Bilosome – A Bile Salt Based Novel Carrier System

Gaining Interest in Pharmaceutical Research

PROJECT WORK submitted to

SAVITRIBAI PHULE PUNE UNIVERSITY

PROJECT WORK COURSE

IN
FINAL YEAR B. PHARMACY SEMESTER -VIII

DOMAIN-PHARMACEUTICS

Submitted by :
1. Abhinandan Golechha
2. Neha Gawande
3. Shubhangi Ghanawate
4. Pooja Gawade
5. Mangesh Galbale

Under the guidance of-

Prof. K. D. Lone

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 CERTIFICATE
This is to certify that the review work described in the project work entitled,“Bilosome – A Bile
Salt Based Novel Carrier System Gaining Interest in Pharmaceutical Research” is done by
Miss Neha Gawande
Miss Pooja Gawade
Miss Shubhangi Ghanwate
Mr Mangesh Galbale
Mr Abhinandan Golechha
In the partial fulfilment of work done for the award of degree of Bachelor of Pharmacy has
been carried out in the department of Pharmaceutics , JSPM’s Rajarshi Shahu College Of
Pharmacy and Research, Tathawade, Pune-33, under my guidance and supervision. This project
work is now ready for examination.

Project Guide : Principal :

Prof. K.D.Lone Prof. Dr K.R.Khandelwal
JSPM’s RSCOP&R JSPM’s RSCOP&R
Tathawade, Pune- 411033 Tathawade, Pune- 411033

Place : Tathawade, Pune

Date : 09/05/23

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 Acknowledgement

“Keep your dreams alive”, Understand and achieve anything that requires faith, belief in
yourself, vision, hard work, determination and dedication. Remember all things are possible for
those who believe in themselves. I take this privilege to acknowledge the contributions of many
individuals who have been aspiring me throughout the work and endowed me with the most
precious knowledge to see success in my endeavour.

With profound feeling of immense gratitude and affection, I would like to thank my guide
respected Prof. Krishna Lone for continuous support, motivation, enthusiasm, guidance, keen
interest and inspiration.

I am immensely thankful to Dr. K.R. Khandelwal, Principal, JSPMs Rajarshi shahu college of
pharmacy , for providing necessary facilities for my practice school work.
I will be failing in duty if I do not acknowledge with grateful thanks to the authors of the references
and other literatures referred to in this practice school report.

Last but not the least I am very much thankful to my parents who guided me in every step which
I took.

Name of Student
a. Abhinandan Golechha
b. Neha Gawande
c. Shubhangi Ghanawate
d. Pooja Gawade
e. Mangesh Galbale

Date:
Place: Pune

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 INDEX

Sr.No. Contents Page

1 Abstract 4

2 Introduction 5

3 Advantages of bilosomes 6

4 Limitations of bilosomes 7

5 Varieties of bilosomes 7

6 Formulation variables 8

7 Methods of preparation 9

8 Evaluation Parameter of Bilosomes 11

9 Bile Salt Integrated Delivery System 12

8 Conclusion and future prospective

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1.ABSTRACT

Vesicular drug delivery systems are promising agents with diverse applications in
pharmaceuticals, cosmeceuticals and cosmetics. Bilosomes are bilayered vesicles of non-ionic
surfactants and bile salts which are similar to niosomes. Bile salts are endogenous surfactants
which act as a double-edged sword by increasing the aqueous solubility and permeability of active
pharmaceutical ingredient. These vesicular structures increase the solubility of lipophilic drugs
and increase the stability of the formulation in the gastrointestinal tract. Bilosomes are
ultradeformable flexible structures which increases the stratum corneum permeability. Thus these
have applications in both oral and transdermal drug delivery. These vesicles are utilised for topical
drug delivery like ocular and intranasal drug delivery. In addition, bile salt integrated
nanomedicines like probilosomes, surface engineered bilosomes and non-oral bilosomes have
been surveyed. Tremendous research in the last decade has made bilosomes a potential carrier
system. The extensive search has been presented related to the formulation and characterisation of
bilosomes. Bilosomal systems have profound application in biological therapeutics and vaccine
delivery. This review offers a comprehensive and informative data focusing on the great potential
of bile acid and their salts for therapeutic application. In conclusion, bilosomes are superior to
other conventional vesicular carriers (liposome and niosome) with regards to the stability, low
toxicity and bioavailability.

