Bile salts-containing vesicles: promising pharmaceutical carriers for oral delivery of poorly...

http://informahealthcare.com/drdISSN: 1071-7544 (print), 1521-0464 (electronic)

Drug Deliv, Early Online: 1–21! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.976892

REVIEW ARTICLE

Bile salts-containing vesicles: promising pharmaceutical carriers for oraldelivery of poorly water-soluble drugs and peptide/protein-basedtherapeutics or vaccines

Mona Hassan Aburahma

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo, Egypt

Abstract

Most of the new drugs, biological therapeutics (proteins/peptides) and vaccines have poorperformance after oral administration due to poor solubility or degradation in thegastrointestinal tract (GIT). Though, vesicular carriers exemplified by liposomes or niosomescan protect the entrapped agent to a certain extent from degradation. Nevertheless, the harshGIT environment exemplified by low pH, presence of bile salts and enzymes limits theircapabilities by destabilizing them. In response to that, more resistant bile salts-containingvesicles (BS-vesicles) were developed by inclusion of bile salts into lipid bilayers constructs. Theeffectiveness of orally administrated BS-vesicles in improving the performance of vesicles hasbeen demonstrated in researches. Yet, these attempts did not gain considerable attention. Thisis the first review that provides a comprehensive overview of utilizing BS-vesicles as a promisingpharmaceutical carrier with a special focus on their successful applications in oral delivery oftherapeutic macromolecules and vaccines. Insights on the possible mechanisms by which BS-vesicles improve the oral bioavailability of the encapsulated drug or immunological response ofentrapped vaccine are explained. In addition, methods adopted to prepare and characterize BS-vesicles are described. Finally, the gap in the scientific researches tackling BS-vesicles thatneeds to be addressed is highlighted.

Keywords

Bile salts, bile salts-containing liposomes,bilosomes, immune response, lipophilicdrugs, proteins and peptides, vesicularcarriers

History

Received 24 September 2014Revised 10 October 2014Accepted 11 October 2014

Introduction

To improve drug–receptor interactions, many new designed

potent drug molecules based on contemporary drug discovery

programs possess high degree of lipophilicity that unfortu-

nately causes poor solubility in GI fluids accompanied by low

and variable bioavailability (Boyd, 2008). Furthermore, the

progress in biotechnological engineering has led to the

evolvement of new biological-based therapeutics and vaccines

that are usually characterized by limited ability to cross cell

membranes accompanied by poor in vivo performance due to

degradation in the gastrointestinal tract (GIT) (Boyd, 2008;

Li et al., 2012). Different strategies that include encapsulating

therapeutic agents or vaccines into nano-colloidal delivery

systems (polymeric or lipid-based nanoparticles, nanoemul-

sions, nanostructured lipid carriers, mixed micelles and

vesicular structures) have been adopted by researchers to

cope with impediments associated with delivering these

challenging agents to specific target tissues (Malik et al.,

2007; Tiwari et al., 2010; Li et al., 2012; Gupta et al., 2013;

Demetzos & Pippa, 2014). Among different classes of

colloidal systems, vesicular carriers have gained particular

attention in delivering poorly soluble drugs and proteins/

peptides (Torchilin, 2005). Vesicular carriers comprise of

unilamellar or multilamellar spherical structures formed of

lipid molecules gathered into bilayers orientation and capable

of encapsulating drug molecules (Sinico & Fadda, 2009).

Conventional vesicular systems exemplified by liposomes and

niosomes have demonstrated an appealing potential in

augmenting the oral bioavailability of therapeutic agents

and immunogenic response of vaccines (Torchilin, 2005;

Bramwell & Perrie, 2006). Nevertheless, the efficacy of

conventional vesicles has been compromised by their instabil-

ity in the GIT, which necessitated modification in their

bilayers constructs to improve their in vivo resistance

(Andrieux et al., 2009). Different research efforts conducted

over the past decade has demonstrated the effectiveness of

including bile salts into vesicular systems bilayers in

improving their in vivo resistance and performance after

oral administration (Chen et al., 2009; Niu et al., 2014).

However, these attempts have not gained considerable atten-

tion in relation to their demonstrated positive outcomes.

Accordingly, the aim of this review is to provide a compre-

hensive overview of the fundamentals of bile salts-containing

vesicles (BS-vesicles) with a focus on their successful

applications in oral delivery of poorly water soluble drugs,

therapeutic proteins/peptides and vaccines. Some insights on

the possible mechanisms by which BS-vesicles improve the

Address for correspondence: Dr. Mona Hassan Aburahma, Phd,Department of Pharmaceutics and Industrial Pharmacy, Faculty ofPharmacy, Cairo, Egypt. Email: [email protected]

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oral bioavailability of the entrapped drug or in case of

vaccine delivery, protect the antigen from the hostile GIT

environment are vividly explained and diagrammatically

illustrated. In addition, different methods adopted to prepare

and characterize BS-vesicles are described. Finally, this

article concludes by highlighting the gap in scientific

researches tackling BS-vesicles that needs to be addressed

in the near future.

Bile salts in drug delivery systems

Bile salts, ionized form of bile acids, are distinctive amphi-

pathic molecules that consist of a steroid nucleus with a

hydrophilic side encompassing hydroxyl groups (concave

a-side) and a hydrophobic side containing methyl groups

(convex b-side) (Holm et al., 2013). This makes bile salts

dissimilar from traditional pharmaceutical surfactants that are

formed of a hydrophilic polar head group and a long

hydrophobic tail group (Holm et al., 2013). The chemical

structure of bile salts commonly used in BS-vesicles and a

representation of the hydrophilic and hydrophobic sides of

bile salts are depicted in Figure 1.

Bile salts and acids play a major role in emulsifying and

solubilizing dietary fats through the formation of mixed

micelles (Holm et al., 2013). In the latter, bile salts are

compressed between the phospholipids (PL) polar heads

with their hydrophilic sides facing the aqueous medium

(Hofmann & Hagey, 2008). The ability of bile salts to

improve the passage of lipophilic drug molecules across

biological membranes and thus augment their oral bioavail-

ability has been demonstrated for many drug candidates

(Mikov et al., 2006). It was reported that the absorption of

sulfadiazine and indomethacin increased when bile secretion

was simulated (Nightingale et al., 1971; Miyazaki et al.,

1981). Taking advantage of the aforementioned property,

various mixed micelles systems based on bile acids and salts

were successfully employed for improving the solubility of

extremely lipophilic drugs. Mixed micelles composed of

sodium cholate with PL, linoleic acid and lecithin were

found to improve the solubility and intestinal absorption of

silybin (Yu et al., 2010), clofazimine (O’Reilly et al., 1994)

and Cyclosporine A (Guo et al., 2005), respectively.

Furthermore, the applicability of bile salts in the preparation

of nanoparticulate systems was demonstrated in different

researches. Zhang et al. (2013) reported that the incorpor-

ation of sodium cholate improved the intestinal absorption

of candesartan from cilexetil-loaded lipid nanocarriers. Liu

et al. (2007) employed sodium cholate in solid lipid

nanoparticles to improve the lipo-solubility of insulin.

Zeng et al. (2013) attempted to functionalize nanoparticles

with cholic acid to improve docetaxel delivery to cervical

cancer. Furthermore, deoxycholic acid was used to augment

the oral bioavailability of biodegradable nanoparticles

(Samstein et al., 2008) and to impart negative charge to

nanoemulsions designed for enhancing the oral bioavailabil-

ity of paclitaxel (Tiwari & Amiji, 2006). Significant

improvement in the bioavailability of itraconazole from

pegylated bile acids self-emulsifying system was reported by

Le Devedec et al. (2013). On the other hand, transdermally,

bile salts were employed as potent penetration enhancers for

topically administrated drugs due to the membrane destabi-

lizing activity of these agents. Sodium cholate and combin-

ations of taurocholate and glycholate were found to enhance

the percutaneous penetration of indomethacin (Chiang et al.,

1991) and elcatonin (Ogiso et al., 1991), respectively.

Figure 1. (A) Chemical structure of bile salts commonly used in bile salts-containing vesicles and (B) an illustration depicting the hydrophilic side andhydrophobic side of a bile salt molecule.

2 M. H. Aburahma Drug Deliv, Early Online: 1–21

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In addition to that, the applicability of utilizing bile salts as

nasal absorption promoters was investigated. Chavanpatil &

Vavia (2005) confirmed the effectivity of sodium deoxycho-

late (SDC) in enhancing the nasal absorption of sumatriptan

succinate. Different research groups have utilized bile salts/

fatty acids mixed micelles to promoting nasal absorption

insulin (Tengamnuay & Mitra, 1990), acyclovir (Shao &

Mitra, 1994) and gentamicin (Duchateau et al., 1986).

Likewise, the influence of bile salts on conjunctival

penetration of drug molecules have been highlighted.

Morimoto et al. (1987) demonstrated the effectiveness of

sodium taurodeoxycholate (STDC) in increasing the ocular

adsorption of hydrophilic compounds. Regarding the buccal

route of administration, sodium glycocholate (SGC) was

capable of enhancing the buccal absorption of the ionized

form of flecainide (Deneer et al., 2002). Furthermore,

Mahalingam el al. (2007) reported that sodium deoxygly-

cocholate and SGC increased the buccal permeation of the

decitabine about 40-fold. Regarding the inhaled delivery

systems, sodium taurocholate (STC) was added to inhaled

itraconazole dry nanoparticles powders to minimize the

irreversible aggregation of the nanoparticles inducing by

spray-drying. Recently in 2014, Gangadhar et al. (2014)

synthesized SDC sulfate from deoxycholic acid to formulate

lipid-based Amphotericin B nanoparticles with reduced

toxicity for the treatment of lung fungal infections via

inhalation.

In summary, the aforementioned studies presented various

benefits associated with including bile salts in drug delivery

systems administrated through oral, topical, transmucosal and

inhalation routes.

