Bile salts-containing vesicles: promising pharmaceutical carriers for oral delivery of poorly water-soluble drugs and peptide/protein-based therapeutics or vaccines

Figures & data

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

Figure 2. A schematic representation of the behavior of conventional liposome and niosome in comparison to bilosomes in presence of intestinal bile salts. (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 and bile salts for preparing BS-vesicles.

Ingredients
Lipids
Cholesterol (CH)
Dicetyl phosphate (DCP)
Soybean phosphatidyl choline (SPC)
Monopalmitoyl glycerol (MPG)
Dipalmitoyl phosphatidylethanolamine (DPPE)
Distearyl phosphatidyl ethanolamine (DSPE)
Surfactants
Sorbitan tristearate (STS)
Sorbitan monooleate (SMO)
Bile salts
Sodium glycocholate (SGC)
Sodium taurocholate (STC)
Sodium deoxycholate (SDC)
Sodium taurodeoxycholate (STDC)

Abbreviations of ingredients are present between parentheses.

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

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

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

Figure 6. Schematic representation of the uptake of bilosomes containing 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) Uptake of bilosomes by M-cells and dendritic cells.

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 2. Formulation details, preparation methods, and vesicles characteristics of bile salt-containing liposomes (BS-liposomes).

Vesicular carrier	Composition (mol/mol)	Drug/nature	Preparation method	Vesicle size reduction	PS, PI, ZP, EE%	References
BS-liposomes	SPC:SDC (4:1)	Fenofibrate/ Poorly water soluble drug	Thin film hydration method	Sonication followed by high-pressure homogenization	PS ≈ 159 nm EE% ≈ 88.23%	Chen et al., 2009
BS-liposomes	SPC:SDC (3:1)	Cyclosporine A/ poorly water soluble drug	Thin film hydration method	High-pressure homogenization	PS = 85.6 ± 1.0 nm PI = 0.064 ± 0.013 ZP = −35.2 ± 4.42 mV EE% = 94.05 ± 2.76%	Guan et al., 2011
BS-liposomes	SPC:STC (5:1)	Tacrolimus/ Poorly water solubility drug	Thin film hydration method	Ultrasonication	PS = 97.3 ± 1.6 nm PI = 0.267 ± 0.033 ZP = 25.2 ± 3.8 mV EE% = 95.15 ± 1.91%	Dai et al., 2013
BS-liposomes	PC:SDC (2:1)	Hexamethylmelamine/ Lipophilic drug	Reverse phase evaporation method		PS = 229 ± 20 nm PI = 0.354 ± 0.070 EE% = 43.85 ± 1.87%	Sun et al., 2010
BS-liposomes	SPC:SGC (4:1)	Porcine insulin/ Protein drug	Reverse phase evaporation method	High-pressure homogenization	PS = 178.4 ± 6.2 nm PI = 0.346 ± 0.022 ZP = −51.3 ± 0.8 mV EE% = 30.23 ± 0.84%	Hu et al., 2013
BS-liposomes	SPC:SGC (4:1)	Recombinant human insulin/ Protein drug	Reverse phase evaporation method	High-pressure homogenization	PS = 154 ± 18 nm EE% = 30 ± 2%	Niu et al., 2011
BS-liposomes	SPC:bile salts (SGC/STC/SDC) (4:1)	Recombinant human insulin/ Protein drug	Reverse phase evaporation method	High-pressure homogenization	PS ≈ 150 nm EE% ≈ 30%	Niu et al., 2014
Pro-BS-liposomes	PC:STDC (4:1) Loaded on sorbitol (4 g)	Salmon calcitonin/ Peptide drug	Proliposomal method; loading liposomes on porous carrier		PS = 741 ± 76.9 nm EE% = 54.9 ± 4.1%	Song et al., 2005

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

Table 3. Formulation details, preparation methods and vesicles characteristics of bilosomes.