Key words: Vesicular drug delivery system, bilosome, endogenous surfactants, aqueous
solubility, permeability, ultradeformable, nanomedicines, biological therapeutics, vaccine
delivery, bioavailability

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2.INTRODUCTION

Pharmaceutical nanotechnology is the most innovative and highly specialized field which will
revolutionize the pharmaceutical industry in near future. Nanocarriers have been used to
circumvent problems associated with the conventional drug delivery systems. Vesicular carriers
like liposome and niosome have gained great attention during the last few biomedicine

1. Even though they have many advantages, they have disadvantages also . It includes
problems associated with stability and leakage of the encapsulated drug. Bilosome a novel
vesicular carrier composed of non - ionic surfactant and bile salt are very useful in
overcoming the limitations of the conventional vesicular carriers
2. Bilosomes were first described by Conacher et al
3. It was stated that 70 % of new drug candidates were discarded in early stages due to poor
solubility in water . Poor solubility leads to problems with bioavailability
4. These bile salts stabilized vesicles are capable of enhancing the solubility of poorly water-
soluble drugs thereby enhancing the bioavailability. Increased solubility is due to bile salt
micelle formation at critical bile salt micellar concentration. Due to their amphiphilic
property, they can associate in water and form micelle when their concentration is greater
than critical micellar concentration. The stability of the bile salt vesicles in gastrointestinal
tract make it as wonderful carrier for the delivery of proteins, peptides and antigens
5. The stability in gastrointestinal environment is due to repulsion between bile salts in the
vesicle and the external bile salts in solution. Bilosomes have profound application not

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 only in oral drug delivery. It is an effective transdermal drug delivery carrier. These are
nano sized formulations which is optimum for transdermal delivery
6. The conventional bilosomes contain span 60 as non - ionic surfactant and sodium
deoxycholate as bile salt . Bile acids are endogenous surfactants synthesised in liver and
stored in gall bladder and exists as salts. They have surface active and interfacial
properties. They adopt flat conformation at interfaces with rigid steroidal backbone
parallel to the interface allow hydroxyl groups with the aqueous environment. They have
emulsifying, solubilising, and wetting properties
7. It acts as a penetration enhancer in topical dosage forms including buccal, ocular , nasal ,
transdermal routes of administration . Negatively charged bile salts increases the stability
of the formed vesicular system. It has fluidizing effect which enhances the permeability
of the drug. Thus, bile salt stabilized vesicles improve stratum corneum permeability
8. Sodium deoxycholate is the most commonly used bile salt due to it's lipophilic, anionic
nature and high of membrane permeability
9. Span 60 is used in the conventional bilosomal systems because they have higher alkyl
chain length and saturated thus imparting increased surfactant lipophilicity and thus
increasing the efficiency of encapsulation of the drug . Bilosomal systems show biphasic
release profiles which shows an initial burst followed by sustained release of drug
10. Actually these bilosomes are modifications of the conventional vesicular carriers . The
basic difference between bilosomes and conventional vesicles is their composition and
structure.

3. ADVANTAGES OF BILOSOMES

1. Cholesterol Bile salts.Enhanced chemical and storage stability Enhanced stability in

gastrointestinal tract Improves permeability of drug Small particle size Increased
encapsulation efficiency Alters stratum corneum permeability by Sodium deoxycholate
Sodium taurocholate Sodium lithocholate Sodium tauroglycocholate Sodium glycocholate
.
2. Increases aqueous solubility of poorly water soluble drugs loosening intercellular lipid
barriers Highly deformable Prolonged drug release Extended duration of action Reduced
dose , dosing interval of drug Both hydrophilic and lipophilic drugs can be encapsulated

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 3. Bioavailability of drug from bilosome is greater than liposome and micronized form of
drug
4. Easy availability of excipients, low cost Oral, ocular, buccal, nasal, transdermal delivery is
possible
5. Topical application helps to overcome systemic side effects, prevents first pass, avoids
degradation of drugs in the stomach by acids and enzymes

4.LIMITATIONS OF BILOSOMES

1. Poor in vitro in vivo correlation due to lack of in vitro method that mimic in vivo condition
thereby increasing the difficulty in characterization
2. Incorporation of anionic hydrophilic drug cause migration of drug to external space thereby
decreasing the encapsulation efficiency of drug
3. Minor irritation

5.VARIETIES OF BILOSOMES

a) Probilosomes

These are dry, free flowing granular products that instantaneously form bilosomal dispersion on
ingestion. Due to its dry nature their stability is high. They have high permeability also. Formation
of a lipophilic ion pair between the drug and the bile salt increases the encapsulation efficiency of
drug.

b) Non oral bilosomes

Transdermal permeation of drug is increased by bile salt containing vesicles. Ultra deformable
liposomes for transdermal application are called as transferosomes. These are otherwise referred
as non-oral bilosomes because these ultra-deformable structures contain bile salts.