Vesicular carriers

Pharmaceutical vesicular carriers are composed of water-filled

colloidal unilamellar or multilamellar vesicles comprising

lipid amphiphilic molecules arranged in a bilayers conform-

ation (Honeywell-Nguyen & Bouwstra, 2005; Torchilin, 2005;

Sinico & Fadda, 2009). Hydrophilic drugs are encapsulated

within the internal aqueous compartment of the vesicles,

whereas hydrophobic drugs are associated with the hydropho-

bic lipid bilayers (Honeywell-Nguyen & Bouwstra, 2005;

Torchilin, 2005). Broadly, vesicular carriers are divided into

two main classes: liposomes, which represent the ground-

breaking discovery of Alec Bangham in the 1960s and are

composed mainly of phosphatidylcholine (PC) and cholesterol

(CH) bilayers, and niosomes that were revealed later in 1970,

by L’Oreal, and denote non-ionic surfactant vesicles

(Torchilin, 2005). Following that, newer forms of lipid vesicles

were presented by various researchers and includes tranfero-

somes (Bragagni et al., 2012), emulsomes (Vyas et al., 2006),

enzymosomes (Gaspar et al., 2003), ethosomes (Pandey et al.,

2014) and pharmacosomes (Semalty et al., 2009).

In this review, the term ‘‘conventional liposomes’’ is used

to describe traditional PL- and CH-based liposomes, whereas

the term ‘‘conventional vesicles’’ is employed to collectively

denote traditional liposomes and niosomes. In general, any

changes undertaken in the composition of the vesicles’

bilayers influence their physicochemical characteristics that

include size, charge, bilayer elasticity and stability, which

simultaneously impact their in vivo behavior and effectiveness

as pharmaceutical carriers (Sinico & Fadda, 2009; Torchilin,

2005).

Though, oral vesicular carriers have been widely used to

improve drugs bioavailability, yet, the pH gradient, enzymes

and physiological bile salts can easily cause vesicles’

destabilization (Martins et al., 2007). Nevertheless, the

extent to which the vesicular carriers preserve their structural

integrity after oral administration during transfer through the

GIT is extremely crucial to ensuring effective protection of

the entrapped therapeutic agent or antigen in case of vaccine

immunization (Martins et al., 2007; Neutra & Kozlowski,

2006).

Vesicles destruction in GIT

The ability of bile salts to interrupt lipid bilayers by

intercalating amongst the PL and forming mixed micelles is

well demonstrated (Andrieux et al., 2009). It is hypothesized

that upon reaching the small intestine and exposing to bile

salts, conventional vesicles are effectively destroyed due to

inclusion of bile salts monomers into the lipid bilayers of the

vesicles (Schubert et al., 1983; Wilkhu et al., 2013). Further

increase in bile salt concentration induces vesicle–micelle

transition (Andrieux et al., 2009) followed by formation of

new aggregates. The latters have served as vehicles for

enhancing the absorption of poorly water-soluble drug

molecules in the GIT (Nightingale et al., 1971; Miyazaki

et al., 1981).

Based on the research published by Subuddhi & Mishra

(2007), vesicles solubilization by bile salts occurs in three

stages depending on bile salt concentrations. At low bile salts

concentration, the surfactants partition into the lipid bilayer.

When the concentration of bile salts increase, vesicles

and micelles co-exist and the bilayer permeability for the

bile salts intensifies allowing more bile salts to associate with

the bilayer converting them into mixed micelles. In the last

stage, surfactants are at their CMC, disrupting the vesicles’

bilayers completely and solubilizing them into mixed

micelles.

BS-vesicles

Conventional vesicular carriers can protect peptides and

proteins to a certain extent from degradation in the GIT

(Bramwell & Perrie, 2006). Yet, the existence of intestinal

bile salts limit the capabilities of these carriers by cause

membrane deformation and vesicles lysis resulting in the

release of entrapped macromolecules from the vesicle

preceding reaching its planned site of action (Wilkhu et al.,

2013). In response to this, modified stabilized vesicles were

developed by inclusion of bile salts into the lipid bilayers

making them more resistant against bile salts disruption in the

GIT. The stability imposed by BS-vesicles was attributed to

the repulsion between the bile salts already present in the

vesicle bilayer and the intestinal bile salts in the GIT

(Conacher et al., 2001). Hence, providing protection for the

entrapped agents from the hostile environment in the GIT

(Beg et al., 2013; Sahdev et al., 2014) (Figure 2). Different

ingredients utilized in the preparation of BS-vesicles are

collectively listed in Table 1.

DOI: 10.3109/10717544.2014.976892 Bile salts-containing vesicles 3

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Bile salts-containing liposomes (BS-liposomes)

Many efforts have been undertaken to increase the stability

and integrity of liposomes in the GIT either by coating the

vesicles with polymers, such as chitosan, polyethylene glycol

and pectin (Immordino et al., 2006; Nguyen et al., 2011;

Parmentier et al., 2011; Kowapradit et al., 2012), or by

inclusion of tetraether lipids or gangliosides within their

structures (Torchilin, 2005). Recently, BS-liposomes have

been investigated as stable carriers for delivering sensitive

therapeutic agents and proteins by replacing a fraction of the

CH in the lipid bilayers with bile salts (Guan et al., 2011; Niu

et al., 2011, 2012, 2014). Schematic illustrations of different

types of liposomes comprising bile salts in comparison to

conventional liposome are presented in Figure 3. Several

studies have demonstrated the superiority of BS-liposomes to

conventional liposomes in improving the oral absorption of

different therapeutic agents and proteins (Chen et al., 2009;

Niu et al., 2011). Although the exact underlying mechanisms

of oral absorption enhancement of drugs or proteins loaded in

BS-liposomes have not been comprehensively elucidated,

several authors acknowledged the capability of intact BS-

liposomes to permeate across the unstirred water layer

followed by uptake of the vesicles by membranous epithelial

cells (M-cells) in the Payer’s patch (Aramaki et al., 1993) and

transcellular through enterocytes (Niu et al., 2014) in addition

to the facilitated transport through the paracellular pathway

(Chen et al., 2009; Niu et al., 2012). Furthermore, it is

expected that upon contact with the intestinal epithelial, the

included bile salts may increase the membrane fluidity

followed by membrane-destabilizing effect, which facilitate

internalization of the vesicles (Chen et al., 2009). Chen et al.

(2009) related the ultra-deformability of liposomes due to the

inclusion of bile salts to the enhanced carrier-mediated

transmembrane absorption. Furthermore, BS-liposomes can

freely convert into mixed micelles in the GI environment and

hence enhance transmembrane drug absorption (Chen et al.,

2009). Using imaging method, Niu et al. (2014) demonstrated

that the in vivo residence time of oral administrated BS-

liposomes in the GIT was longer than its conventional

counterpart. The transenterocytic internalization of BS-lipo-

somes in Caco-2 cells was confirmed using fluorescence

imaging (Niu et al., 2014). The contribution of paracellular

permeability to oral absorption from BS-liposomes was

predicted by measurement of transepithelial electrical resist-

ance (TEER) (Niu et al., 2014). The TEER of Caco-2 cell

monolayers is significantly reduced when tight junctions are

opened. BS-liposomes significantly reduction the TEER

Figure 2. A schematic representation of the behavior of conventional liposome and niosome in comparison to bilosomes in presence of intestinal bilesalts. (A) Disruption and lysis of conventional vesicle by intestinal bile salts and (B) The bilosome remains stable in presence of intestinal bile salts(redrawn from Henriksen-Lacey & Perrie, 2013).

Table 1. Commonly used lipids, surfactants andbile salts for preparing BS-vesicles.

Ingredients

LipidsCholesterol (CH)Dicetyl phosphate (DCP)Soybean phosphatidyl choline (SPC)Monopalmitoyl glycerol (MPG)Dipalmitoyl phosphatidylethanolamine (DPPE)Distearyl phosphatidyl ethanolamine (DSPE)

SurfactantsSorbitan tristearate (STS)Sorbitan monooleate (SMO)

Bile saltsSodium glycocholate (SGC)Sodium taurocholate (STC)Sodium deoxycholate (SDC)Sodium taurodeoxycholate (STDC)

Abbreviations of ingredients are present betweenparentheses.


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compared to conventional liposomes, indicating better para-

cellular permeability by reversibly opening the epithelial tight

junctions (Ward et al., 2000). Schematic illustration of the

mechanisms of drug absorption via BS-liposomes is presented

in Figure 4.

Bile salts-containing niosomes (bilosomes)

Bilosomes, first described by Conacher et al. (2001), are

closed bilayer structures of non-ionic amphiphiles closely

correlated to non-ionic surfactant vesicles (niosomes) but

incorporating bile salts. Several research groups have

demonstrated the potential of utilizing bilosomes in facilitat-

ing successful oral vaccine delivery (Mann et al., 2006, 2009;

Shukla et al., 2008, 2010a,b, 2011). Schematic illustrations of

different types of bilosomes are depicted in Figure 5. The

mechanisms of oral immunization that take place in the

intestinal mucosa differ from that of the oral bioavailability

enhancement of protein drugs loaded in BS-liposomes. In the

GIT, a single layer of epithelial cells, linked by tight junctions

and containing gut-associated lymphoid tissues (GALT), face

the complex intestinal luminal environment (McGhee et al.,

1992; Neutra & Kozlowski, 2006). Orally administrated

vaccines face defense mechanisms in GIT similar to that

imposed on microbial pathogens. The vaccines are diluted and

arrested in the mucus gels, denaturated by enzymes and

excluded by epithelial barriers (Neutra & Kozlowski, 2006;

Jain et al., 2011). Feeble immune responses associated with

oral vaccines arise due to inadequate absorption of antigens

by the Peyer’s patches (Neutra & Kozlowski, 2006; Jain et al.,

2011), which nessicates the administration of relatively large

doses of vaccine. Bilosomes address these challenges by

offering several advantages for oral vaccine delivery. It is

postulated that bilosomes protect the vaccine antigen enclosed

within the lipid bilayer from premature release and/or

degradation in the hostile environment of GI tract (Mann

et al., 2006, 2009; Shukla et al., 2008, 2010a,b, 2011). Using

radiolabeled I-125 insulin entrapped in bilosomes, Mann et al.