Vesicular carrier	Composition (mol/mol)	Antigen	Preparation method	Vesicle size reduction	*PS, PI, ZP, EE%	Reference
Bilosomes	STS:CH:DCP (7:3:1), 100 mg of SDC	Diphtheria toxoid (DTx)	Thin film hydration method	Extrusion through 200 nm pore membrane	PS = 206 ± 20 nm EE% = 20–24%	Shukla et al., 2011
Bilosomes	STS:CH:DCP (7:3:1), 100 mg of SDC	Recombinant baculovirus- VP1 (Bac-VP1)	Thin film hydration method	Homogenization	EE% = 50% nm	Premanand et al., 2013
Bilosomes	STS:CH:DCP (7:3:1), 100 mg of SDC	Hepatitis B Antigen (HBsAg)	Thin film hydration method	Extrusion through 200 nm pore membrane	PS = 204 ± 18 nm EE% = 18–22%	Shukla et al., 2008
Bilosomes	MPG:CH:DCP (5:4:1), 100 mg of SDC	BSA and measles peptide	Thin film hydration method			Conacher et al., 2001
		Influenza A antigen (H3N2 sub-unit protein)			PS = 3 µm
Bilosomes	MPG:CH: DCP (5:4:1), 100 mg of SDC	Tetanus toxoid (TTx)	Thin film hydration method			Mann et al., 2006
Bilosomes	MPG:CH:DCP (5:4:1), 100 mg of SDC	Influenza A antigen	Hot homogenization method		PS = 250 nm EE% = 50%	Mann et al., 2009
			Thin film hydration method		PS = 980 nm EE% = 50%
Bilosomes	MPG:CH:DCP (5:4:1) 100 mM of bile salt	Recombinant influenza antigen (HA or H3N2 sub-unit protein)	Hot homogenization method		PS = 6.44 ± 0.05 μm ZP = −125 mV EE% = 32%	Wilkhu et al., 2013
Bilosomes	MPG:CH:DCP (5:4:1) 100 mM of SDC	Gonadotropin-releasing hormone (GnRH) immunogen	Hot homogenization method	Homogenization	PS = 130 ± 14 nm PI = 0.25 ZP = −34 ± 2 mV EE% = 35 ± 2%	Gebril et al., 2014

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

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

Vesicular carrier	Composition	Antigen	Preparation method	Vesicle size reduction	*PS, PI, ZP, EE%	References
CTB- bilosomes	STS:CH:modified DPPE (7:3:1); 100 mg of SDC, Conjugation of CTB to bilosomes	Hepatitis B surface Antigen (HBsAg)	Thin film hydration method	Extrusion through 200 nm pore membrane	PS = 202 ± 20 nm EE% = 20–24%	Shukla et al., 2010b
CTB- bilosomes	STS:CH:modified DPPE (7:3:1) 20 mg SDC, Conjugation of CTB to bilosomes	Bovine serum albumin (BSA)	Thin film hydration method	Extrusion through 400 nm then 100 nm pore membrane	PS = 108.6 ± 11.4 nm EE% = 28.2 ± 3.4%	Singh et al., 2004
GM-bilosomes	SMO:CH:SDC (2:1:0.1) GM-OCM-DSPE (10% w/w of total lipid content)	Bovine serum albumin (BSA)	Thin film hydration method	Sonication	PS = 157 ± 3 nm PI = 0.287 ± 0.045 ZP = −21.8 ± 2.01 mV EE% = 71.3 ± 4.3%	Jain et al., 2014b
GM-bilosomes	SMO:CH:SDC (2:1:0.1) GM-OCM-DSPE (10% w/w of total lipid content)	Tetanus toxoid (TTx)	Thin film hydration method	Sonication	PS = 164 ± 10 nm PI = 0.258 ± 0.041 ZP = −17.9 ± 2.8 mV EE = 75.8 ± 3.3%	Jain et al., 2014a

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

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; Sun et 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; Shukla et 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., 2014
	Freeze fracture electron microscopy (FFEM)	Mann et al., 2006
Separation of free drug from  drug-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., 2013
	Bicinchoninic acid (BCA) method	Gebril et al., 2014
	Micro bicinchoninic acid (MicroBCA) method	Jain et al., 2014a,b; Shukla et al., 2011
	Modified 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-2 cells)	Song et al., 2005; Niu et al., 2014
	Spontaneously derived human corneal epithelial cells (SDHCECs)	Dai et al., 2013
	Murine macrophage cell line (RAW 264.7)	Jain et al., 2014a,b

