Note: Descriptions are shown in the official language in which they were submitted.
WO 95/09610
PCT/GB94/02191
1
The present invention relates to liposome preparations capable of use
. in administration of organic solvent labile materials, such as whole
live or attenuated cells, to human or animal bodies. Such preparations
have utility in delivery of labile bioactive materials whereby a slow
release is provided which may be targeted to specific body areas. A
method for the manufacture of such preparations is also provided.
The use of liposomes in the administration of vaccine agents is well
known, and their adjuvant activity has been demonstrated by numerous
studies into immunopotentiation of a large variety of bacterial,
viral, protozoan, protein and peptide vaccines; see reviews by
Gregoriandis G (1990) Immunol Today, 11, 89-9'7 and Alving C R (1991)
J Immunol Meth, 140, pl-13.
These studies have all been carried out using liposomes produced by
techniques which generate vesicles of submicron average diameter (see
Gregoriadis G (ed) (1993) Liposome Technology, 2nd Edition, Volumes
I-III CRC Press, Boca Raton, 1993) which are capable of accomodating
peptides and proteins, but not capable of efficiently carrying larger
vaccines. Such larger vaccines include a number of attenuated or
killed viruses and bacteria such as measles, polio virus, Bordetella
pertussis, Bacille Calmette-Guerin and Salmonella tyQhi (see Mimms C A
et al (1893) Medical Microbiology, Chapter 36, Mosby).
Although most of these vaccines are highly immunogenic, there are
circumstances where their administration in sufficiently large
liposomes may be a preferred alternative. For instance, in the case
of multiple vaccines consisting of a mixture of soluble and
particulate (eg. microbial) antigens or vaccine formulations also
containing cytokines, simultaneous presentation of all materials to
immunocompetant cells via a common liposome carrier may be
advantageous in terms of improving the immunogenicity to antigens.
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2
Furthermore, liposomes incorporating antigenic material in their
aqueous phase are known to prevent interaction of the antigen with its
antibodies in pre-immunized animals and ensuing allergic reactions or
antigen neutralisation (Gregoriadis and Allison (1974) FEBS Lett., 45,
71-74. It can thus be seen that liposomes could be beneficial if
employed as carriers for administration of vaccines to infants for
prophylaxis against agents for which maternal antibodies were present,
eg, such as measles, or to individuals with hypersensitivity to
vaccine contaminants.
It is known to incorporate particulate materials into large liposomes
having average diameter up to 9.2 um by methods wherein solvents such
as chloroform are formed into spherules containing smaller water
droplets (see Kim and Martin (1981) Biochimica et Biophysica Acta,
646, 1-9). Using this technique materials such as Collagen, DNA and
bacteria (Streptoco » alivar~~m ) were entrapped, but it was noted
that labile globular proteins such as serum albumen and haemoglobin
did not allow liposome formation, presumably due to surface
denaturation, and that protein denaturation occurred. Such method is
unsuitable for the encapsulation of labile materials due to the
damaging and cytotoxic effects of the organic solvent, and certainly
unsuitable for the encapsulation of whole (live) or attenuated
bacteria, protozoa, viruses or multicellular animal or plant cells.
Methods for entrapping soluble materials in liposomes without use of
organic solvents in the encapsulation step have been known for several
years (see Kirby and Gregoriadis (1984) Liposome Technology, Vol I,
Gregoriadis G (ed), CRC Press, Inc Boca Raton, FL, ppl9-28; Deamer
and Uster (1983) Liposomes, Ostro M J (ed) Marcel Dekker, Inc, NY.
pp27-51; Deamer and Barchfield (1982) J Mol Evol 18, 203-206), and
are based upon a method which dehydrates preformed liposomes then
rehydrates them in the presence of the soluble materials. In these
methods the soluble materials enter with water as the liposomes fuse
together resulting in material being entrapped in multilamella
WO 95109610 ~ ~ ~ PCT/GB94/02191
3
liposomes. The liposomes used were 40 to 80 nm in diameter before
freeze drying and the multilamellar product vesicle volume resulting
was still smaller. Such volume and structure are unsuitable for
encapsulating micrometer size and/or living materials, and entrapment
levels for soluble drugs are not as high as for unilamella liposomes
due to relatively low surface area for entry into the vesicles. The
same technique has also been applied to small unilammela liposomes for
the purpose of encapsulating aqueous solutions (see EP 0171710).
