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ARCHAEOSOMES

Archae bacteria derived


novel liposomes
CONTENTS
Introduction
Archaeosomes
Structural features
Stability
Applications

Conclusion
References
Phospholipids

Polar Head Groups
Lipid bilayers surrounding
an aqueous system
Immuno
liposomes

Hydrophilic
S
lip tealt
os
om h
es

Hydrophobic

Water -soluble
Shortcomings of
conventional liposomes
 Susceptibility for chemical and enzymatic
hydrolysis, auto-oxidation, pH and thermal
instability
 Suboptimal immune response to encapsulated
antigen (lack of depot effect):- adjuvants
required
 Wide gap between the proportion of research at
lab scale and products reaching market.
 Need to develop new formulations to circumvent
the shortcomings
Archaeosomes
Liposomes made from the polar ether
lipids of Archaea
 Provide formulary advantages
 Exhibit excellent physico-chemical
stability
 High efficacy as self-adjuvant in
vaccine delivery vesicles.
Archaeobacteria
and its lipids
Archaea
Highly diverse and abundant,
Differ from eukarya and bacteria
 Genetic, biochemical, structural feature
‘Extremophiles‘

Extra-tough and highly stable membranes help


the microbes survive because the membranes'
lipids are biochemically resistant to breakdown
by oxidation, heat, cold, acid, alkali or salt
THERMOPHILES

HALOPHILES
METHANOGENS

ARCHAE
BACTERIA
EUKARYOTES

EUBACTERIA
Cells of
Methanobrevibacter smithii
Growth requirements of
archaeobacteria are relatively
extreme compared with
eubacteria

Fermentation unit
Total lipids of Archaeobacteria- 5% of cell dry
weight
Core lipids :-Branched, 5-carbon repeating
units forming phytanyl chains (fully saturated)
that are linked via ether bonds to the sn-2,3
carbons of the glycerol backbone.
 Polar head- Phospho,glyco, hydroxy,polyol,amino,
Bra
Sa nche
tur
ate d
Ethe d
r

Archaeosomes Un
Un bra
sa nch
Este tu
ra ed
r ted

Conventional liposomes
Genus Core lipid content

1.Methanogens
a.Methanobacteriales Archaeol lipid(15-90%)

b.Methanococcales Caldarcheol
c.Methanomicrobiales (0-65%)
2 .Halophiles
a. Halobacterials Archaeol lipid(100%)
3.Thermophiles (s-metabolizing)
a.sulfolabales Mainly
b.thermoproteals Caldarchaeol
4.Intermediate group
a.Thermoplasmales Miscellaneous
b.Archaeglobales combination
c.Thermococcales
Transition temperature
 Ester lipids have well-defined phase transition
temperatures (Tc) ⇒ (0 to 60°C.)
 Archaeol lipids-no known phase transition
temperature in -60 to +80°C(lipids in fluid state)
 Attributed to the branched nature of the phytanyl
chains
 Caldarchaeol may undergo phase transition
(-30to+ 15°C), but enthalpy changes are7 fold lower
than those seen with ester lipids, & the presence of
archaeol lipids may quench the phase transition
Lipid membrane of archaeosomes
 Entirely bilayer (exclusively from archaeol lipids /
with mixtures of archaeols & monopolar lipids)
 Monolayer (exclusively from caldarchaeol lipids)
 Combination of mono and bilayers (caldarchaeol
and archaeols)
Particle size
Size of archaeosomes depends on the method used
for their formation &on lipid composition
Inner radius of vesicles increases, as the amount of
caldarchaeol lipid increases
 Caldarchaeol lipids larger vesicles
 Archeol lipids smaller vesicles

Therefore, it is possible to

prepare archaeosomes in various


size ranges as required for
different applications by
 Transmission Electron
Microscopic image

Negatively stained archaeosomes Freeze fractured archaeosomes


Formulation considerations
 Made by methods developed for conventional liposome
 For conventional lipids
 Hydration &liposome formation to be done at a
temperature above the Tc of the bulk lipid
 Archaeobacterial polar ether lipids
In liquid crystalline-like or fluid state at
ambient temperature, and hydrated at room
temperature
Simplifies the archaeosome
preparation protocol
Annealing of Liposomes
Conventional liposomes
 Incubated (for a few hours) at greater than Tc
of the bulk lipid immediately after formation
 Equalization of the packing density between the
opposite sides of the lipid bilayer.
Archaeosomes
 Annealed even at refrigeration temperatures
 Encapsulating compounds that are heat labile
and/or require refrigeration for preservation.
Shelf-life of 1.5-2 yrs
 Auto oxidation-unsaturated fatty acyl chain ester lipids