c) Surface engineered bilosomes

The surface of the bilosomes is modified for drug targeting and to improve the stability of the drug.
Glucomannan modified bilosomes are used for oral administration of Tetanus Toxoid. This
enhances the chemical and conformational stability of entrapped Tetanus Toxoid. The
immunological response of the encapsulated drug also can be improved. The stability of
Glucomannan against digestive enzymes is attributed by its polymeric nature. Better immune

8
response was elicited after oral administration of Glucomannan bilosomes compared to
conventional bilosomes. This was related to the existence of high density of mannose molecules
over the bilosome surface that resulted in more precise recognition and binding of bilosomes to
mannose receptors.

d) Bile salt liposome

Liposomes are vesicular carriers commonly used for drug delivery. Orally administered liposomes
have low solubility in the gastrointestinal tract because of the acids and enzymes present in the
stomach. The physiological bile salts have membrane disruptive effects thereby producing
premature drug release prior to intestinal absorption. Thus, liposomal vesicles are stabilized by
addition of bile salts. The increased stability is due to repulsion between bile salts present in the
vesicles and external bile salts

6.FORMULATION VARIABLES

a) Type of non-ionic surfactant Span type surfactant is commonly used. This is because they have
high phase transition temperature and thus increases the efficiency of encapsulation. Among
the span type surfactant span 60 is commonly used. Span 60 has higher alkyl chain length than
span 40 and thus will have increased surfactant lipophilicity and increased encapsulation
efficiency. Span 80 has unsaturation and thus it's vesicle permeability is high and reduces the
encapsulation efficiency. Absolute zeta potential of bilosomes prepared by using span 60 as
surfactant is high
b) Amount of lipid Cholesterol is the lipid component. It also increases the flexibility of the
vesicle and imparts fluidizing effect. The amount of cholesterol determines the particle size
and encapsulation efficiency. When the amount of cholesterol is high there is increased
interaction of cholesterol with hydrophobic tail of span 60 which result in close packing and
decreases the particle size of vesicles. Due to this close packing the encapsulation efficiency
is also increased. But when the cholesterol concentration is increased further it causes
perturbation of bilayer and expulsion of drug from vesicles. Increased concentration of
cholesterol reduces drug leakage thereby decreasing the percentage drug release .
c) Type and amount of bile salt
d) Anionic bile salts are commonly employed because they can produce stabilized vesicles.
Sodium deoxycholate is commonly used because it is more anionic, lipophilic and can form

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 ion pair with drugs. Bile salts increases the flexibility of bilosomes and reduces the membrane
tension resulting in small particle size. It also reduces leakage of the encapsulated drug.
Incorporation of bile salts produce a sustained release of drug. Anionic bile salts improves
stability by reducing
e) Sonication time
f) Increasing sonication time reduces vesicle size increasing the lamellar diffusion area thereby
increasing the drug release. Sonication method also affects the particle size.

7.METHODS OF PREPARATION

a) Hot homogenization

method It is also called as melt method. Monopalmitoyl glycerol, cholesterol, diacetyl phosphate
were heated at 130°C. Bile salts like sodium deoxycholate in buffer was taken and added to the
above mixture and vortexed. Non entrapped bilosomes were removed by centrifugation and
entrapped one's were resuspended in appropriate buffer .

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b)Thin film hydration

It is often referred as lipid film hydration. Drug, non- ionic surfactant and cholesterol were taken
in round bottomed flask and dissolved in suitable organic solvent. The organic solvent was
evaporated using rota-evaporator to obtain a thin film of lipid. Ensure complete evaporation of the
solvent. The formed film is hydrated using distilled water or buffer containing the bile salt and
stirred in a magnetic stirrer to obtain bilosomal dispersion. The particle size was further reduced
by sonication. The dispersion is stored at 4°C until characterisation. Multilamellar vesicles
prepared by thin film hydration can be converted into small unilamellar vesicles by using
membrane extrusion. ultrasonication, homogenization.

c)Ethanol injection

In this method Drug, non-ionic surfactant and cholesterol were dissolved in ethanol in a water
bath. The ethanolic solution was slowly injected into phosphate buffer pH 7.4 and is magnetically
stirred. Bile salts and other edge activators are added formerly to aqueous phase. Bilosome
dispersions are formed which are indicated by turbidity of solution. Stirring was continued to
ensure complete volatilization of ethanol. Dispersions are cooled at room temperature and
sonicated.

d)Reverse phase evaporation

In this method phosphatidyl choline and bile salt were dissolved in ethyl ether. Buffer solution
containing the drug was added to it. It is subjected to ultrasonication. It results in the formation of
reverse water in oil emulsion. The solvent is removed by rota-evaporator under reduced pressure.
Dried lipid thus obtained is hydrated by buffer to form homogenous aqueous vehicle dispersion
that was extruded through a high-pressure homogenizer.

e) Probilosomal method

In this method sorbitol particles were located in a round bottomed flask. This is then vacuum dried
in a rotary evaporator. Then the solution of phosphatidyl choline, bile salt and drug were dissolved
in organic solvent were added in drop wise manner into the round bottomed flask to load them
onto sorbitol particles. Loaded sorbitol particles are freeze dried to obtain probilosomal powder
which is then converted into bilosome by manual agitation in water.