The aforesaid process is relatively mild and has been used to
successfully encapsulate labile solutes such as factor VIII (see Kirby
and Gregoriadis (1984) Biotechnology, 2, 979-984) and tetanus toxoid
(Gregoriadis et al (1987) Vaccine, Vol 5, p145-151). It relies upon
solute entering the liposomes as they form while rehydration water
enters. Despite such work on solutes, there has still not been
provided a method for the encapsulation of whole (live) or attenuated
organisms, cells or other insoluble structures bearing labile
entities, without damaging them; whether bacterial, protozoan, viral
or otherwise.
Furthermore, no method has yet been provided for encapsulating water
labile soluble materials in larger liposomes, whether unilamellar or
multilamella, that would allow targeting at specific tissues with
still higher quantities of material.
The present inventors have now surprisingly found that dehydration
Jrehydration is capable of successful encapsulation of insoluble
particulates such as whole live or attenuated organisms, cells, or
microscopic water insoluble structures having organic solvent labile
activity, whereby organisms are not killed and activity is retained.
The invention allows micrometer sized unilamella and multilamella
liposomes to be produced, (ie. 0.1-50um diameter liposomes) which in
contrast with the liposomes of the prior art, are capable of
entrapping micrometer size and/or living material, and have inner
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4
vesicles of relatively high capacity, being similar in size
to their outer diameter in the case of the unilamellar giant
liposomes.
It is particularly surprising that (i) when micrometer sized
liposomes are dehydrated then rehydrated in this manner,
unilamellar liposome structure is retained which offers
improved capacity for soluble material as well as the
ability to retain particulates described above and
(ii) where conditions are used such that multilamellar
liposomes are formed containing insoluble or undissolved
material they are of micron size rather than the previously
obtained 40 to 80 nm in diameter.
The present invention provides a method for forming
liposomes of greater than 0.1 ~m diameter containing
undissolved or water insoluble particulate biologically,
chemically or physically active material comprising (a)
forming a suspension of liposomes (b) freeze-drying the
liposomes so formed and then (c) rehydrating the
freeze-dried liposomes in intimate admixture with the
particulate material to be contained therein.
In another aspect the present invention provides a method
for l~he formation of unilamellar liposomes containing
undissolved or water insoluble particulate material as
desc=ribed herein comprising (a) forming a suspension of
unilamellar liposomes of size sufficiently large to
accommodate the particulate material to be included therein,
(b) ~=reeze-drying the liposomes so formed and then (c)
rehydrating the freeze-dried liposomes in intimate admixture
with the particulate material.
In another aspect the present invention provides a method
for the formation of multilamellar liposomes containing
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4a
mixture of the liposomes and material to be entrapped and
may be carried out by known methods for freeze-drying
liposomes. The rehydration step is preferably controlled
such that the number of liposomes destroyed by osmotic
pressures induced by solute concentrations generated by
water entering the vesicles is minimised.
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undissolved or water insoluble particulate material as
described herein comprising (a) forming a suspension of
unilamellar liposomes of diameter less than that of the
multilamellar liposome to be produced (b) freeze-drying the
5 liposomes so formed in the presence of the particulate
material to be contained therein and then (c) rehydrating
the freeze dried liposomes and particulate material.
Where unilamellar liposomes are to be produced step (a)
forms liposomes of greater than 0.1 ~m in diameter and uses
these in step (b). Where multilamellar liposomes are to be
produced the size of the liposomes need not be fixed in
step (a), but determined by the undissolved or insoluble
material with which they are preferably freeze-dried in
step (b) prior to rehydration in step (c).
The freeze-drying step is, in the case of both unilamellar
and multilamellar liposomes, preferably carried out on a
mixture of the liposomes and material to be entrapped and
may :be carried out by known methods for freeze-drying
liposomes. The rehydration step is preferably controlled
such that the number of liposomes destroyed by osmotic
pressures induced by solute concentrations generated by
water entering the vesicles is minimised.
In a further aspect the present invention further provides
liposomes produced by the method of the first aspect of the
invention, and particularly provides liposomes characterised
in that they are over O.l~m, preferably over lam, in
diameter and contain biologically, chemically or physically
active materials that would have their activity damaged or
destroyed by contact with organic solvents.
It i;~ particularly preferred that substantially all of any
organic solvent used in the step of liposome preparation (a)
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5a
is removed prior to the rehydration step (c), most
conveniently before the freeze drying step (b).
