Bacterial Ghost as Drug Delivery System

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S.K. Pansare


Bacterial ghosts are non-denatured bacterial cell envelopes that are produced
by the plasmid encoded gene E mediated lysis.1

Bacterial ghosts
are usually obtained from Gram-negative bacteria with fully intact surface
structures for specific attachment to mammalian cells.

They have a natural outer surface make-up which provides them with the original
targeting functions of the bacteria they are derived from and are thus able
to bind to and/or are taken up by specific cells or tissues of animal, human
or plant origin The loaded drug is usually non-covalently associated with
the bacterial ghosts and the drug delivery vehicles themselves represent a
slow release system2-3.



Particularly preferably, the ghosts are derived from Gram-negative bacteria
which are selected, for example, from Escherichia coli, Klebsiella, Salmonella,
Enterobacter, Pseudomonas, Vibrio, Actinobacillus, Haemophillus, Pasteurella,
Bordetella, Helicobacter, Francisella, Brambamella, Erwinia, Pantoea, Streptomyces,
Frankia, Serratia, Agrobacterium, Azotobacter, Bradyrhizobium, Burkholderia,
Rhizobium, Rhizomonas and Sphingomonas
. Particularly preferred examples
of Gram-positive bacteria are Staphyloccoccus, Streptococcus and Bacillus4.

Expression of cloned PhiX174 gene E in bacteria results in lysis of the bacteria
by introducing a transmembrane tunnel structure through the cell envelope
complex. The resulting bacterial ghost have intact envelope structures devoid
of cytoplasmic content including genetic material5.

Advantages of Bacterial Ghost:

1)Bacterial ghosts offer several advantages over traditional vaccines including
the ability to express recombinant proteins in a variety of locations on the
Ghost, their inherent adjuvant properties, their long room temperature storage
life after lyophilization which eliminates the need for a cold-chain distribution
system, and their low cost of production.6

2) Bacterial ghost can be effectively administered orally and aerogenically
as drug carriers. The new system is an alternative to liposomes and may have
an advantage due to its higher specificity for targeting specific tissues,
its easy method of production and its versatility in entrapping and packaging
various compounds in different compartments of the carriers.

3)The main advantages of bacterial ghost as carriers of subunit vaccines include
their ability to stimulate a high immune response and to target the carrier
itself to primary antigen-presenting cells. The intrinsic adjuvant properties
of bacterial ghost enhance the immune response to target antigens, including
T-cell activation and mucosal immunity.

4) Native and foreign antigens can be carried in the envelope complex of bacterial
ghosts, combination vaccines with multiple antigens of diverse origin can
be presented to the immune system simultaneously. Beside the capacity of bacterial
ghost to function as carriers of protein antigens, they also have a high loading
capacity for DNA. Thus, loading bacterial ghost with recombinant DNA takes
advantage of the excellent bioavailability for DNA-based vaccines and the
high expression rates of the DNA-encoded antigens in target cell types such
as macrophages and dendritic cells.

5) There are many spaces within bacterial ghost including the inner and outer
membranes, the periplasmic space and the internal lumen which can carry antigens,
DNA or mediators of the immune response. All can be used for subunit antigen
to design new vaccine candidates with particle presentation technology7.

6) Bacterial ghost can also carry piggyback large-size foreign antigen particles,
increases the technologic usefulness of bacterial ghosts as combination vaccines
against viral and bacterial pathogens. Furthermore, the bacterial ghost antigen
carriers can be stored as freeze-dried preparations at room temperature for
extended periods without loss of efficacy.

7) The potency, safety and relatively low production cost of bacterial ghost
offer a significant technical advantage over currently utilized vaccine technologies.8

8)Advantage of the bacterial ghosts is many antigenic epitopes of the cell
wall complex are presented by the bacterial ghosts. In addition, the lipopolysaccharide
present in the bacterial envelope acts as a mitogen and also triggers a signal
for cell division. As a result, one achieves an effective stimulation of the
B-cell specific production of immunoglobulins9.

(9)An adjuvant effect is achieved due to the bacterial ghost envelopes, which
themselves already have an immunogenic effect

10) The active compound which is enclosed in the ghost is protected from breakdown
by physiological processes, e.g. by enzymes such as proteases, nucleases or

11)In bacterial ghost as drug delivery system it is possible to combine the
active compound with other active compounds


Method of Preparation:

1)E. coli ghosts were filled with the reporter substance calcein and sealed
by fusion with membrane vesicles. By flow cytometry and fluorescence microscopy
it was shown that bacterial ghosts can be filled with calcein, and that the
bacterial ghosts can be sealed by restoring the membranes integrity. The adherence
and uptake studies showed that almost all murine macrophages and a lower proportion
of human colorectal adenocarcinoma cells took up fluorescence labeled bacterial
ghosts. Moreover, these cells also took up effectively sealed E. coli ghosts
filled with calcein, which then was released within the cells10.