(2006) confirmed the aforementioned and proved that

Figure 3. Structural composition of different types of liposomes. (A) Conventional liposome, (B) Bile salts-containing liposome and (C) Bile saltscoated liposome.

Figure 4. Schematic representation of the uptake of bile salts liposomesby intestinal epithelium after oral administration. (A) Uptake of thevesicles by M-cells in the Payer’s patch, (B) Transport of the vesiclesthrough the paracellular pathway and (C) Transcellular uptake ofvesicles through the enterocytes.


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bilosomes are stable in the stomach and intestine. Induction of

mucosal immune responses require efficient uptake of antigen

by GALT that consist primarily of Peyer’s patches overlying

M-cells present between the mucosal epithelial and intrae-

pithelial dendritic cells (DCs). The M-cell take up the

bilosomes by endocytosis and transport them to the antigen-

presenting cells (APCs), such as macrophages and DCs, that

present the antigen to B or T cell initiating antigen-specific

mucosal and systemic immune responses (McGhee et al.,

1992; Neutra & Kozlowski, 2006; Jain et al., 2011). In this

context, fluorescence microscopy studies conducted by

Shukla et al. (2011, 2008) established effective uptake of

bilosomes by the M-Cells, thereby confirming the capability

of bilosomes to transport the vaccine antigens to Peyer’s

patches.

Additional mechanisms, such as direct intracellular deliv-

ery of antigens through phagocytosis of particles have also

been proposed to explain the potent immunological adjuvant

activity of bilosomes (Mann et al., 2009). Schematic illustra-

tion of the mechanisms of immune response associated with

bilosomes is presented in Figure 6.

Modified forms of bile salts vesicles (BS-vesicles)

Modified versions of BS-vesicles via anchoring specific

ligands to the vesicles surface were endeavored by several

research groups to enhance their uptake by certain target cells

(Figure 3C, 5B and C) (Singh et al., 2004; Shukla et al.,

2010a; Jain et al., 2014a,b).

Cholera toxin B-modified bilosomes. The adjuvanticity of

cholera toxin B subunit (CTB) is related to their affinity to

Glucomannan-modified (GM)1 ganglioside receptors on M-

cells (Dertzbaugh & Elson, 1993). To accomplish efficient

systemic and mucosal antibody responses following oral

administration, CTB was conjugated to the surface of

bilosomes to permit them to specifically bind to GM1

ganglioside receptors on M-cells membrane of Peyer’s

patches (Singh et al., 2004; Shukla et al., 2010a). The

Figure 5. Structural composition of different types of bilosomes. (A) Conventional bilosome, (B) Glucomannan bilosome and (C) Cholera toxinB-bilosome.

Figure 6. Schematic representation of the uptake of bilosomes contain-ing antigen through the intestinal epithelium after oral administration.(A) Transcellular uptake of bilosomes through the enterocytes, (B)Transport of the bilosomes through paracellular pathway and (C) Uptakeof bilosomes by M-cells and dendritic cells.


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superiority of CTB-conjugated bilosomes in terms of effective

localization in the M-cells of the GALT compared to non-

conjugated bilosomes was verified using fluorescence micros-

copy (Shukla et al., 2010a).

GM bilosomes. To increase the stability of bilosomes

and enhance their uptake through the mannosyl receptors

of APCs, Jain et al. (2014a,b) decorated the bilosomes

surface with glucomannan (GM). Results confirmed

that GM-bilosomes were stable against digestive enzymes

due the polymeric nature of the ligand. Furthermore,

better immune response was elicited after oral administra-

tion of GM-bilosomes compared to conventional bilo-

somes. This was related to the existence of high density

of mannose molecules over the bilosomes surface that

resulted in more precise recognition and binding of

bilosomes to mannose receptors, thereby, enhance uptake

through APCs.

Bile salt-coated liposomes (BSC-liposomes). Bile salts are

taken from the blood stream into the hepatocytes with the aid

of a well-defined transport system. Putz et al. (2005) took

advantage of this property and examined the capability of bile

salts in serving as ligands to target liposomes to the

hepatocytes. For that purpose, the bile salt lithocholyltaurine

was covalently linked with PL moiety. The formed bile salt-

coated liposomes (BSC-liposomes) having the covalently

linked bile salt–lipid conjugate showed hepatocyte-specific

targeting, thereby opened promising possibilities for hepato-

cyte-specific drug delivery.

Preparation of BS-vesicles

Based on the reviewed literature, BS-vesicles were prepared

using different methods collectively presented in Tables 2–4.

Brief overview of these methods are presented hereafter and

schematically illustrated in Figure 7.

Reverse-phase evaporation method

Reverse-phase evaporation method was used to formulate BS-

liposomes loaded with insulin (Niu et al., 2011, 2012, 2014)

or hexamethylmelamine (Sun et al., 2010). In this approach,

soybean phosphatidylcholine (SPC) and bile salts were

dissolved in ethyl ether to which buffered solution containing

insulin was added followed by ultra-sonication to form reverse

w/o emulsion (Niu et al., 2011, 2012, 2014). The solvent was

then removed from the emulsion using a rota-evaporator

under reduced pressure. The dried lipids formed were

eventually hydrated by buffer to form homogenous aqueous

vesicles dispersion that was extruded through a high pressure

homogenizer to obtain BS-liposomes entrapping protein. For

loading the poorly water-soluble drug, hexamethylmelamine,

it was dissolved with the lipid components in the organic

solvent in first step.

Thin film hydration method

This method was adopted to prepare both BS-liposomes and

bilosomes. For preparing drug-loaded BS-liposomes, the drug

and SPC were dissolved in organic solvent, which was then

evaporated in a rota-evaporator. The formed thin film of SPC

on the wall of the flask was hydrated with buffered bile salt

solution and then rotated to obtain a crude dispersion of

multilamellar liposomes. The latter was then sonicated

followed by homogenization to obtain small unilamellar

structure of BS-liposomes (Chen et al., 2009). Slight differ-

ences in the preparation steps were found between different

research groups where, in some cases, all the components of

liposomes namely, SPC, bile salts and drug, were initially

dissolved in the organic solvent, then followed the same

aforementioned procedures (Guan et al., 2011; Dai et al.,

2013).

For preparing antigen-loaded bilosomes using thin-film

hydration method, the lipid components, surfactant, CH and

dicetylphosphate (DCP) were dissolved in an organic solvent

in a round-bottomed flask followed by solvent evaporation

under reduced pressure using rota-evaporator. The thin film

formed on the glass wall of the round-bottomed flask was

then hydrated with buffer containing the bile salt and the

antigen to form large multilamellar vesicles that were

transformed to small unilamellar vesicles by ultrasonication

(Dai et al., 2013) or extrusion (Shukla et al., 2008, 2011).

Thin film hydration method was used to prepare bilosomes

loaded with diphtheria toxoid (DTx) (Shukla et al., 2011),

hepatitis B surface antigen (HBsAg) (Shukla et al., 2011) and

bovine serum albumin (BSA) (Jain et al., 2014b).

Hot homogenization method

For preparing bilosomes using this approach, the lipid

components, namely, monopalmitoyl glycerol, CH and DCP

were melted at 140 �C for 5 min and subsequently hydrated

with buffered solution. The lipid mixture was then homo-

genized followed by the addition of bile salt solution to form

dispersion containing empty vesicles that was homogenized.

Thereafter, the antigen buffered solution was added to the

homogenate, and protein entrapment was achieved by further

homogenization (Gebril et al., 2014) or several freeze–thaw

cycles (Mann et al., 2009). This method was used to entrap

influenza A antigen (Gebril et al., 2014) and gonadotropin-

releasing hormone (GnRH) immunogens (Gebril et al., 2014)

to bilosomes. It is worth mentioning that the antigen was

added at the final stage to minimize long exposure to

homogenization (Wilkhu et al., 2013).

Proniosomal method

Preparation of bile salts-containing proniosomes was

initiated one decade ago by Song et al. (2005) to facilitate

handling of liposomes and improve their long-term stability

by converting them to powder form. In this method, the

carrier, usually sorbitol particles, was located in a round-

bottomed flask and vacuum dried on the rota-evaporator.

Then, solution of PC, bile salt and drug dissolved in organic

solvent were added drop wise into the flask to load them

onto sorbitol particles. Following that, the loaded sorbitol

particles were freeze-dried to confirm the complete evapor-

ation of any organic solvents residues. The resultant

proliposomal powder was converted to liposomes following

manual agitation in water.


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Tab

le2

.F

orm

ula

tio

nd

etai

ls,

pre

par

atio

nm

eth

od

s,an

dves

icle

sch

arac

teri

stic

so

fb

ile

salt

-co

nta

inin

gli

po

som

es(B

S-l

ipo

som

es).