 Archaeobacterial lipids with saturated phytanyl


chains-Not oxidized in air
 Archeal Lipids can be stored in chloroform / methanol
for years at room temperature.
 Archaeosomes -Stored for upto 16 months (at 5°C) in
the presence of air
ayer structure that spans the vesicle mem
hese structures are more rigid and stabl
 Superior thermal stability & lower proton
permeability of archaeobacterial ether lipid
vesicles were reported on comparison with
vesicles made from ester lipids
 Presence of branched phytanyl chains &
bipolar caldarchaeol lipids
Requirement for in vivo applications.
Liposomes made from ester phospholipids
Heat sterilization-aggregation of vesicles,release of water
soluble compounds,hydrolysis of the ester lipids
Filter sterilization - may not remove virus contaminants &
slow process (large-scale preparation)
Archaeosomes sterilized by autoclaving
without alteration in vesicle (size, stabilty) , no chemical
changes in the polar lipids
Also be sterilized empty subsequently loaded with filter-
sterilized compounds that may be heat labile
High-density lipoproteins (HDL) can remove the
phospholipid from the outer leaflet of the ester lipid-
liposome bilayer- vesicle destabilization , leakage
Archaeosomes made from archaeol lipids alone indicated
that the in vitro stability in serum was similar to that of
liposomes
Stability of caldarchaeol-containing archaeosomes was
much better
The stability of ester lipid liposomes in serum could be
improved by supplementing with caldarchaeol lipids
pH stability
 Poor stability in harsh GI environment
 Limited success in improvements in the stability
of liposomes in the GI tract
 Archaeosomes were more stable (loss 0f 25%)
than conventional Liposomes (60 % loss )of
encapsulated material during storage at pH 2.0
for 21 days & majority of the archaeosomes
appeared to be intact.
Action of lipases