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8.EVALUATION PARAMETER OF BILOSOMES

Particle size

Polydispersity index, Zeta potential Bilosome dispersion is diluted to 10 fold using distilled water
and measured the particle size, poly dispersity index and zeta potential using zeta sizer. The
measurements are taken in triplicate.

Encapsulation efficiency

Iml of the bilosome dispersion was taken and centrifuged at 15,000 rpm for 2hour at 4°C. The
supernatant was taken, suitably diluted and determined spectrophotometrically for the drug

Transmission electron

microsc transmission electron micros selected bilosome dispersion was ausorbed copper grid,
negatively stained with 1% phosphotungistic acid and then air dried at room temperature for 10
min before TEM observation.

Differential Scanning Calorimetry (DSC)

Apparatus was calibrated. Bile salts, cholesterol, non-ionic surfactant, drug loaded bilosomes and
blank bilosomes are investigated. 3mg samples were placed in standard aluminium pans and heated
from 10°C to 200°C at a scanning rate of 10°C/min.

Invitro drug release studies

Dialysis bag method is used. Dialysis bag was overnight immersed in the release medium. The
medium is 50 ml phosphate buffer pH 6.8 and maintained at 37+0.5°C. 3ml samples were
withdrawn at 1, 2, 4, 6, 8, 10 and 24 hour and substituted with equal volume of medium. Withdrawn
samples are suitably diluted and analysed spectrophotometrically to determine the drug release.

Storage stability

The prepared bilosomes are stored at refrigerated temperature (4+0.5°C) and at room temperature
(25+2°C) at 70% relative humidity for 90 days. The stability of the prepared bilosomes was
evaluated at 0, 45, 90 days.

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9. BILE SALT INTEGRATED DELIVERY SYSTEMS

Recently, BSs have been exploited as a core for versatilenanosystems. From a review of the
literature, major categories of BS-integrated nanocarriers can be classified as follows. Figure 1
summarizes the four major BS- integrated nanosystems.

9.1. Size-tunable cholate nanocarrier

Recently, a unique class of amphiphilic copolymers was introduced into the field of cancer
targeting and gene delivery based on BS core. Such polymers are composed of a hydrophilic linear
hydrophilic biodegradable branch such as PEG [polyethylene glycol], dextran, pullan, chitosan,
and PLGA [poly (lactic-co-glycolic acid)] and a flexible two-arm linear oligomer of cholic acids

as a hydrophobic core-forming block. Owing to hydrophilicity of the surface, such amphiphilic

structures would spontaneously form self-nanoaggregates in water promoted by intra- and/or
intermolecular association between hydrophobicsegments to minimize interfacial free
energyFurthermore, the resultant nanocarriers exhibit tunable properties owing to the rigidity of
the steroidal polycyclic backbone and the amphiphilic properties of bile acids. Consequently,
biodegradable nanocarriers based on bile acids are of captivating potential in the field of drug
delivery.

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9.2. Bile salt-phospholipid mixed micelles

Bile acids can form mixed micelles when combined with polar lipids, conventional surfactants or
amphiphilic drugs, usually with lower CMC and better solubilization capacity than the individual
components due to synergistic interactions. Additionally, mixed micellar systems can be prepared
without the aid of organic solvents and are applied in pharmaceutical formulations to improve the
bioavailability of poorly water soluble drugs 28-29. Moreover, phospholipids such as lecithin
buffer the detergent properties of bile salts and attenuate their cytotoxic effects due to mixed
micelle formation 30. A commercial formulation for intravenous administration of diazepam, a
lipophilic benzodiazepine, employs mixed micelles of glycocholic acid and soy lecithin as
solubilising agent 30. The antifungal drug amphotericin B, a hydrophobic polyene antibiotic, is
commercialized as micellar dispersion with sodium deoxycholate for parenteral administration.
The formulation takes advantage of the facial amphiphilic nature of both the drug and the bile acid,
which promotes their association