Ves

icu

lar

carr

ier

Co

mp

osi

tio

n(m

ol/

mo

l)D

rug

/nat

ure

Pre

par

atio

nm

eth

od

Ves

icle

size

red

uct

ion

PS

,P

I,Z

P,

EE

%R

efer

ence

s

BS

-lip

oso

mes

SP

C:S

DC

(4:1

)F

eno

fib

rate

/P

oo

rly

wat

erso

lub

led

rug

Th

infi

lmhy

dra

tio

nm

eth

od

So

nic

atio

nfo

llow

edby

hig

h-p

ress

ure

ho

mo

gen

izat

ion

PS&

15

9n

mE

E%&

88

.23

%C

hen

etal

.,2

00

9

BS

-lip

oso

mes

SP

C:S

DC

(3:1

)C

ycl

osp

ori

ne

A/

po

orl

yw

ater

solu

ble

dru

gT

hin

film

hy

dra

tio

nm

eth

od

Hig

h-p

ress

ure

ho

mo

gen

izat

ion

PS¼

85

.6±

1.0

nm

PI¼

0.0

64

±0

.01

3Z

P¼�

35

.2±

4.4

2m

VE

E%¼

94

.05

±2

.76

%

Gu

anet

al.,

20

11

BS

-lip

oso

mes

SP

C:S

TC

(5:1

)T

acro

lim

us/

Po

orl

yw

ater

solu

bil

ity

dru

gT

hin

film

hy

dra

tio

nm

eth

od

Ult

raso

nic

atio

nP

S¼

97

.3±

1.6

nm

PI¼

0.2

67

±0

.03

3Z

P¼

25

.2±

3.8

mV

EE

%¼

95

.15

±1

.91

%

Dai

etal

.,2

01

3

BS

-lip

oso

mes

PC

:SD

C(2

:1)

Hex

amet

hylm

elam

ine/

Lip

op

hil

icd

rug

Rev

erse

ph

ase

evap

or-

atio

nm

eth

od

PS¼

22

9±

20

nm

PI¼

0.3

54

±0

.07

0E

E%¼

43

.85

±1

.87

%

Su

net

al.,

20

10

BS

-lip

oso

mes

SP

C:S

GC

(4:1

)P

orc

ine

insu

lin

/P

rote

ind

rug

Rev

erse

ph

ase

evap

or-

atio

nm

eth

od

Hig

h-p

ress

ure

ho

mo

gen

izat

ion

PS¼

17

8.4

±6

.2n

mP

I¼

0.3

46

±0

.02

2Z

P¼�

51

.3±

0.8

mV

EE

%¼

30

.23

±0

.84

%

Hu

etal

.,2

01

3

BS

-lip

oso

mes

SP

C:S

GC

(4:1

)R

eco

mb

inan

th

um

anin

suli

n/

Pro

tein

dru

gR

ever

sep

has

eev

apo

r-at

ion

met

ho

dH

igh

-pre

ssu

reh

om

ogen

izat

ion

PS¼

15

4±

18

nm

EE

%¼

30

±2

%N

iuet

al.,

20

11

BS

-lip

oso

mes

SP

C:b

ile

salt

s(S

GC

/ST

C/S

DC

)(4

:1)

Rec

om

bin

ant

hu

man

insu

lin

/P

rote

ind

rug

Rev

erse

ph

ase

evap

or-

atio

nm

eth

od

Hig

h-p

ress

ure

ho

mo

gen

izat

ion

PS&

15

0n

mE

E%&

30

%N

iuet

al.,

20

14

Pro

-BS

-lip

oso

mes

PC

:ST

DC

(4:1

)L

oad

edo

nso

rbit

ol

(4g

)S

alm

on

calc

ito

nin

/P

epti

de

dru

gP

roli

po

som

alm

eth

od

;lo

adin

gli

po

som

eso

np

oro

us

carr

ier

PS¼

74

1±

76

.9n

mE

E%¼

54

.9±

4.1

%S

on

get

al.,

20

05

PS

,p

arti

cle

size

;P

I,p

oly

dis

per

sity

ind

ex;

ZP

,ze

tap

ote

nti

al;

and

EE

%,

entr

apm

ent

effi

cien

cyp

erce

nt.


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Tab

le3

.F

orm

ula

tio

nd

etai

ls,

pre

par

atio

nm

eth

od

san

dves

icle

sch

arac

teri

stic

so

fb

ilo

som

es.

Ves

icu

lar

carr

ier

Co

mp

osi

tio

n(m

ol/

mo

l)A

nti

gen

Pre

par

atio

nm

eth

od

Ves

icle

size

red

uct

ion

*P

S,

PI,

ZP,

EE

%R

efer

ence

Bil

oso

mes

ST

S:C

H:D

CP

(7:3

:1),

10

0m

go

fS

DC

Dip

hth

eria

toxo

id(D

Tx

)T

hin

film

hy

dra

tio

nm

eth

od

Ex

tru

sio

nth

rou

gh

20

0n

mp

ore

mem

bra

ne

PS¼

20

6±

20

nm

EE

%¼

20

–2

4%

Sh

uk

laet

al.,

20

11

Bil

oso

mes

ST

S:C

H:D

CP

(7:3

:1),

10

0m

go

fS

DC

Rec

om

bin

ant

bac

ulo

vir

us-

VP

1(B

ac-V

P1

)T

hin

film

hy

dra

tio

nm

eth

od

Ho

mo

gen

izat

ion

EE

%¼

50

%n

mP

rem

anan

det

al.,

20

13

Bil

oso

mes

ST

S:C

H:D

CP

(7:3

:1),

10

0m

go

fS

DC

Hep

atit

isB

An

tigen

(HB

sAg

)T

hin

film

hy

dra

tio

nm

eth

od

Ex

tru

sio

nth

rou

gh

20

0n

mp

ore

mem

bra

ne

PS¼

20

4±

18

nm

EE

%¼

18

–2

2%

Sh

uk

laet

al.,

20

08

Bil

oso

mes

MP

G:C

H:D

CP

(5:4

:1),

10

0m

go

fS

DC

BS

Aan

dm

easl

esp

epti

de

Th

infi

lmhy

dra

tio

nm

eth

od

Co

nac

her

etal

.,2

00

1

Infl

uen

zaA

anti

gen

(H3

N2

sub

-un

itp

rote

in)

PS¼

3mm

Bil

oso

mes

MP

G:C

H:

DC

P(5

:4:1

),1

00

mg

of

SD

CT

etan

us

toxo

id(T

Tx

)T

hin

film

hy

dra

tio

nm

eth

od

Man

net

al.,

20

06

Bil

oso

mes

MP

G:C

H:D

CP

(5:4

:1),

10

0m

go

fS

DC

Infl

uen

zaA

anti

gen

Ho

th

om

ogen

izat

ion

met

ho

dP

S¼

25

0n

mE

E%¼

50

%M

ann

etal

.,2

00

9

Th

infi

lmhy

dra

tio

nm

eth

od

PS¼

98

0n

mE

E%¼

50

%

Bil

oso

mes

MP

G:C

H:D

CP

(5:4

:1)

10

0m

Mo

fb

ile

salt

Rec

om

bin

ant

infl

uen

zaan

tigen

(HA

or

H3

N2

sub

-un

itp

rote

in)

Ho

th

om

ogen

izat

ion

met

ho

dP

S¼

6.4

4±

0.0

5mm

ZP¼�

12

5m

VE

E%¼

32

%

Wil

kh

uet

al.,

20

13

Bil

oso

mes

MP

G:C

H:D

CP

(5:4

:1)

10

0m

Mo

fS

DC

Go

nad

otr

op

in-r

elea

sin

gh

or-

mo

ne

(Gn

RH

)im

mu

no

gen

Ho

th

om

ogen

izat

ion

met

ho

dH

om

ogen

izat

ion

PS¼

13

0±

14

nm

PI¼

0.2

5Z

P¼�

34

±2

mV

EE

%¼

35

±2

%

Geb

ril

etal

.,2

01

4

*P

S:

par

ticl

esi

ze;

PI,

po

lyd

isp

ersi

tyin

dex

;Z

P:

zeta

po

ten

tial

;an

dE

E%

,en

trap

men

tef

fici

ency

per

cen

t.


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Figure 7. Representation of different methods adopted for preparation of bile salts-containing vesicles. (A) Reverse phase evaporation method, (B)Thin film hydration method and (C) Hot homogenization method.

Table 4. Formulation details, preparation methods and vesicles characteristics of modified bilosomes.

Vesicular carrier Composition AntigenPreparation

methodVesicle size

reduction *PS, PI, ZP, EE% References

CTB- bilosomes STS:CH:modifiedDPPE (7:3:1);100 mg of SDC,Conjugation ofCTB to bilosomes

Hepatitis B sur-face Antigen(HBsAg)

Thin film hydra-tion method

Extrusionthrough200 nm poremembrane

PS¼ 202 ± 20 nmEE%¼ 20–24%

Shukla et al., 2010b

CTB- bilosomes STS:CH:modifiedDPPE (7:3:1)20 mg SDC,Conjugation ofCTB to bilosomes

Bovine serumalbumin(BSA)


Extrusionthrough400 nm then100 nm poremembrane

PS¼ 108.6 ± 11.4 nmEE%¼ 28.2 ± 3.4%

Singh et al., 2004

GM-bilosomes SMO:CH:SDC(2:1:0.1)GM-OCM-DSPE(10% w/w of totallipid content)

Bovine serumalbumin(BSA)


Sonication PS¼ 157 ± 3 nmPI¼ 0.287 ± 0.045ZP¼�21.8 ± 2.01 mVEE%¼ 71.3 ± 4.3%

Jain et al., 2014b

GM-bilosomes SMO:CH:SDC(2:1:0.1)GM-OCM-DSPE(10% w/w of totallipid content)

Tetanus toxoid(TTx)


Sonication PS¼ 164 ± 10 nmPI¼ 0.258 ± 0.041ZP¼�17.9 ± 2.8 mVEE¼ 75.8 ± 3.3%

Jain et al., 2014a

*PS: particle size; PI, polydispersity index; ZP, zeta potential; and EE%: entrapment efficiency percent.


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In vitro characterizations and variables influencingBS-vesicles

The in vitro characterizations of BS-vesicles included deter-

mination of the vesicle size, polydispersity index (PI), zeta

potential, vesicles morphology, entrapment efficiency, in vitro

release and cellular uptake studies. The characterization

techniques identified in the published literature are listed in

Table 5. A brief description of the characterization techniques

and variables influencing some properties of BS-vesicles are

provided hereafter.