2) Step-I) Preparing E. coli Ghosts

E. coli cells were transformed with the lysis plasmid.The
transformants were cultured at 28° C. in LB medium (10 g of tryptone/l, 5
g of yeast extract/l, 5 g of NaCl/l) containing antibiotic. 1 lit medium was
inoculated with an overnight culture, which was derived from a single transformant
colony, and used as a preliminary culture for a fermenter The bacteria were
cultured in the fermenter in a volume of 10 lit, while aerating and stirring,
until an optical density at 600 nm of 0.4 had been reached. After 30 min,
0.2 M MgSO4 was added and, 20 min after that, expression of the
lysis protein E was induced by increasing the temperature from 28° C to 42°
C. After 1 hr, the cells were harvested by being centrifuged at 4000 g. Resuspension
of the pellets in distilled water (final volume 5 l) led to immediate lysis.
The ghosts were washed twice in a large volume of Tris-buffered salt solution
(TBS) and subsequently lyophilized.


Step-II) Preparing Membrane Lipid Vesicles

E. coli cells were cultured at 37° C. in LB medium and harvested
in the late logarithmic phase of growth. After having been washed three times
with phosphate-buffered salt solution (PBS), pH 7.4, the cells were resuspended
in an aliquot of PBS (protein concentration, 26 mg/ml) and frozen at -70°
C. After having been thawed, 10 ml of the suspension were pressed in a French
press at 900 psi (large chamber). Cell residues and relatively large fragments
were removed by centrifugation (6000 rpm, 10 min). The vesicles which were
present in the supernatant were pelleted by ultracentrifugation at 285,000
g (60 min) and, after that, taken up in tris buffer, pH 7.5 (protein concentration,
approx. 4 mg/ml). The vesicle suspension can be stored at 4° C. for approx.
1 week

Step-III) Filling Bacterial Ghosts with Active Compounds

75 μl of a ghost suspension were pelleted at 13,000 rpm (5 min)
and taken up in 75 μl of fusion buffer (100 mM NaCl, 10 mM sodium acetate,
10 mM Hepes, pH 5, 6 or 7), with the active compound with which the ghosts
were to be filled already having been dissolved in the buffer.

Step-IV) Method of fusuion of bacterial ghost:

The membrane fusion
comprises a fusion between the membrane of the bacterial ghosts and the
membrane of lipid vesicles. The lipid vesicles can be derived from natural or
synthetic sources and preferably contain a lipid double layer which contains
phospholipids such as phosphatidylethanolamine. For example, it is possible to
use vesicles which are formed when cells, in particular bacterial cells, are
homogenized, for example by means of ultrasonication or in a French press. On
the other hand, it is also possible to use synthetic lipid vesicles, such as

The bacterial ghosts
are preferably fused with the lipid vesicles under conditions under which both
the membrane of the bacterial ghosts and the membrane of the lipid vesicles are
in a fluid state, e.g. at a temperature of ≧30° C., e.g. 37° C. In
order to achieve a fusion, the membranes are brought into intimate contact,
such that electrostatic repulsion forces between the bacterial ghosts and the
lipid vesicles are overcome and the membranes in the starting materials are destabilized.
Such conditions are achieved, for example, during an ultracentrifugation. Other
methods for overcoming electrostatic repulsion forces are using chemical
fusogens, such as polyethylene glycol, glycerol, DMSO and/or polyhistidine,
which bring about a decrease in the surface potential and consequently a
decrease in the electrostatic repulsion. When phospholipid-containing vesicles
are used, the fusion can be improved by adding calcium. When viral lipid
vesicles are used, the fusion can be improved by the presence of proteins, such
as influenza hemagglutinin, Sendai F protein, Semliki Forest spike glycoprotein,
vesicular stomatitis virus (VSV)-VSVG protein, etc., in the viral membrane11.


1)Bacterial ghost as oral vaccine:

Enterohemorrhagic Escherichia coli
(EHEC) is a bacterial pathogen that is associated with several life-threatening
diseases for humans. The combination of protein E-mediated cell lysis to
produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the
development of an oral EHEC vaccine12.