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

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 containing fenofibrate	Beagle dogs	Compare the oral bioavailability of fenofibrate loaded BS-liposomes to that of conventional liposomes.	SDC-liposomes exhibited 1.57-fold increase in bioavailability relative to conventional liposomes.	Chen et al., 2009
BS-liposomes containing Cyclosporine A (CyA)	Male Wistar rats	Compare the oral bioavailability of CyA-loaded BS-liposomes to that of conventional liposomes and Sandimmune Neoral as the reference.	The relative oral bioavailabilities of CyA-loaded SPC-liposomes and conventional liposomes were 120.3% and 98.6%, respectively, with Sandimmune Neoral as the reference.	Guan et al., 2011
BS-liposomes containing recombinant human insulin (rhINS)	Male Kunming mice	Confirm the bioactivity of rhINs loaded in BS-liposomes after disruption of liposomes and administration of rhINS subcutaneously.	The bioactivity of insulin was well preserved with hypoglycemic percentage of 45.2% ± 6.5% equivalent to free rhINS solution.	Niu et al., 2011
BS-liposomes containing recombinant human insulin (rhINS)	Wistar rats	Identify the GI residence profiles of rhINS after oral administration of BS-liposomes using in vivo imaging.	BS-liposomes improved in vivo residence time of rhINS and enhanced permeation across the biomembranes.	Niu et al., 2014
BS-liposomes containing Tacrolimus	Albino New Zealand rabbits	Investigate the in vivo corneal uptake of tacrolimus from BS-liposomal formulations containing different types of (STC, SGC and SDC).	STC and SGC-liposomes showed low toxicity and improved permeability compared to SDC-liposomes that caused irritation.	Dai et al., 2013
BS-liposomes containing recombinant human insulin (rhINS)	Male Wistar rats	Explore the hypoglycemic activities and oral bioavailability of different rhINS-loaded BS-liposomes in both diabetic and non-diabetic rats.	SGC-liposomes showed higher oral bioavailability than other BS-liposomes (STC and SDC) or conventional liposomes. SGC-liposomes showed oral bioavailability of 8.5% and 11.0% compared to SC insulin in non-diabetic and diabetic rats, respectively.	Niu et al., 2012
Pro-BS liposomes containing salmon calcitonin	Sprague Dawley rats	Compare the pharmacokinetic parameters of intra-duodenal administrated salmon calcitonin-loaded STDC-proliposomes to that of conventional proliposomes and drug solution.	A 7.1-fold increase in the bioavailability of salmon calcitonin was achieved from the STDC proliposomes compared to drug solution.	Song et al., 2005
BS-liposomes containing Hexamethylmelamine (HMM)	Female Wistar rats	Compare the pharmacokinetic parameters of HMM loaded SDC-liposomes to that of conventional liposomes and drug solution.	The bioavailability of HMM SDC-liposomes was be ∼ 9.76- and 1.21-fold higher than that of HMM solution and HMM liposomes, respectively.	Sun et al., 2010

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. In vivo performances of bilosomes in animal models.