Particle size, PI, morphology and zeta potential

Particle size of BS-vesicles exerts substantial impact on their

in vitro and in vivo performances (Sun et al., 2008; Mann

et al., 2009). Analytical instruments constructed on dynamic

light scattering principle basis, i.e. photon correlation spec-

troscopy, were used to estimate the particle size and PI of the

BS-vesicles dispersions (Chen et al., 2009; Dai et al., 2013).

The vesicle size of BS-vehicles ranged from 97.3 ± 1.6 nm

(Dai et al., 2013) to around 3 mm (Conacher et al., 2001).

Based on the published literature, several variables affect

the particle size and PI of BS-vesicles. These variables

include bile salts content, pH of the hydration buffer,

homogenization (Chen et al., 2009) or ultrasonication condi-

tions adopted during vesicles preparation (Dai et al., 2013).

Regarding the influence of bile salts content, substantial

decrease in particle size and PI of BS-vesicles was associated

with the increase in bile salt contents due to the enhanced

flexibility and decrease in surface tension of the vesicles

(Chen et al., 2009; Guan et al., 2011; Wilkhu et al., 2013; Dai

et al., 2013). However, further increase in bile salts content

caused vesicles enlargement bound with increase in PI due to

the increase in medium viscosity (Guan et al., 2011). The pH

of the hydration medium also influences the particle size of

the vesicles due to the ionization of bile salts at higher pH

which in turn increase its surface activity (Chen et al., 2009).

Likewise, different processing variables influenced the ves-

icles particle size. The multilamellar micron-size BS-vesicles

produced by thin film dispersion method were converted into

small unilamellar nano-sized vesicles using extrusion through

membrane (Shukla et al., 2008, 2011), ultrasonication (Dai

et al., 2013) or homogenization (Guan et al., 2011). It was

Table 5. Major methods for BS-vesicles characterizations.

Characteristics Methodology References

Particle size Dynamic light scattering instrument Conacher et al., 2001; Shukla et al., 2008; Chen et al.,2009; Mann et al., 2009; Shukla et al., 2010a; Sunet al., 2010; Guan et al., 2011; Niu et al., 2011, 2012,2014; Dai et al., 2013; Gebril et al., 2014; Jain et al.,2014a,b

Laser diffraction particle size analyzer Song et al., 2005; Wilkhu et al., 2013

Polydispersty index Dynamic light scattering instrument Sun et al., 2010; Jain et al., 2014a

Zeta potential Dynamic light scattering instrument Dai et al., 2013; Wilkhu et al., 2013; Gebril et al., 2014;Jain et al., 2014a,b

Electrophoretic mobility (EPM) measurements

Morphology Transmission electron microscopy (TEM) Shukla et al., 2008; Mann et al., 2009; Shukla et al.,2010a; Guan et al., 2011; Niu et al., 2011; Shuklaet al., 2011; Dai et al., 2013; Premanand et al., 2013;Jain et al., 2014a,b

Cryogenic-transmission electron microscopy(Cryo-TEM)

Chen et al., 2009

Scanning electron microscopy (SEM) Gebril et al., 2014Freeze fracture electron microscopy (FFEM) Mann et al., 2006

Separation of free drug fromdrug-loaded vesicles

Molecular exclusion chromatography Singh et al., 2004; Shukla et al., 2008, 2011; Chen et al.,2009; Sun et al., 2010; Guan et al., 2011; Niu et al.,2011, 2012, 2014; Dai et al., 2013; Hu et al., 2013;Jain et al., 2014a,b

Ultracentrifugation Conacher et al., 2001; Song et al., 2005; Mann et al.,2009; Premanand et al., 2013; Gebril et al., 2014

Entrapment efficiency percent High performance liquid chromatography (HPLC) Chen et al., 2009; Sun et al., 2010; Niu et al., 2011,2012, 2014; Hu et al., 2013

UV spectrophotometer Guan et al., 2011; Dai et al., 2013Bicinchoninic acid (BCA) method Gebril et al., 2014Micro bicinchoninic acid (MicroBCA) method Jain et al., 2014a,b; Shukla et al., 2011Modified ninhydrin assay Conacher et al., 2001; Mann et al., 2006, 2009;

Premanand et al., 2013

In vitro release Dynamic dialysis method Chen et al., 2009; Guan et al., 2011; Dai et al., 2013

Cellular uptake Human colon adenocarcinoma cell line (Caco-2cells)

Song et al., 2005; Niu et al., 2014

Spontaneously derived human corneal epithelialcells (SDHCECs)

Dai et al., 2013

Murine macrophage cell line (RAW 264.7) Jain et al., 2014a,b


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reported that increasing either homogenization pressure or

cycles reduced the size of BS-liposomes (Chen et al., 2009;

Niu et al., 2011). Oppositely, extremely high homogenization

pressure increased particle size and PI of BS-vesicles due to

rupturing and combination of the vesicles under high

homogenization force (Guan et al., 2011).

The preparation method also influences the vesicle particle

size. Mann et al. (2009) reported that homogenization method

produced single population of small vesicles; however, thin-

film method produced a mixture of large vesicles combined

with smaller ones.

It is worth mentioning that the uptake of bilosomes

by Peyer’s patches and mesenteric lymph tissue is some-

how dependent on the vesicles size (Mann et al., 2009).

Bilosomes with size between 5 and 10 mm are retained within

the Peyer’s patches offering mucosal immunoglobulin

A (IgA) response, whereas particles less than 5 mm are

conveyed into the efferent lymphatics inside macrophages

inducing predominantly circulating antibody response

(Eldridge et al., 1990).

Likewise, particle size of BS-liposomes influences their

in vivo cellular uptake via endocytosis. Niu et al. (2014)

reported that SGC-liposomes with particle size of 80–400 nm

showed more cellular uptake in comparison to their counter-

part with size of 2000 nm. Endocytosis is an energy-

dependent process, and the greater extent of uptake shown

for smaller particle is related to the less energy required

during the endocytosis process (Gaumet et al., 2009; Niu

et al., 2014).

Transmission electron microscopy (TEM) was used to

ascertain the formation of BS-vesicles and visualize their

morphology (Chen et al., 2009). The TEM micrographs

presented in different studies indicates that BS-vesicles were

spherical or nearly spherical in nature. Discernable lamella

was observed for SGC-liposomes loaded with insulin (Niu

et al., 2011). Likewise, the bilosomes appeared to be

unilamellar vesicles utilizing TEM. Other microscopic tech-

niques that include cryogenic TEM (cryo-TEM) (Chen et al.,

2009) and freeze fracture electron microscopy (Mann et al.,

2006) were employed for visualization of BS-vesicles.

Zeta potential is the measure of overall charges acquired by

particles in a particular medium. In general, vesicles with

surface charge are more stable against accumulation than

uncharged ones. Opposing conventional liposomes that

showed a tendency to agglomerate, BS-liposomes were

negatively charged due the presence of bile salts that

stimulated the zeta potential and hindered the aggregation

of the vesicles (Dai et al., 2013). Regarding in vivo perform-

ance, it is reported that negatively charged vesicles are

favorably taken up by the Peyer’s patches (Wilkhu et al.,

2013).

Entrapment efficiency percent

For the use of vesicles in pharmaceutical applications, one of

the most important parameter to assess is the entrapment

efficiency percent (EE%). After preparation of BS-vesicles

suspension, the unencapsulated drug/protein was separated

from the total amount of drug by techniques such as ultra-

centrifugation (Song et al., 2005; Mann et al., 2009; Gebril

et al., 2014) or size exclusion chromatography using

Sephadex column (Chen et al., 2009; Dai et al., 2013; Niu

et al., 2014). The amount of encapsulated agents was

quantified by HPLC (Niu et al., 2011, 2012, 2014), UV

spectrophotometer (Guan et al., 2011; Dai et al., 2013) or

using different protein analysis methods like bicinchoninic

acid (Shukla et al., 2011; Jain et al., 2014a,b) and modified

ninhydrin assay (Mann et al., 2006; Mann et al., 2009;

Conacher et al., 2001; Premanand et al., 2013).

Similar to the particle size, a variety of variables affected

drug or protein entrapment in BS liposomes (Niu et al., 2011).

It was observed that the entrapment of different drugs

(hexamethylmelamine, Cyclosporine A and fenofibrate)

increased with the increase in bile salt contents in the vesicles.

Bile salts, with surface-active properties, can integrate

perpendicularly into the exterior of the bilayer membrane,

perturbating the acyl chains of the lipid matrix, and thereby

increasing the elasticity and solubility of drug in the

membrane (Chen et al., 2009; Sun et al., 2010). However,

further increase in the content of bile salts simultaneously

increases the drug solubility in the dispersion medium, thereby

compromising the entrapment efficiency (Niu et al., 2014).

Furthermore, the fluidizing effect of bile salts on the lipid

bilayers causes leakage of the entrapped drug (Niu et al.,

2011). The PC content in the vesicles as well contributes to the

EE. Significant increase in the amount of cyclosporine A and

hexamethylmelamine entrapped was bound with the increase

of SPC content, which was attributable to the fact that higher

lipid content propose more space for drug molecules (Sun

et al., 2010; Guan et al., 2011). The hydration pH affects the

EE% as its affects the bile salt dissociation, which in turn

affects the particle size and ability of the bile salts to hold the

drug molecules (Chen et al., 2009; Niu et al., 2014).