9.3. Bile acid-polymer nanocarriers

Polymeric nanoparticles represent a new generation of therapeutic delivery platforms with several
advantages over lipid-based carriers, such as micelles or liposomes, due to their smaller size and
higher cell-penetrating capacity, showing enhanced circulation time and preferential accumulation
at the target site via the enhanced permeability and retention effect 31. In this context, lipid-
polymer hybrid amphiphiles obtained by conjugation of bile acids with hydrophilic and
biodegradable polymers constitute promising drug delivery systems. These hybrid amphiphiles
can self-assemble in aqueous environments to form nano-sized micelles with a unique core-shell
structure, made of a hydrophobic bile salt core stabilized by a hydrophilic corona 26. The size of
both core and corona-forming blocks is relevant for drug loading, and higher entrapment efficiency
of hydrophobic drugs is associated with larger cores, whereas the increase in polymer length of
the hydrophilic corona-forming block contributes to increasing the CMC. Some of these systems
formed micelles with sizes between 10–100 nm, which has been reported as the optimum size for
passive tumor targeting with minimum liver uptake 26. The solubilization efficiency of PEGylated
bile acids was found to depend on the nature of the bile acid and on the length and number of
grafted PEG chains 32. Self-emulsifying drug delivery systems (SEDDSs) based on PEGylated
bile acids and oleic acid enhanced the solubilization and absorption of itraconazole, a poorly water-

14
soluble antifungal agent, providing a controlled release system with significant improvement of
itraconazole bioavailability in rats 33

9.4. Bile acid-drug conjugates

Bile acid-drug conjugates act as Trojan horses taking advantage of the organotropism of bile acids
in the enterohepatic circulation for specific drug targeting to the liver or to improve metabolic
stability and enhance intestinal absorption of poorly water soluble drugs making use of the bile
acid transport systems 34Conjugation of therapeutic peptides, proteins and oligonucleotides to bile
acids increases metabolic stability and improves intestinal absorption and systemic bioavailability

of the macromolecules. Recombinant human insulin has been covalently attached to deoxycholic
acid to obtain orally active insulin analogues . Conjugation of cytotoxic agents with bile acids can
also improve the pharmacokinetic properties of the drug while targeting it to the liver. For example,

15
conjugation of cytarabin with cholic acid showed potent antitumor activities on cytarabine-
sensitive HL60 cells, excellent targeting to the liver, good absorption and longer half-life in vivo
compared to cytarabine alone

9.5.1 Oral drug candidates

Amenability of bilosomes for enhancing oral availability was not confined to hydrophilic drugs
entrapped in hydrophilic core. Few hydrophobic actives have been successful candidates for
bilosomes as well where the drug would be entrapped in the phospholipid (PL)bilayer of the
vesicles. In this context, Chen et al have investigated the oral bioavailability of fenofibrate-loaded
bilosomes and conventional liposomes compared with the micronized form of the drug.
Bioavailability from bilosomes was significantly higher than that from liposomes and the
micronized form. Guan et al. loaded Cyclosporine in bilosomes in comparison with conventional
liposomes and marketed a microemulsion product (Sandimmune® and Neoral® [Novartis
Pharmaceuticals Corporation, East Hanover, NJ, USA]). Among others, bilosomes exhibited the
lowest in vitro drug release but the highest oral bioavailability.

9.5.2. Oral Immunization

Oral immunization is always preferred overconventional immunization achieved through

theparenteral route because of the associated highproduction costs associated with the need for a
sterileenvironment, pain at the site of administration,uneasiness to the patient and risk of
diseasetransmission. Moreover, oral immunization isconsidered superior because it induces
mucosal immuneresponse (i.e. induction of sIgA), which is thepredominant entry site for most of
the infectiouspathogens

Bilosomes in oral immunization with model antigen

Conacher et al. reported oral immunization using BSA. The orally administered bilosomal
formulationcontaining BSA induced high antibody titers against it,which were found to be
equivalent to those generatedafter systemic immunization. Singh et al. formulatedBSA-loaded and
CTB-conjugated bilosomes to enhancetheir affinity toward M cells of Peyer’s patches

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Bilosomes in oral immunization against influenza

Conacher et al. reported oral immunization using aninfluenza subunit vaccine and demonstrated
that orallyadministered formulation induced high antibody titersand cell-mediated response as
compared with systemicimmunization. T helper type 1 and type 2 responseswere found to be
induced Mann et al. optimizedbilosomes containing A/Panama influenza hemagglutinin and
demonstrated that bilosome-entrapped influenza HA not only induced significant specific systemic
antibody production but also mucosal IgA. Whereas parenterally administered inactivated
influenza vaccines resulted in protection from homologous viral infection by induction of serum
anti-HA IgG antibodies, they were relatively inefficient inprotection against variant strains arising
during theinfluenza season

Bilosomes in oral immunization against hepatitis B

Arora et al. reported oral immunization against hepatitisB virus using mannosylated bilosomes.
Immuneresponse was found to be significantly higher along withenhanced sIgA level at all local
and distal mucosal sitesas compared with bilosomes alone, whereas parenteralvaccine was
unsuccessful at providing any considerable cell-mediated response. Shukla et al. reported
oraldelivery of recombinant HBsAg using bilosomes