Carrier type	Animal	Purpose of in vivo study	Achievement	References
Bilosomes containing Diphtheria toxoid (Dtx)	Female BALB/c mice	Measurement of specific IgG and IgA responses after oral administration of DTx bilosomes in comparison to IM alum-adsorbed DTx.	Oral Dtx-bilosomes produced comparable serum antibody titres to IM DTx, at a fourfold higher dose. Oral DTx-bilosomes stimulated systemic (IgG) and mucosal (IgA) immune responses.	Shukla et al., 2011
Bilosomes containing Tetanus toxoid (TTx)	Female BALB/c mice	Compare between the immunization responses induced by oral TTx- bilosomes with SC TTx.	Bilosomes induce both systemic and mucosal immunity against bacterial protein antigen. Higher entrapped TTx dose (200 µg) induce IgG1 similar to subcutaneous un-entrapped TT at the lower (< 50 µg) dose.	Mann et al., 2006
Bilosomes containing Hepatitis B antigen (HBsAg)	Female BALB/c mice	Measurement of specific IgG and IgA responses from oral HBsAg bilosomes in comparison to IM alum-adsorbed DTx.	Oral administrated HBsAg -bilosomes produced systemic and mucosal antibody responses. Oral bilosomes with a five times higher dose produced comparable serum antibody titres to those of IM immunization.	Shukla et al., 2008
Bilosomes containing and bovine serum albumin (BSA)	Female BALB/c mice	Compare oral vaccination with BSA-bilosomes with BSA alone or BSA in niosomes.	Vaccination with BSA or BSA in niosomes didn’t induced measurable BSA specific IgG response in contrast to BSA-bilosome that induced IgG immune response.	Conacher et al., 2001
Bilosomes containing inactivated influenza surface antigen (A.Texas)	Female BALB/c mice	Compare oral vaccination with A. Texas-bilosomes with SC immunization of A. Texas.	Oral administration of bilosomes incorporating influenza sub-unit vaccine induce antibody response comparable to the parenterally administered vaccine containing the same quantity of antigen.	Conacher et al., 2001
Bilosomes containing Tetanus toxoid (TTx) and Hepatitis B antigen (HBsAg)	BALB/c mice	Estimation of IgG and sIgA following oral immunization with combinations of HBsAg- and TTx-loaded bilosomes.	High dose of HBsAg-bilosomes (50 µg) and TTx-bilosomes (5Lf) combination produced high anti-IgG levels comparable to IM alum-adsorbed HBsAg (10 µg) and (0.5 Lf) TTx alone or in combination. High-dose bilosomal combitorial formulation elicited measurable sIgA in mucosal secretions.	Shukla et al., 2010b
Bilosomes associated with baculovirus-VP1 (Bac-VP1)	BALB/c mice	Compare between oral Bac-VP1- bilosomes in comparison to bilosomes non-associated with Bac-VP1 and subcutaneously immunization with live Bac-VP1.	Bac-VP1- bilosomes elicited significantly higher antibody levels compared to bilosomes non-associated with Bac-VP1. Subcutaneous immunization with live Bac-VP1 had significantly enhanced specific serum IgG levels compared to orally immunization with live Bac-VP1 alone or associated with bilosomes.	Premanand et al., 2013
Bilosomes containing recombinant HA or the H3N2 sub-unit protein	Female BALB/c mice	To study the impact of vesicle size on bilosomes distribution.	Larger bilosomes vesicles (∼6 μm versus 2 μm in diameter) increased uptake within the Peyer’s patches. Significantly higher level of antigen was measured within the mesenteric lymph tissue when delivered using the bilosome carrier compared to antigen given alone without a carrier system.	Wilkhu et al., 2013
Bilosomes containing Influenza A antigen	Female BALB/c mice	Determine the influence of orally administrated bilosomes size on Th1/Th2 bias in the immune response.	Bilosomes with two populations (60–350 and 400–2500 nm, Z-average 980 nm) generated significantly greater Th1 bias immune response than those containing a single population (range 10–100 nm, Z-average diameter 250 nm).	Mann et al., 2009
Bilosomes containing GnRH immunogen	Female BALB/c mice	Investigate the capability of mucosal GnRH immunogens of inducing anti-GnRH antibody titers.	Orally administrated GnRH in bilosomes failed in inducing an antibody response.	Gebril et al., 2014
CTB-bilosomes containing Hepatitis B surface antigen (HBsAg)	Female BALB/c mice	Compare oral immunization with CTB-conjugated bilosomes loaded with HBsAg (20 µg dose) with IM HBsAg (10 µg).	CTB-bilosomes (20 µg antigen dose) produce comparable systemic antibody response to non-conjugated bilosomes containing 50 µg antigen. CTB-bilosomes (20 µg antigen dose) produced IgG antibody titre response comparable to IM injection of 10 µg of alum-adsorbed HBsAg. All the bilosomal preparations elicited measurable sIgA in relation to negligible response with IM administered HBsAg.	Shukla et al., 2010a
CTB-bilosomes containing bovine serum albumin (BSA)	Female BALB/c mice	Examine the efficiency of oral administrated CTB in comparison to normal bilosomes and Freund’s complete adjuvant (FCA).	A single oral dose of CTB-conjugated bilosomes produced equivalent immune response compared to parenteral administration of antigen with Freund’s complete adjuvant. Bilosomes conjugated with CTB significantly increased IgG levels compared to unconjugated bilosomes.	Singh et al., 2004
GM-bilosomes containing bovine serum albumin (BSA)	Male BALB/c mice	Compare the immune response elicited by GM-bilosomes administered orally to that of IM administrated alum-adsorbed BSA suspension and oral bilosomes.	GM-bilosomes induced mucosal and cell mediated immune response, which were not induced by IM alum adsorbed BSA. Significantly higher systemic immune response (serum IgG level) was observed with GM-bilosomes in comparison with bilosomes and alum-adsorbed BSA following oral administration. The immune response observed in case of GM-bilosomes was similar to IM alum-adsorbed BSA without any significant difference.	Jain et al., 2014b
GM-bilosomes containing tetanus toxoid (TTx)	Male BALB/c mice	Examine the efficiency of the immune response obtained after oral administration of GM-bilosomes in relation to IM alum-adsorbed TTx and oral niosomes and bilosomes.	GM-bilosomes showed higher systemic immune response (serum IgG level) compared to orally administered bilosomes, niosomes and alum-adsorbed TTx. GM-bilosomes elicit systemic, mucosal and cellular immune responses.	Jain et al., 2014a