In vitro release and cellular uptake studies

Regarding BS-vesicles, the in vitro release study was

evaluated by dynamic dialysis method to circumvent the

leakage of vesicles into the external release medium (Chen

et al., 2009; Guan et al., 2011; Dai et al., 2013). In vitro release

studies are essential indicators for in vivo performance of a

drug delivery system. It is worth stating that their main

drawbacks are the absence of complete mimicking of in vivo

digestion and failing to take into account the probability of

vesicular disruption in physiological conditions (Andrieux

et al., 2009). In dynamic dialysis study, compared to conven-

tional liposomes, BS-liposomes released more drug due to the

enhance lipid bilayer fluidity that allowed drug leakage from

the vesicles (Chen et al., 2009). Besides, the uptake of BS-

vesicles was assessed in different biological cells like human

colon adenocarcinoma cell line (Caco-2 cells) (Song et al.,

2005; Niu et al., 2014), spontaneously derived human corneal

epithelial cells (Dai et al., 2013), and murine macrophage cell

line (RAW 264.7) (Jain et al., 2014a,b).

In vivo performance of bile salt vesicles (BS-vesicles)

Improvement in oral drug bioavailability

Based on the published literature, incorporation of drugs or

proteins into BS-liposomes substantialy enhanced their


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bioavailability and in vivo efficacy as depicted in Table 6 and

diagrammatically illustrated in Figure 8. Chen et al. (2009)

reported that orally administrated SDC-liposomes loaded with

fenofibrate exhibited 1.57-fold increase in bioavailability

compared to conventional liposomes in beagle dogs (Chen

et al., 2009). The observed enhancement was related to the

uptake of the vesicles through M-cells in the Peyer’s patch

and facilitated transmembrane absorption of fenofibrate due

to the ultra-deformability of BS-liposomes. Likewise, the

superiority of BS-liposomes in enhancing the oral bioavail-

ability of cyclosporine A relative to the marketed micro-

emulsion formulation (Sandimmune Neoral) and conventional

liposomes was confirmed by Guan et al. (2011). Facilitated

absorption of intact BS-liposomes vesicles rather than

enhancing drug solubility and release rate was the main

reason for the increase in cyclosporine A bioavailability.

Besides, Sun et al. (2010) achieved about 9.76- and 1.21-fold

higher bioavailability form hexamethylmelamine-loaded SDC

liposomes in animal model relative to drug solution and drug

loaded in conventional liposomes, respectively. The oral

delivery of insulin loaded in BS-liposomes experimented in

animal models generated encouraging results indicating that

this carrier system may be an interesting oral alternative for

parenteral insulin in the near future. Using luminescence

imaging, Niu et al. (2014) confirmed that orally administrated

SGC-liposomes loaded with recombinant human insulin

(rhINS) were more stable in vivo than conventional liposomes

due to the dual protective effects imposed by SGC vesicles

against damaging effects of bile salts and enzymes in the GIT.

Besides, the enhanced absorption of rhINS-loaded SGC-

liposomes was related to the improved transenterocytic

internalization and permeation of rhINS across the biomem-

branes. As for selecting the bile salt with more protective

action, Niu et al. (2012) observed that the hypogly-

cemic efficiency and oral bioavailability of insulin

from different BS-liposomes were in the sequence of

SGC4STC4CH4SDC indicating that SGC in liposomes

offer better protection against enzymatic degradation.

The suitability of utilizing different bile salts as absorption

enhancers in free-flowing proliposomal powder loaded with

Table 6. In vivo performances of BS-liposomes in different animal models.

Carrier loaded with drug Animal Purpose of in vivo study Achievement References

BS-liposomes containingfenofibrate

Beagle dogs Compare the oral bioavailability offenofibrate loaded BS-lipo-somes to that of conventionalliposomes.

SDC-liposomes exhibited1.57-fold increase in bioavail-ability relative to conventionalliposomes.

Chen et al., 2009

BS-liposomes containingCyclosporine A (CyA)

Male Wistar rats Compare the oral bioavailability ofCyA-loaded BS-liposomes tothat of conventional liposomesand Sandimmune Neoral as thereference.

The relative oral bioavailabilitiesof CyA-loaded SPC-liposomesand conventional liposomeswere 120.3% and 98.6%,respectively, with SandimmuneNeoral as the reference.

Guan et al., 2011

BS-liposomes containingrecombinant humaninsulin (rhINS)

Male Kunming mice Confirm the bioactivity of rhINsloaded in BS-liposomes afterdisruption of liposomes andadministration of rhINSsubcutaneously.

The bioactivity of insulin was wellpreserved with hypoglycemicpercentage of 45.2% ± 6.5%equivalent to free rhINSsolution.

Niu et al., 2011


Wistar rats Identify the GI residence profilesof rhINS after oral administra-tion of BS-liposomes usingin vivo imaging.

BS-liposomes improved in vivoresidence time of rhINS andenhanced permeation across thebiomembranes.

Niu et al., 2014

BS-liposomes containingTacrolimus

Albino New Zealandrabbits

Investigate the in vivo cornealuptake of tacrolimus fromBS-liposomal formulationscontaining different types of(STC, SGC and SDC).

STC and SGC-liposomes showedlow toxicity and improvedpermeability compared toSDC-liposomes that causedirritation.

Dai et al., 2013


Male Wistar rats Explore the hypoglycemic activ-ities and oral bioavailability ofdifferent rhINS-loadedBS-liposomes in both diabeticand non-diabetic rats.

SGC-liposomes showed higheroral bioavailability than otherBS-liposomes (STC and SDC)or conventional liposomes.

SGC-liposomes showed oral bio-availability of 8.5% and 11.0%compared to SC insulin in non-diabetic and diabetic rats,respectively.

Niu et al., 2012

Pro-BS liposomes contain-ing salmon calcitonin

Sprague Dawley rats Compare the pharmacokineticparameters of intra-duodenaladministrated salmon calci-tonin-loaded STDC-prolipo-somes to that of conventionalproliposomes and drug solution.

A 7.1-fold increase in the bio-availability of salmon calcitoninwas achieved from the STDCproliposomes compared to drugsolution.

Song et al., 2005

BS-liposomes containingHexamethylmelamine(HMM)

Female Wistar rats Compare the pharmacokineticparameters of HMM loadedSDC-liposomes to that ofconventional liposomes anddrug solution.

The bioavailability of HMM SDC-liposomes was be � 9.76- and1.21-fold higher than that ofHMM solution and HMMliposomes, respectively.

Sun et al., 2010


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salmon calcitonin was examined by Song et al. (2005). STDC-

proliposomes exhibited balanced permeation enhancement

across Caco-2 cell monolayers and minimized intestinal

epithelial cells damage. Besides, STDC-proniosomes resulted

in 7.1-fold increase in salmon calcitonin bioavailability when

administered duodenally to rats compared to proliposomes

without bile salts. In addition to the intrinsic ability of bile

salts to fluidize biological membranes, STDC was capable of

forming a lipophilic ion-pair complex with salmon calcitonin

characterized by increase permeability across biological

membranes.

Enhancement of vaccine immunogenicity

After introducing bilosomes by Conacher et al. in 2001,

considerable evidence in literature, collectively listed in

Table 7, affirmed the suitability of employing bilosomes as

carrier for vaccines to provide transmucosal immunization

(Figure 9).

Conacher et al. (2001) reported that orally administrated

bilosomes induced cell-mediated responses against synthetic

peptides and high antibody titers against protein antigens

comparable to those engendered succeeding systemic

immunization.

The potential of utilizing SDC-bilosomes loaded with

either HBsAg or DTx in providing transmucosal immuniza-

tion was investigated by Shukla et al. (2008, 2011). In both

studies, orally administrated bilosomes loaded with high

dose of antigen produced systemic immunoglobulin G (IgG)

response in mice comparable to those induced by intramus-

cular (IM) administrated antigens. In addition, bilosomes

elicited measurable secretory IgA in mucosal secretions that

were not induced by IM administrated antigens. Similarly,

the combinatorial bilosomes formulation containing high

dose of HBsAg and tetanus toxoid (TTx), prepared by

Shukla et al. (2010b), produced anti-HBsAg-IgG and

anti-TTx-IgG levels mice serum similar to IM administered

HBsAg and TTx. Likewise, Mann et al. (2006) reported

positive results regarding the effectiveness of the bilosomes

containing TTx in inducing significant systemic and muco-

sal immune response after oral administration. The obtained

end-point antibody titers from TTx-loaded bilosomes were

superior to the oral administration of the un-entrapped

antigen, but analogous to parenterally administrated ones.

Premanand et al. (2013) confirmed that baculovirus

displaying VP1 (Bac-VP1) linked with bilosomes provoked

significantly higher immune responses relative to bilosomes

non-linked with Bac-VP1 suggesting that bilosomes have

intrinsic adjuvant characters when linked with antigen.

Wilkhu et al. (2013) confirmed the protective property of

bilosomes to antigens as low levels of antigen (39%) were

measured across the GIT after oral administration of free

antigen to mice in compared to 55% for antigen-loaded

bilosomes. The influence of bilosomes particle size on the

associated immune response after oral administration was

assessed by Mann et al. (2009). Results indicated that mixed

population of small and large vesicles entrapped influenza A

antigen caused higher protection measured as symptom

score and higher responder number than bilosomes with

smaller vesicle size.

To increase their concentration in the intestinal immune

cells, targeted bilosomes with surface attached ligands

capable of recognizing and linking to the cells of interest

have been examined. In this context, Singh et al. (2004)

depicted that the oral dose of CTB-conjugated bilosomes

produced nearly comparable immune reaction to that of

parenteral administrated antigen containing Freund’s com-

plete adjuvant. Using the same approach, Shukla et al.

(2010b) showed that HBsAg-loaded CTB-conjugated bilo-

somes (20 mg) produced anti-HBsAg IgG antibody titre

response comparable to either IM alum-adsorbed HBsAg

(10 mg) or HBsAg-loaded bilosomes (50mg).

As far as the current literature is concerned, Jain et al.