Bilosomes in oral immunization against tetanus

Mann et al. reported the significant systemic andmucosal immunity on oral immunization with
Tetanus toxoid-loaded bilosomes The Tetanus toxoidentrapped in bilosomes was found capable of
inducing The response characterized by systemic IgG1. A cleardose-dependency was observed
with specific Tetanus toxoid IgG1 antibody titers, which was induced only with a higher
concentration of Tetanus toxoid (200mg/dose), not with the lower one (40 mg/dose). In addition
to antibody production, only the Tetanus toxoidentrapped in bilosomes resulted in SIgA
antibodies. The endpoint antibody titers were superior to oral administration of the un-entrapped
antigen, but comparable to parenterally delivered Tetanus toxoid.Only Th2 and IgA responses
were induced with orally delivered, entrapped antigen

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Bilosomes in oral immunization against diphtheria

Katare and coworkers developed nano-bilosomes containing Diphtheria toxoid prepared by thin
film hydration that was characterized in vitro for their shape,size, percent antigen entrapment and
stability and in vivofor anti-Diphtheria toxoid IgG and anti-Diphtheria toxoid sIgA response in
serum and in various body secretions, following oral immunization with different doses of
Diphtheria toxoid entrapped in nano-bilosomes

The highdose-loaded nano-bilosomes (Diphtheria toxoidNB3, 2 Lf) produced comparable anti-

Diphtheria toxoid IgG levels in serum to those induced by IM-administered alum-adsorbed
Diphtheria toxoid (0.5 Lf).All nano-bilosomal formulations elicited a measurableanti-Diphtheria
toxoid sIgA response in mucosal secretions, whereas IM-administered alum-adsorbedDiphtheria
toxoid (0.5 Lf) was devoid of this response.The orally administered nano-bilosomal Diphtheria
toxoid formulation produced comparable serumantibody titers to IM administered alum-
adsorbedDiphtheria toxoid at a fourfold higher dose and withoutthe induction of tolerance.

9.6. Nonoral bilosomes

In a paradoxical effect for oral bilosomes where BS content strengthens the wall against digestion,
BS-modified liposomes have been utilized via other routes as permeation enhancers due to
elasticity. In this context, transdermal transferosomes have been investigated in numerous research
articles for enhancing percutaneous drug absorption. Transferosomes are ultradeformable
liposomes that contain different edge activators including BSs Transferosomes containing BSs
demonstrated efficacy in improving transdermal permeation of cosmetics and pharmaceutical
molecules when used in concentrations lower than 0.2% [Sodium cholate demonstrated efficacy
as edge activator as equal as Tween-80 and Span-80 in improving transdermal permeation of
estradiol. The application of bilosomes in corneal penetration is a novel field that has not so far
been fully investigated. Dai et al have considered elaboration and corneal permeation of bilosomes
as novel ocular delivery systems for tacrolimus. A previous study has indicated that liposomes
loaded with tacrolimus can facilitate penetration of the drug across the cornea into the aqueous
humor However, transcorneal permeation from liposomal suspension was still too small to
achievea therapeutic effect. Realizing poor corneal permeation of drugs and delivery systems, the
authors investigated ocular tacrolimus bilosomes containing different types of BSs as permeation
enhancers.

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9.7. Probilosomes

Nanocarrier preconcentrates can spontaneously form the cgorresponding liquid-state

nanomedicine upon ingestion. Because they are deprived of water, self-nanocarriers possess higher
physical stability compared with liquid nanocarriers. Proliposomes are dry, free-flowing granular
products that instantaneously form multilamellar liposomal dispersion upon ingestion. It was
reported that oral delivery of liposomes could be improved by enhancing their ability to retain their
integrity at the site of absorption, which could be achieved by formulating them into proliposomes

. In this context, Song et al. have developed proliposomes using STDC (2.5%, w/w) for the oral
delivery of salmon calcitonin. They reported a 7.1-fold increase in the calcitonin bioavailability
from probilosomes. They assumed that proliposomes are superior in protecting calcitonin against
possible degradation by gastric fluid. Furthermore, entrapment efficiency and permeation of
probilosomes were higher compared with the proliposomes that could be attributed to the
formation of a lipophilic ion pair between the drug and BS 54