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

Bilosome formulation	Simulated gastric fluid	Simulated intestinal fluid	5 mM bile salt solution	20 mM bile salt solution	References
Bilosomes containing TTX	75.3 ± 2.2%	68.9 ± 3.2%	71.6 ± 2.7%	66.1 ± 3.4%	Jain et al., 2014a
Bilosomes containing HBsAg	90%	95%	95%	85%	Shukla et al., 2008
Bilosomes containing BSA			90%	85%	Conacher et al., 2001
Bilosomes containing DTx	88%	92%	93%	83%	Shukla et al., 2011
Bilosomes containing BSA	72.3 ± 1.9%	65.4 ± 2.5%	67.8 ± 3.4	60.3 ± 2.3%	Jain et al., 2014b
CTB-bilosomes containing HBsAg	86%	91%	91%	80%	Shukla et al., 2010a
CTB-bilosomes containing BSA			92–95%	84%	Singh et al., 2004
GM-bilosomes containing TTx	84.4 ± 3.1%	78.3 ± 2.2%	77.3 ± 2.1%	73.5 ± 3.0%	Jain et al., 2014a
GM-bilosomes containing BSA	81.6 ± 1.5%	74.2 ± 2.6%	72.6 ± 3.2%	69.8 ± 2.8%	Jain et al., 2014b

Henriksen-Lacey M, Perrie Y. (2013). Designing liposomes as vaccine adjuvants. In: Flower DR, Perrie Y, eds. Immunomic discovery of adjuvants and candidate subunit vaccines. New York: Springer, Chapter 10, 181–203

 Google Scholar

Chen Y, Lu Y, Chen J, et al. (2009). Enhanced bioavailability of the poorly water-soluble drug fenofibrate by using liposomes containing a bile salt. Int J Pharm 376:153–60

 PubMed Web of Science ®Google Scholar

Guan P, Lu Y, Qi J, et al. (2011). Enhanced oral bioavailability of cyclosporine A by liposomes containing a bile salt. Int J Nanomedicine 6:965–74

 PubMed Web of Science ®Google Scholar

Dai Y, Zhou R, Liu L, et al. (2013). Liposomes containing bile salts as novel ocular delivery systems for tacrolimus (FK506): in vitro characterization and improved corneal permeation. Int J Nanomedicine 8:1921–33

 PubMed Web of Science ®Google Scholar

Sun J, Deng Y, Wang S, et al. (2010). Liposomes incorporating sodium deoxycholate for hexamethylmelamine (HMM) oral delivery: development, characterization, and in vivo evaluation. Drug Deliv 17:164–70