(2014a,b) reported the foremost studies that examined the

Figure 8. In vivo performance of orally administrated bile salts-containing liposomes in comparison to conventional liposomes. (A) Fenofibrate-loadedbile salts-containing liposomes (Chen et al., 2009) and (B) Recombinant insulin-loaded bile salts-containing liposomes (Niu et al., 2014).


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Table 7. In vivo performances of bilosomes in animal models.

Carrier type Animal Purpose of in vivo study Achievement References

Bilosomes containingDiphtheria toxoid (Dtx)

Female BALB/c mice Measurement of specificIgG and IgA responsesafter oral administrationof DTx bilosomes incomparison to IM alum-adsorbed DTx.

Oral Dtx-bilosomes producedcomparable serum antibodytitres to IM DTx, at a four-fold higher dose.Oral DTx-bilosomes stimu-lated systemic (IgG) andmucosal (IgA) immuneresponses.

Shukla et al., 2011

Bilosomes containingTetanus toxoid (TTx)

Female BALB/c mice Compare between theimmunization responsesinduced by oral

TTx- bilosomes with SCTTx.

Bilosomes induce both sys-temic and mucosal immun-ity against bacterial proteinantigen.

Higher entrapped TTx dose(200 mg) induce IgG1 simi-lar to subcutaneous un-entrapped TT at the lower(550mg) dose.

Mann et al., 2006

Bilosomes containingHepatitis B antigen(HBsAg)

Female BALB/c mice Measurement of specificIgG and IgA responsesfrom oral HBsAg bilo-somes in comparison toIM alum-adsorbed DTx.

Oral administrated HBsAg-bilosomes produced sys-temic and mucosal antibodyresponses.

Oral bilosomes with a fivetimes higher dose producedcomparable serum antibodytitres to those of IMimmunization.

Shukla et al., 2008

Bilosomes containing andbovine serum albumin(BSA)

Female BALB/c mice Compare oral vaccinationwith BSA-bilosomeswith BSA alone or BSAin niosomes.

Vaccination with BSA or BSAin niosomes didn’t inducedmeasurable BSA specificIgG response in contrast toBSA-bilosome that inducedIgG immune response.

Conacher et al., 2001

Bilosomes containing inac-tivated influenza surfaceantigen (A.Texas)

Female BALB/c mice Compare oral vaccinationwith A. Texas-bilosomeswith SC immunization ofA. Texas.

Oral administration ofbilosomes incorporatinginfluenza sub-unit vaccineinduce antibody responsecomparable to the paren-terally administered vaccinecontaining the same quantityof antigen.

Conacher et al., 2001

Bilosomes containingTetanus toxoid (TTx)and Hepatitis B antigen(HBsAg)

BALB/c mice Estimation of IgG and sIgAfollowing oral immun-ization with combin-ations of HBsAg- andTTx-loaded bilosomes.

High dose of HBsAg-bilo-somes (50 mg) and

TTx-bilosomes (5Lf)combination produced highanti-IgG levels comparableto IM alum-adsorbedHBsAg (10 mg) and (0.5 Lf)TTx alone or in combin-ation.

High-dose bilosomal combitor-ial formulation elicitedmeasurable sIgA in mucosalsecretions.

Shukla et al., 2010b

Bilosomes associated withbaculovirus-VP1 (Bac-VP1)

BALB/c mice Compare between oralBac-VP1- bilosomes in

comparison to bilosomesnon-associated with

Bac-VP1 and subcutane-ously immunization withlive Bac-VP1.

Bac-VP1- bilosomes elicitedsignificantly higher anti-body levels compared tobilosomes non-associatedwith Bac-VP1.

Subcutaneous immunizationwith live Bac-VP1 had sig-nificantly enhanced specificserum IgG levels comparedto orally immunization withlive Bac-VP1 alone or asso-ciated with bilosomes.

Premanand et al., 2013

(continued )


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Table 7. Continued


Bilosomes containingrecombinant HA or theH3N2 sub-unit protein

Female BALB/c mice To study the impact ofvesicle size on bilosomesdistribution.

Larger bilosomes vesicles(�6 mm versus 2 mm indiameter) increased uptakewithin the Peyer’s patches.

Significantly higher level ofantigen was measuredwithin the mesenteric lymphtissue when delivered usingthe bilosome carrier com-pared to antigen given alonewithout a carrier system.

Wilkhu et al., 2013

Bilosomes containingInfluenza A antigen

Female BALB/c mice Determine the influence oforally administratedbilosomes size on Th1/Th2 bias in the immuneresponse.

Bilosomes with two popula-tions (60–350 and 400–2500 nm, Z-average 980 nm)generated significantlygreater Th1 bias immuneresponse than those con-taining a single population(range 10–100 nm, Z-aver-age diameter 250 nm).

Mann et al., 2009

Bilosomes containingGnRH immunogen

Female BALB/c mice Investigate the capability ofmucosal GnRH immu-nogens of inducing anti-GnRH antibody titers.

Orally administrated GnRH inbilosomes failed in inducingan antibody response.

Gebril et al., 2014

CTB-bilosomes containingHepatitis B surface anti-gen (HBsAg)

Female BALB/c mice Compare oral immunizationwith CTB-conjugatedbilosomes loaded withHBsAg (20 mg dose)with IM HBsAg (10 mg).

CTB-bilosomes (20 mg antigendose) produce comparablesystemic antibody responseto non-conjugated bilo-somes containing 50mgantigen.

CTB-bilosomes (20 mg antigendose) produced IgG anti-body titre response compar-able to IM injection of 10mgof alum-adsorbed HBsAg.

All the bilosomal preparationselicited measurable sIgA in

relation to negligibleresponse with IM adminis-tered HBsAg.

Shukla et al., 2010a

CTB-bilosomes containingbovine serum albumin(BSA)

Female BALB/c mice Examine the efficiency oforal administrated CTBin comparison to normalbilosomes and Freund’scomplete adjuvant(FCA).

A single oral dose ofCTB-conjugated bilosomes

produced equivalentimmune response comparedto parenteral administrationof antigen with Freund’scomplete adjuvant.

Bilosomesconjugated with CTB signifi-

cantly increased IgG levelscompared to unconjugatedbilosomes.

Singh et al., 2004

GM-bilosomes containingbovine serum albumin(BSA)

Male BALB/c mice Compare the immuneresponse elicited by GM-bilosomes administeredorally to that of IMadministrated alum-adsorbed BSA suspen-sion and oral bilosomes.

GM-bilosomes inducedmucosal and cell mediated

immune response, whichwere not induced by IMalum adsorbed BSA.Significantly highersystemic immune response(serum IgG level) wasobserved with GM-bilo-somes in comparison withbilosomes and alum-adsorbed BSA followingoral administration.

The immune responseobserved in case of GM-bilosomes was similar to IMalum-adsorbed BSA withoutany significant difference.

Jain et al., 2014b

(continued )


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efficiency of anchoring GM polymer to bilosomes encapsu-

lating TTx or BSA. In both cases, orally administrated

GM-bilosomes superseded niosomes and conventional bilo-

somes in eliciting better immune response due to their

selective and enhanced uptake through APCs in mucosal

linings.

Opposing the aforementioned positive outcomes associat-

ing with loading the antigen into bilosomes, recently, negative

result was reported by Gebril et al. (2014) where orally

administrated bilosomes entrapping GnRH immunogen failed

in inducing any antibody response. This indicated that non-

pathogenic origin peptides such as GnRH are not suitably

delivered by bilosomes.

Stability considerations of BS-vesicles

Stability studies (whether in-process, in simulated fluids or

during storage) are of vital prominence for successful devel-

opment of pharmaceutical carrier systems. Decomposition of

entrapped therapeutic agents may lead to decrease in their

potency, whereas in the case of immunological preparations,

denaturation of antigens may cause inadequate immune

response making the subject prone to diseases.

In processing stability

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Different research groups utilized sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) to examine

the chemical stability of entrapped peptides (rhINS) or

antigens (DTx, TTx and BSA) in BS-vesicles after being

exposed to the preparation stress conditions (Niu et al., 2011;

Shukla et al., 2011; Jain et al., 2014a,b). SDS-PAGE is a

widely used procedure to isolated proteins according to their

electrophoretic mobility. Sodium dodecyl sulfate (SDS), as

an anionic surfactant, was added to protein specimens to

linearize proteins and impart negative charge thus fractionate

them according to estimated size through electrophoresis

(Rath et al., 2009). In all the published studies, symmetrical

position of bands between the pure as well as extracted

antigens/proteins along with the absence of additional bands

were evident confirming that the preparation method, and the

Figure 9. In vivo performance of orally administrated bilosomes. (A) Confocal laser scanning microscopy (CLSM) of the intestine (Shukla et al., 2011)and (B) Immunological study and measurement of specific IgA and IgG.

Table 7. Continued


GM-bilosomes containingtetanus toxoid (TTx)

Male BALB/c mice Examine the efficiency ofthe immune responseobtained after oraladministration of

GM-bilosomes in relationto IM alum-adsorbedTTx and oral niosomesand bilosomes.

GM-bilosomes showed highersystemic immune response(serum IgG level) comparedto orally administeredbilosomes, niosomes andalum-adsorbed TTx.

GM-bilosomes elicit systemic,mucosal and cellularimmune responses.

Jain et al., 2014a


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quantity of bile salts included did induce any irreversible

accumulation or decomposition of the entrapped agents.

Circular dichroism spectroscopy analysis

Extracted antigens and proteins from BS-vesicles samples

were subjected to conformational stability studies using

circular dichroism (CD) spectroscopy (Niu et al., 2011,

2012; Jain et al., 2014a,b). CD is displayed by molecules

because of their levorotary and dextrorotary constituents and

CD spectroscopy analysis is used to study the conformations

adopted by proteins as they have CD spectral signatures

representative of their structures (Martin & Schilstra, 2008).