9.8. Surface-engineered bilosomes

Surface modification of bilosomes has been recently proposed in a few research articles attempting
to increase stability and targeting efficiency of the nanovesicles. In the field of oral immunization,
surface-modified vesicles were reported by anchoring a suitable ligand for a variety of receptors
(such as mannosyl galactosyl, folic acid and fibronectin) preferentially and abundantly present on
the cell surface of antigen-presenting cells in mucosal linings. In this context, Jain et al developed
novel glucomannan-modified (GM) bilosomes for eliciting an immune response following the oral
administration of tetanus toxoid. The authors compared three types of vesicular systems, niosomes,
bilosomes, and GM-bilosomes. All vesicular systems exhibited comparable in vitro quality
attributes (particle size, zeta potential, and entrapment efficiency). Nevertheless, GM-bilosomes
showed a higher immunological response parallel to maintained chemical and conformation
stability of the tetanus toxoid entrapped. Results revealed significant immunological superiority of
GM-bilosomes to conventional bilosomes and superiority of both formulations to niosomes, oral
and IM peptides. The authors attributed this improvement to the polymeric nature of GM that
increases the surface functionality by increasing mannose molecules density on the surface, which

19
can enhance the uptake of mannosylated bilosomes by the antigen-presenting cells. Furthermore,
the polymeric nature of GM can also provide stability against digestive enzymes. The ability of
glucomannosylated bilosomes to improve targeting efficiency for oral immunization with bovine
serum albumin has also been reported

PREPARATION OF BS-VESICLES

Based on the reviewed literature, BS-vesicles were prepared using different methods are
schematically illustrated in examples of various bilosomes prepared by different methods
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

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2. Enhancement of vaccine immunogenicity

It is 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 Diphtheria toxoid in providing transmucosal immunization was investigated by Shukla
et al . 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 intramuscular
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, prepared by Shukla et
al, produced anti-HBsAg-IgG and anti-Tetanus toxoid-IgG levels mice serum similar to IM
administered HBsAg and Tetanus toxoid

7. 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 . However,

21
some researchers have shown that PL can reduce the noxiousness of bile salts . In this domain, Niu
evaluated the cytotoxicity of BS-liposomes in concentration range of 0.25– 6.25mmol/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 concentrations. Similarly, Dai et alevaluated 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 (54mg/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

8. TRANSPORTATION OF BILOSOMES TO PEYER’S PATCHES (M CELLS)

The M cells of Peyer’s patches can transport macromolecules, particles and microorganisms
directly into intestinal Peyer’s patches. The apical surface of M cells differs from intestinal
absorptive cells because they lack a brush border. M cells also contain a large number of endocytic
vesicles to uptake and transport lumen contents across the epithelial layer . A unique feature of M
cells is the presence of an intraepithelial pocket in which transcytosis particles and macromolecules
are delivered. The pocket contains lymphocytes and a few macrophages, which interact with the
transported antigen or microorganisms. Thus the transport of soluble and particulate antigen by M
cells is an important primary step in producing a mucosal immune response Additionally, the
cellular processes of the M cells, which extend into the underlying lymphoid tissue, provide
potential contacts with resident lymphocytes and dendritic cells. The M cells can also secrete IL-
1, which indicates that M cells could provide co-stimulatory signals, such as cytokines and cell
surface molecules, to T cells and B cells in the microenvironment of Peyer’s patches. salts
liposomes by intestinal epithelium after oral administration CLSM studies of transportation of
bilosomes .

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GALT

The CLSM studies by Shukla et al. revealed that, after administration of R-123-loaded
bilosomes, the localized fluorescence in the GALT region was found to be much higher as
compared with the unentrapped R-123 on oral administration

. This study confirmed the efficient uptake of bilosomes by the GALT and demonstrated the
capability of bilosomes for transporting the vaccine antigens to the Peyer’s patches containing M
cells. Bilosomes protected the vaccine antigen during its passage through the hostile GIT

The CLSM study

assured the nano-bilosomes mediated effective uptake of vaccine antigens by GALT on oral
administration, which eventually escorted the comprehensive immune response.

9. TRANSLATIONAL MODIFICATIONSAND INDUSTRIAL SCALE-UP OF

BILOSOMES

In translational research, from laboratory bench scale-up, it is necessary to optimize the

manufacturing process to improve efficiency and simplify production, giving a more economical
end-product. Bennett et al. tested two simplified production methods along with two different
storage methods (lyophilized and non lyophilized) as well as looking at the effect of buffer pH 67.
The formulations were assessed in a murine system for immunogenicity,

alongside characterization in terms of size and antigen entrapment, with the stability of these
aspects assessed with respect to time. Lyophilized and non lyophilized 3-step formulations induced
significant IgG1, IgG2a and IgA titers, with the lyophilized version showing stable size and
antigen entrapment for up to 9 months.