 PubMed Web of Science ®Google Scholar

Niu M, Lu Y, Hovgaard L, Wu W. (2011). Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation. Int J Nanomedicine 6:1155–66

 PubMed Web of Science ®Google Scholar

Niu M, Tan Y, Guan P, et al. (2014). Enhanced oral absorption of insulin-loaded liposomes containing bile salts: a mechanistic study. Int J Pharm 460:119–30

 PubMed Web of Science ®Google Scholar

Song KH, Chung SJ, Shim CK. (2005). Enhanced intestinal absorption of salmon calcitonin (sCT) from proliposomes containing bile salts. J Control Release 106:298–308

 PubMed Web of Science ®Google Scholar

Shukla A, Singh B, Katare OP. (2011). Significant systemic and mucosal immune response induced on oral delivery of diphtheria toxoid using nano-bilosomes. Br J Pharmacol 164:820–7

 PubMed Web of Science ®Google Scholar

Premanand B, Prabakaran M, Kiener TK, Kwang J. (2013). Recombinant baculovirus associated with bilosomes as an oral vaccine candidate against HEV71 infection in mice. PLoS One 8:e55536

 PubMed Web of Science ®Google Scholar

Shukla A, Khatri K, Gupta PN, et al. (2008). Oral immunization against hepatitis B using bile salt stabilized vesicles (bilosomes). J Pharm Pharm Sci 11:59–66

 PubMed Web of Science ®Google Scholar

Conacher M, Alexander J, Brewer JM. (2001). Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes). Vaccine 19:2965–74

 PubMed Web of Science ®Google Scholar

Mann JF, Scales HE, Shakir E, et al. (2006). Oral delivery of tetanus toxoid using vesicles containing bile salts (bilosomes) induces significant systemic and mucosal immunity. Methods 38:90–5

 PubMed Web of Science ®Google Scholar

Mann JF, Shakir E, Carter KC, et al. (2009). Lipid vesicle size of an oral influenza vaccine delivery vehicle influences the Th1/Th2 bias in the immune response and protection against infection. Vaccine 27:3643–9

 PubMed Web of Science ®Google Scholar

Gebril AM, Lamprou DA, Alsaadi MM, et al. (2014). Assessment of the antigen-specific antibody response induced by mucosal administration of a GnRH conjugate entrapped in lipid nanoparticles. Nanomedicine 10:971–9

 PubMed Web of Science ®Google Scholar

Shukla A, Bhatia A, Amarji B, et al. (2010b). Nano-bilosomes as potential vaccine delivery system for effective combined oral immunization against tetanus and hepatitis B. J Biotech 150:98–9

 Web of Science ®Google Scholar

Singh P, Prabakaran D, Jain S, et al. (2004). Cholera toxin B subunit conjugated bile salt stabilized vesicles (bilosomes) for oral immunization. Int J Pharm 278:379–90

 PubMed Web of Science ®Google Scholar

Jain S, Indulkar A, Harde H, Agrawal AK. (2014b). Oral mucosal immunization using glucomannosylated bilosomes. J Biomed Nanotechnol 10:932–47

 PubMed Web of Science ®Google Scholar

Jain S, Harde H, Indulkar A, Agrawal AK. (2014a). Improved stability and immunological potential of tetanus toxoid containing surface engineered bilosomes following oral administration. Nanomedicine 10:431–40

 PubMed Web of Science ®Google Scholar

Shukla A, Katare OP, Singh B, Vyas SP. (2010a). M-cell targeted delivery of recombinant hepatitis B surface antigen using cholera toxin B subunit conjugated bilosomes. Int J Pharm 385:47–52

 PubMed Web of Science ®Google Scholar

Niu M, Lu Y, Hovgaard L, et al. (2012). Hypoglycemic activity and oral bioavailability of insulin-loaded liposomes containing bile salts in rats: the effect of cholate type, particle size and administered dose. Eur J Pharm Biopharm 81:265–72

 PubMed Web of Science ®Google Scholar