The entrapped agents were found conformationally stable

throughout the preparation process as evident by the super-

imposed CD spectra of test samples with that of standard,

thus, further supporting the appropriateness of BS-vesicles in

delivering antigens and therapeutic proteins.

Storage stability

Stability studies were carried out to explore the leaching of

the entrapped agent from the vesicles during storage

(Table 8). Shukla et al. (2011) examined the amount of

DTx retained in the bilosomes after storage at refrigerated

(5 ± 3 �C) and room temperature (25 ± 2 �C) at 70% relative

humidity. After one-month storage, around 94% of the antigen

remained in bilosomes stored at room temperature, whereas

more than 98% of the antigen was found in samples stored at

refrigerated conditions. The stability of the formulations was

credited to the negative charge induced by DCP on the

bilosomes surface that caused electrostatic repulsion prevent-

ing fusion and aggregation of vesicles upon storage.

Stability in simulated biological milieu

It is crucial to interpret the stability of vesicular carriers and

encapsulated peptides and proteins when confronted by

gastric pH, bile salts and GI enzymes as the intact fraction

of the vesicles predominantly influences the associated in vivo

response. However, it is somehow challenging, to perceive the

fate of the vesicles in the GI tract by sampling or

imaging tools. As a result, different research groups took an

alternative easier approach by incubating the BS-vesicles in

in vitro-simulated GI media and bile salts solutions of

concentration between 5 and 20 mM, similar to the bile salts

concentration range in healthy humans (Westergaard, 1977).

The superiority of bilosomes to niosomes, affirmed in terms

of their ability to protect the loaded antigen in simulated

biological fluids, was attributed to repulsion between the bile

salts in the vesicle bilayer and the external bile salts in the

solution (Conacher et al., 2001). In this domain, Conacher

et al. (2001) reported that niosomes lost all of their initially

entrapped BSA content in bile salt concentration of 20 mM in

contrast to bilosomes that retained 85% of its initial BSA,

thereby, confirming the stabilizing role of bile salts in vesicle

constructs. In accordance with the aforementioned result,

Shukla et al. (2008, 2011) reported that bilosomes loaded

with either HBsAg or DTx retained considerable amount of

entrapped antigen during stability studies conducted in

simulated GIF and bile salts solutions (5 mM and 20 mM

concentration). Following a comparable approach, Wilkhu

et al. (2013) examined the changes in antigen load of

bilosomes-incubated GIF. The initial antigen loaded (32%)

was retained in gastric media; however, it decreased to around

8.5–9.5% when the bilosomes were simultaneously trans-

ferred to intestinal medium due to the degradation of antigen

located on the surface of bilosomes.

Different authors have compared the stability of

BS-liposomes in simulated GI media in comparison to

conventional liposomes. Hu et al. (2013) reported that SGC-

liposomes retained most of the insulin load as compared with

conventional liposomes in either simulated GI fluids (includ-

ing pancreatin or pepsin) or in ex vivo GI fluids obtained from

rats. The protective effect was credited to the enzyme-

inhibiting ability of SGC and its associated membrane

stabilization aptitude. The influence of incorporating different

bile salts (SGC, STC, SDC) on the integrity of rhINS-loaded

BS-liposomes against different protease enzymes (pepsin,

trypsin and a-chymotrypsin) was tested by Niu et al. (2011)

who further affirmed the superiority of SGC in formulating

BS-liposomes due to its enzyme-inhibiting ability compared

to the other investigated bile salts.

Regarding the modified bilosomes, Singh et al. (2004)

reported that CTB-bilosomes exhibited better stability than

niosomes incubated at 20 mM bile salt solution as they

retained about 84% of its initial BSA-load compared to

niosomes that retained around 52% of its initial load.

Similarly, GM-bilosomes exhibited enhanced stability, in

terms of significant higher percentage of TTx or BSA retained

within the system, compared to bilosomes and niosomes due

to the additional polymeric coating barrier (Jain et al.,

2014a,b).

Table 8. Percent antigen retained in different bilosomes formulations challenged by simulated biological media.

Bilosome formulationSimulated

gastric fluidSimulated

intestinal fluid5 mM bile

salt solution20 mM bilesalt solution References

Bilosomes containing TTX 75.3 ± 2.2% 68.9 ± 3.2% 71.6 ± 2.7% 66.1 ± 3.4% Jain et al., 2014aBilosomes containing HBsAg 90% 95% 95% 85% Shukla et al., 2008Bilosomes containing BSA 90% 85% Conacher et al., 2001Bilosomes containing DTx 88% 92% 93% 83% Shukla et al., 2011Bilosomes containing BSA 72.3 ± 1.9% 65.4 ± 2.5% 67.8 ± 3.4 60.3 ± 2.3% Jain et al., 2014bCTB-bilosomes containing HBsAg 86% 91% 91% 80% Shukla et al., 2010aCTB-bilosomes containing BSA 92–95% 84% Singh et al., 2004GM-bilosomes containing TTx 84.4 ± 3.1% 78.3 ± 2.2% 77.3 ± 2.1% 73.5 ± 3.0% Jain et al., 2014aGM-bilosomes containing BSA 81.6 ± 1.5% 74.2 ± 2.6% 72.6 ± 3.2% 69.8 ± 2.8% Jain et al., 2014b


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Cytotoxicity of BS-vesicles

Bile salts are reported to cause irritation and toxicity when

used as penetration enhancers because their enhancement

effect is somehow bound to damaging the intestinal epithelial

barrier (Saettone et al., 1996). However, some researches have

shown that PL can reduce the noxiousness of bile salts (Dial

et al., 2008). In this domain, Niu et al. (2014) evaluated the

cytotoxicity of BS-liposomes in concentration range of 0.25–

6.25 mmol/L using cell growth inhibition assay employing

Caco-2 cell monolayers. Although, there was a slight

reduction in cell viability as the liposomes concentration

increased, non-significant difference was observed among

various bile salts (SGC, STC and SDC) used or between

different concentration after incubation for 4 h. The obtained

findings implied that BS-liposomes exhibited non-significant

toxicity toward Caco-2 cells at the investigated concentra-

tions. Cell apoptosis detection was carried out by the same

research group to further evaluate cytotoxicity of BS-

liposomes to Caco-2 cells as their hydrophobicity is

correlated with apoptosis induction. Complementing their

previous result, non-significant difference was found in

apoptosis between BS-liposomes and negative control con-

firming the low cytotoxicity of BS-liposomes. Similarly, Dai

et al. (2013) evaluated the toxicity of BS-liposomes prepared

using SGC, STC and SDC in comparison to conventional

liposomes on the viability of corneal epithelial cells. After

incubation for 12 h, at low lipid concentration (54 mg/mL),

the viability of the cells were more than 85% for all liposomal

formulations; however, at high lipid concentration

(46 mg/mL), both SGC and STC showed low toxicity

affirming their suitability for ocular drug delivery opposite

to SDC-liposomes that had greater toxicity to corneal cells.

Critical opinion

Based on the reviewed literature, BS-liposomes, not only

were they capable of enhancing the bioavailability of poorly

water soluble drugs in animal models but also they

demonstrated the ability to protect the entrapped peptides

and proteins after oral administration. Moreover, after the first

breakthrough of utilizing bilosomes for oral vaccines deliver-

ing by Conacher et al. in 2001, investigations by different

research groups in BALB/c mice confirmed the usefulness of

oral bilosomes entrapping antigens in alerting both mucosal

and systemic immune responses. In addition, surface engin-

eered bilosomes (CTB-bilosomes and GM-liposomes) pre-

pared via anchoring ligands to the bilosomal surface

demonstrated the ability of targeting the antigens into specific

immune cells. Likewise, covalently linked bile salt-lipid

conjugate to liposomes (BSC-liposomes) showed promising

specific hepatocytes targeting. Though much have been

learned from studies conducted in animals about the in vivo

effectiveness of BS-vesicles, endeavoring to scale up the

obtained positive results from animals to humans is usually

problematic. The current challenge that faces the researchers

is applying the gained knowledge to carry out safe trials in

human subjects to systematically monitor all parameters of

the elicited immune response and elucidate the exact immune

mechanism after oral administration of bilosomes. Besides,

the precise mechanisms of facilitated absorption and the

interaction between the BS-liposomes and biological envir-

onment have not yet been explained due to the complexity of

the GI physiology. Nevertheless, it is of remarkable signifi-

cance to explicate the exact underlying mechanisms of

improved absorption of macromolecules by BS-liposomes to

serve as basis for their optimization. Further studies should

also include the assessment of the influence of the ingested

food on the in vivo performance of BS-vesicles.

In addition to the aforementioned, certain criteria must be

fulfilled in BS-vesicles to ensure their acceptance as

pharmaceutical carriers. An efficient and adequate process

for large industrial scale production that ensures high and

reproducible levels of protein/peptide entrapment should be

addressed. Furthermore, the long-term storage stability of

BS-vesicles should be scrutinized in details, which is still yet

far from being accomplished due to the limited researches that

address long-term stability.

Conclusion

Although, conventional lipid-based vesicular carriers, such as

liposomes or niosomes, showed the potential for delivering

different macromolecules, yet, after oral administration, the

intestinal bile salts and enzymes disrupt the bilayers con-

structs of the vesicles, causing the release of entrapped

macromolecules prior to reaching the intended site of action.

In response to that, different forms of bile salts-containing

vesicular carriers, namely BS-liposomes, bilosomes and

surface-engineered BS-vesicles, have been developed as

stable replacements for conventional vesicles that decompos-

ition within the harsh environment in the GIT. The presented

studies in this review confirmed that BS-vesicles have

superior characteristics to conventional liposomes or nio-

somes for enhancing the oral bioavailability of therapeutically

challenging agents and immunological response of vaccines.

In addition, research on BS-vesicles are currently accelerat-

ing, stimulated by the present information regarding their

positive in vivo performance combined with the simplicity in

preparation.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

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