CONCLUSION AND FUTURE PERSPECTIVES

Bilosomes are nanovesicular carriers for drug delivery. They have applications in vaccine delivery,
protein/peptide delivery, direct nose to brain delivery for treatment of brain diseases like
Alzheimer's and migraine. Thus they have biomedical and pharmaceutical application in drug
delivery, cancer therapy and diagnostics. As they improve the solubility and permeability, they
enhance the bioavailability of drug thereby increasing therapeutic effectiveness. They can be given

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by various routes including oral, transdermal, nasal, ocular, buccal. Thus bilosomes have profound
application in drug delivery and therapeutics. Conclusion and Future Perspectives

Vesicular drug delivery systems are emerging with the diverse application in pharmaceuticals,
cosmetics and cosmeceuticals and food industries. The delivery of drug directly to the site of
infection and it’s leading to reduction of drug toxicity with no adverse effects. It also reduces the
cost of therapy by imparting better biopharmaceutical properties to the drug, resulting in improved
bioavailability and especially in case of poorly soluble drugs. Oral administration remains the
favoured method of drug delivery by patients and drug to be successfully absorbed by the body. It
requires the active molecule to transit through the stomach and then to be transferred across the
intestinal wall to the bloodstream. For a majority of vaccines and biologic therapeutics this is not
an option due to a 1% uptake of the dose given. Approximately 1/3 of small molecule drugs also
face oral absorption challenges many of these are focussed on CNS targets.TechnologyScientists
at Strathclyde have developed bilosomes and bile salt stabilised vesicles that act as an envelope to
protect their contents from the harsh environment of the gut enabling oral administration. The
system was well tolerated in vivo toxicology studies.Markets and

Bilosomes has been reported as an effective in the delivery of vaccines (currently under license)
and can be applied to the delivery of biological therapeutics and traditional small molecule

A close examination of the literature accentuates twomajor restraints during bilosomal

development. Poor in vitro/ in vivo correlation is a common drawback with a lack of in vitro
method that mimics the actual conditions. Regular in vitro release methods lack the biological
constituents significantly affecting vesicular digestion (BSs and enzymes). Using ex vivo
permeation modmels and regular cell lines to assess vesicular permeability would lack this factor
as well.

A proposed solution would involve employment of the in vitrodigestion-Caco-2 cell model 68

This would encompass digestive enzymes and BS content in addition to amenability for assessment
of depot liposomal formulations.

Although bilosomes proved to be a successful carrier for cationic water-soluble actives, sufficient
loading of anionic active is still an obstacle 26Taking into consideration negative charge and
hydrophilicity of BSs, incorporation of cationic active would hold the BS in the bilayer to exert its

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membrane-stabilizing effect. Nevertheless, incorporation of anionic hydrophilic drugs would be
accompanied by low entrapment efficiency and migration of both hydrophilic BSs and active to
external phase 9 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 delivering by Conacher et al. in 2001,
investigations by different research groupsin BALB/c mice confirmed the usefulness of oral
bilosomes entrapping antigens in alerting both mucosal and systemic immune responses 37

. In addition, surface engineered bilosomes (CTB-bilosomes and GM-liposomes) prepared via

anchoring ligands to the bilosomal surface demonstrated the ability of targeting the antigens into
specific immune cells.

The availability and low cost of bile acids, which can be easily derivatized, turns these chiral
templates into attractive building blocks for the design of novel drugs and drug carrier systems 69
Realizing the importance of vaccines and their delivery challenges, the 21st century has witnessed
an enormous amount of research work in the domain of vaccine development. The development
of a needle-free,painless, patient-compliant and orally active vaccine suitable for mass vaccination
campaigns in the developing countries is warranted Accomplishing this objective, however, has
been constrained by the fact that purified protein antigens usually induce systemic
nonresponsiveness rather than active immunity by orally administered vaccine antigens

Moreover, acid degradation in the stomach and poor permeability across the gastrointestinal
mucosa further limit the uptake of antigens by M cells, which is an essential step for an immune
response. Therefore, the development of an effective oral delivery system for mucosal vaccines is
asignificant challenge for the immunologists. In this regard, various lipid-based delivery systems
including bilosomes have been increasingly researched and developed for oral immunization.

However, more research is needed to elucidate the mechanisms of selective transport of antigens
to the intestinal lymphatics by bilosomes, specifically processes at the cellular level including
digestion, uptake and intracellular metabolism. Additionally, delivery of a broader range of

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candidate antigens having different physicochemical characteristics and instability in the GIT must
be tested.

Further advanced study of different aspects of bilosomes for lymphatic delivery is requirednot
only to overcome these issues but also to developmore- efficient and smart bilosomal vaccine
deliverysystems for better immunization against different fatal diseases. Use of this biocompatible,
stable, and highly specific carrier for vaccination in the near future would contribute to a global
countermeasure against infectious diseases, and would greatly benefit in the eradication of the
same

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 58. Besides, the
precise mechanisms of facilitated .

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