2) Bacterial ghosts as carrier and targeting systems

Bacterial ghosts are empty cell envelopes
originating from Gram-negative bacteria. They have a natural outer surface
make-up which provides them with the original targeting functions of the
bacteria they are derived from and are thus able to bind to and/or are taken up
by specific cells or tissues of animal, human or plant origin. The extended
bacterial ghost system represents a platform technology for creating new
qualities in non-living carriers which can be used for the specific targeting
of drugs, DNA or other compounds to overcome toxic or non-desired obstacles.

Example: Bacterial ghosts from Mannheimia
haemolytica were used for site-specific delivery of doxorubicin (DOX) to human
colorectal adenocarcinoma cells (Caco-2)13-14.

3) Bacterial ghosts as carriers of enzymes

For specific applications membrane-anchored enzymes like polyhydroxybutyrate
synthase are able to produce a polymer matrix within the ghosts when fed with
the precursor molecule butyryl-CoA. It is envisaged that formation of polymers
within the ghost can be coupled with packaging of chemicals, drugs or nucleic
acids to the matrix material.
Any biotinylated material can be bound within the lumen of ghosts carrying
membrane-anchored streptavidin. When biotinylated alkaline phosphatase was
bound to such ghosts the enzymatic activity of the enzyme was not impaired15.

4) Pharmacologically active materials used in bacterial ghost s a drug delivery

Examples of
pharmacologically active substances are polypeptides such as antibodies,
therapeutically active polypeptides, such as cytokines, interferons,
chemokines, etc., enzymes and immunogenic polypeptides or peptides. Another
example of active compounds is represented by nucleic acids, for example DNA
and/or RNA, in particular therapeutic nucleic acids, for example nucleic acids
for gene therapy, which nucleic acids are preferably present in the form of a
chromosomally integratable vector, or nucleic acids for a nucleic acid
vaccination, antisense nucleic acids or ribozymes. Still other examples of
active compounds are low molecular weight active substances, peptides,
hormones, antibiotics, antitumor agents, steroids, immuno modulators, etc. The
active compounds can be present in the bacterial ghosts in dissolved form, as
suspensions and/or as emulsions, where appropriate in combination with suitable
carrier substances and/or auxiliary substances. Furthermore, the active
compounds can also be diagnostic markers, e.g. fluorescent substances, dyes or
x-ray contrast media.

5) For investigating cellular processes:

Closed bacterial ghosts
which possess metabolic functions, or which have the ability to multiply, but
which contain a functionally limited genome as compared with a natural cell,
can be used when investigating cellular processes

6)Used as a attenuated live vaccine:

The reconstituted
ghosts can be used as an attenuated live vaccine, since the degree of
attenuation can be controlled very simply and reliably on the basis of
appropriate manipulations carried out in the genome.

7)For production of recombinant proteins:

Reconstituted ghosts
can also be used in biotechnology, for example as "reactors" for
producing recombinant proteins, in particular recombinant human proteins, in
industrial-scale processes. The cells according to the invention, possessing a
retarded genome, have an intermediary metabolism which is substantially less
complex than that of the starting cell and can therefore be manipulated
selectively for achieving higher levels of production11.

8) For production of T cell specific cell immune response:

Antigen-presenting cells
have the capacity to stimulate specific T cells after the internalization and
processing of Actinobacillus ghosts, as demonstrated by a strong specific
T-cell response generated against the ghost antigens16.

Patents for the Bacterial Ghost

Title: Closure of bacterial ghosts (6951756)

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Assignee: Rothwell, Figg, Ernst and Manbeck



5.William V Holt, Amanda R. Pickard etal; Reproductive Science and integrated
conservation; Cambridge university press; 298,299 -

About authors:

S.K. Pansare

S.K. Pansare

Corresponding Author*

Final Year B.Pharm, MAEER’S Maharashtra Institute of Pharmacy, Pune-411038

P.V. Kamat


Final Year B.Pharm.MAEER’S Maharashtra Institute of Pharmacy, Pune-411038

S.R. Parakh


Principal and Prof. In Pharmaceutics at MAEER’S Maharashtra Institute
of Pharmacy, Pune-411038


Jagdale Swati C

working as Assistant Professor in Pharmaceutics at MAEER’S Maharashtra
Institute of Pharmacy, Pune-411038.

Mrs. Patil R.A.

Mrs.Patil R.A.

working as Lecturer in Pharmacology at MAEER’S Maharashtra Institute
of Pharmacy, Pune-411038.

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