Archaeosomes relatively more stable to attack by lipases,


compared with those made from ester lipids.
Unlike conventional liposomes which lost 80 to 100% of
encapsulated material after 1 to 4 h exposure to
phospholipase A2 at 37°C, archaeosomes made from
archaeol lipids lost less than 20% and those from
caldarchaeol lipids less than 5%, after 4 h exposure
The stability of liposomes could be improved dramatically by
incorporation of archaeobacterial TPL (1:1 ratio by weight)
BILE SALTS
biological detergents
 loss of most of the encapsulated material within 5 to
30 min of incubation at 37°C(in presence of bile)
Exposure of unilamellar archaeosomes to
simulated human bile (SHB) showed similiar
However, work with multilamellar vesicles
indicated that after a 90-min incubation in SHB
about 15% was retained in these vesicles ,
Microscopic observtions–most vesicles appeared
to be intact.
Combination of both
phospholipase and bile salts
 Encountered in GI tract, and this assay condition
would be most relevant in predicting in vivo
stability.
 Not much comparable published work
 Unlike ester phospholipidliposomes , pancreatic
lipase and simulated human bile had no
synergistic effect on the release from
archaeosomes the effect being the same as that of
SHB alone
Uptake by macrophages
 Liposomes rapidly cleared from blood
(t1/2 -few minutes) uptake by the Mononuclear
Phagocytic System
 Delivery of drugs to treat diseases caused by
pathogens residing in intracellular sites
 Advantage for vaccine delivery (most antigen
presenting cells are phagocytic & as such are able to
take up particulate antigens to process and present
to T cells for induction of an immune response)
 The uptake of archaeosomes by phagocytic cells can
be up to 50-fold greater than that of conventional
liposome formulations
Other studies…..
 Tissue distribution studies of archaeosomes
administered via various systemic and peroral
routes indicate potential for targeting to
specific organs.
 Extensivemurine model studies date indicate
that archaeosomes are safe and do not invoke
any noticeable toxicity.
ALL IN A NUT SHELL
Feature Liposome Archaeosome
Source Natural/synthetic Archaeobacteria
lipid
Basic lipid Unsaturated , Regularly
structure unbranched fatty branched,
acyl chains saturated
phytanyl chains
Linkage Ester(sn-2,3type) Ether(sn-1,2 type)
Phase Well defined (0-600c) Not specific
transition
temperature
Feature Liposome Archaeosome
a. Hydration and formation a.Exist in fluid
require temperature state and thus can
above Tc of bulk lipid be hydrated at
Preparation
b. Require incubation room temperature
temperature more than b.Annealed at room
Tc to allow equalization temperature
of packing density
a. Susceptibility to Stable
Stability autooxidation
b. Susceptable to chemincal
and enzymatic hydrolysis
Temp.range-4-65c
c. Thermally & pH unstable
Ph range(2-12)
Vaccine Require co-adjuvants Not require
delivery
In vaccine delivery
 Success of vaccine
Identification of antigen
Elicitation of strong and appropriate immune response
 Limited success of conventional liposomes -Fail to
effectively stimulate immune system in absence of
adjuvants, due to lack of depot effect
 In murine models, systemic administration of
archaeosomes containing encapsulated antigen(s)
elicits strong & sustained antigen-specific antibody
responses comparable to those obtained with
Freund’s adjuvant & more than of conventional or
alum
 Mechanism of action
Ψ Enhance the recruitment and activation of
professional antigen presenting cells in vivo,
and deliver the antigen to both MHC class I
and II pathways for antigen presentation,
Ψ Promoted by phosphatidyl serine receptor
mediated endocytosis
Ψ Variation in induction depends on lipid
composition due to variable receptor-
mediated endocytosis
♥ Archaeosomes compositions can be selected to give
a prolonged, sustained immune response, and the
generation of a memory response
♥ Induce CD8+ cytotoxic T lymphocyte responses
(critical for protection against cancer &
intracellular infections) to entrapped protein
♥ Effective adjuvants for univalent vaccines against
bacterial infections and cancer
♥ Better alternative for developing oral vaccines
delivery system because of better ph stability
♥ Potential for adjuvant for multivalent vaccines
Issues to be resolved
Inability to properly isolate and cultivate
archaebacteria contributing to relative lack
of knowledge.
The cost and availability of archaeobacterial
polar lipids.
Studies are required relating to the efficacy
of archaeosomes for protective immunization
, mechanism of action , and safety.
Summary and conclusion
Novel lipid vesicles from
archaeobacterial membrane lipids
⇒Better stability in harsh
conditions
 Their role in adaptation to
extreme environment makes
them suitable candidate for the
formulation of relatively stable
liposomes that exhibit freedom
from limitations of in-vitro and in-
vivo stability .
References
Girishchandra B. And sprott, G. Dennis (1999),
‘Archaeobacterial ether lipid liposomes (archaeosomes) as
novel vaccine and drug delivery systems', Critical reviews in
biotechnology, 19:4, 317 – 357
Rawat,Saraf ,Archaeosomes –Archaebacteria derived novel
liposomes ,Indian Drugs,44(7)July 2007
Krishnan, lakshmi and sprott, G. Dennis (2003)
'archaeosomes as self-adjuvanting delivery systems for cancer
vaccines*', Journal of drug targeting, 11:8,515 – 524
Girishchandra B., Zhou, hongyan, kuolee, rhonda and chen,
wangxue (2004) Archaeosomes as adjuvants for combination
vaccines ',Journal of liposome research, 14:3, 191 - 202
Krishnan, L., et.al. Archaeosome vaccine adjuvants induce strong
humoral, cell-mediated and memory responses: Comparison to
conventional liposomes and alum. Infection and Immunity 68, 54-
63, 2000.
Krishnan, L, et.al. Archaeosomes induce long-term CD8+ cytolytic
T cell response to entrapped soluble protein by the exogenous
cytosolic pathway, in the absence of CD4+ T cell help. J.
Immunology 165: 5177-5185, 2000.
Patel, Girishchandra B., Omri, abdelwahab, deschatelets, lise and
sprott, G. Dennis (2002) 'Safety of archaeosome adjuvants
evaluated in a mouse model , Journal of liposome research, 12:4,
353
Krishnan, L., et.al. The potent adjuvant activity of Archaeosomes
correlates to the recruitment and activation of macrophages and
dendritic cells in vivo. J. Immunology 166: 1885-1893, 2001