from Ammonium Dihydrogen Phosphate Solution

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Surfactant System Anionic

[27] [29] [31] [24] [32] [33] [34] [35] [63]

[36] [30]

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Journal Pre-proof Table 2: Recent CLA Enzyme immobilization studies.

Anionic/ Cationic/ Nonionic Anionic/ Non-ionic Anionic/Cationic/Non-ionic

Ref

Lipase -Amylase, Galactosidase, Lipase, Lysozyme, Trypsin, Ribonuclease A.

[1] [8]

-Chymotrypsin, Aprotinin, Lipase Bovine Serum Albumin, Ovalbumin, Chymotrypsin, Lysozyme

[64]

[38]

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Non-ionic

Enzyme Immobilized β-galactosidase

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Surfactant System

[9]

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Non-ionic

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Table 3: Formation of polyaphron phases with various solvents differing in functionality [8] [Reprinted with permission].

CH3 CH3 CH2 =CH COO OH CHO CO

0.701 0.701 0.856 -1.251 -1.470 -1.172 -1.643

of

8.8 5.6 5.19 4.9 4 3.68 3.4

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6 mg/ml Yes Yes Yes Yes No Yes Yes

Hydrophobic Fragment constant

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4 mg/ml Yes Yes Yes Yes No Yes Yes

Functional Group

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2 mg/ml Yes Yes Yes Yes Yes Yes Yes

Log P [Po/w]

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Hexadecane Decane Decene Ethyl-decanoate Decanol Decanal 2-Decanone

Stability based on enzyme loading [mg/ml]

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Solvent

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List of Figures

Figure 1: Schematic of a CLA as proposed by Sebba [17]. [Reprinted with permission] Figure 2: Immobilization of Lipase, Aprotinin and -Chymotrypsin as a function of mean polyaphron diameter (fabricated using Tween 20/Tween 80- Mineral oil) and PVR; A) PVR 4, B) PVR 8, C) PVR 10 [Reprinted with permission]. Figure 3: Enhanced mass transfer properties of phenylalanine reactive extraction using Aliquat 336 [24][Reprinted with permission].

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Figure 4: Lysozyme-FITC fluorescence images of polyaphron manufactured at PVR 4. A) Before polyaphron dispersion, B) After polyaphron dispersion, C) After proteolysis. Excitation wavelength 488nm; magnification x63, laser intensity 4.5 %[Reprinted with permission].

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Figure 5: Far-UV Circular Dichroism spectra of Native and Immobilized BSA[9]. [Reprinted with Permission]

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Journal Pre-proof Highlights

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Characterization of CLAs based on surfactant and solvent properties. Role of PVR and its relationship to droplet size and surface area. Applications of CLAs for PSE and Enzyme immobilization. Incorporation of biopolymers within CLA formulations for drug delivery applications.

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   

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