Preferred particulate materials are microorganisms,
including bacteria, protozoa and viruses, plant or animal
cell~~ or water insoluble structures having organic solvent
labi7_e biochemical or immunological activity. It should be
noted however that any water insoluble particulate may be
encapsulated using the method. For example catalysts or
drug~~ that are sparingly soluble may also be so incorporated
such that slow release into a patients body may be achieved.
However, as organic solvents would not be expected to
adversely affect these materials such method would be merely
an option that might be used in place of the known methods;
the rnain advantage of this preferred aspect of the present
method being realised in its application to the organic
solvent sensitive microorganisms, cells and materials, and
in y~~elding increased capacity with multilamella liposomes.
Step (a) of forming the liposomes may use any of the known
methods, including those involving use of solvents in their
manufacture, as these remove such solvents to leave hollow
bodies; the hollows forming the vesicles into which the
solutions, microorganisms, cells or insoluble structures are
to be situated after entrapment. Typically, for unilamella
lipo:~omes production, the step (a) will comprise a method
for i~he formation of so called 'giant liposomes' of
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6
suitable size for encapsulating the material added in
step (c); such method being suitably eg. that of Kim and
Martin described above. Most preferably these will be of
'micrometer' or 'micron' size, ie. herein defined as from
0.1 ~m to 50 ~m in diameter, more preferably 1 ~m to 30 Vim.
For multilamellar liposome production standard
dehydration/rehydration vesicles (DRVs) may be formed.
For most satisfactory encapsulation rates the step of
freeze-drying step (b) is carried out with the material to
be encapsulated already intimately mixed with the liposomes.
In this manner relatively high encapsulation rates have been
achieved whereas when the mixture of liposomes and material
for encapsulation is not intimate enough, little or no
incorporation is more likely. This is not the case where
solutions are being incorporated as in the prior art.
Step (c) may be carried out by any rehydration method that
allows the liposomes to admit the material to be
encapsulated. Conveniently this is found to include a
procedure wherein water in any readily available form, eg.
distilled or tap water or a buffer solution, is added in a
controlled manner to the freeze-dried mixture of liposomes
and material to be encapsulated. Preferably distilled water
is first added in order to avoid still further osmotic
stress to the liposome structure. Conveniently this is
added in small volume sufficient to produce a suspension,
followed after several minutes, preferably 20 to 40 minutes,
eg. 30 minutes, by addition of a similar volume of a buffer
which is suitable for allowing the material to retain its
desired activity; one such suitable buffer being phosphate
buffered saline (PBS) pH 7.4. Again, the buffer is
preferred at this stage in order to balance the high osmotic
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6a
pressure of the solution forming in the vesicles of the
liposomes as the materials present before the drying step
are slowly rehydrated.
The suspension so obtained is preferably mixed with a larger
volume of buffer, eg. PBS, after a further period, again
preferably 20 to 40
WO 95/09610 PCT/GB94/02191
7
minutes, preferably for about 30 minutes. The liposomes are typically
freeze-dried from a suspension of liposomes, and the total volume of
water and saline added in rehydration is conveniently sufficient to
t provide from 1 to 10 times that of the volume of the suspension,
although no particular limits are placed here.
The rehydration step may be carried out at any temperature compatible
with viability or retention of the desired activity of the material
that is to be encapsulated. Thus typically any temperature from 0°C to
60°C might be selected where high melting point lipids are used in
the liposomes and the material to be encapsulated is resistant to
this temperature. Where living materials or proteins are used then
0°C to 40°C would be more usual, preferably 10°C to
30°C. It will be
realised however that certain organisms and proteins will be capable
of treatment at much higher temperatures.
In order to maximise survival of the labile activity and the integrity
of the liposomes in storage it may be advantageous to incorporate a
cryoprotectant to counter the affects of freezing and water loss.
This is preferably added after rehydration step (c) has been effected.
Typical of such protectants are sugars and their derivatives,
particularly sugars such as trehalose (see Crowe and Crowe in Liposome
Technology (1993) V Vol I, pp229-249, CRC Press Inc, Boca Raton), with
techniques for using this being well known to those skilled in the art.
The composition of the preformed liposomes provided in step (a) is
also not particularly limited, but must allow for stable formation of
liposomes having sufficient capacity to hold the material to be
encapsulated. Typical lipid compositions used for formation of so
called 'giant liposomes' and DRVs comprise phosphatidylcholine (PC) or
distearoylphosphatidyl choline (DSPC), and these are optionally
supplemented with components such as cholesterol, phosphatidyl
glycerol (PG) and/or triolein (TO). Other components known in the art
or developments thereof which provide liposome stability or induce
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8
vesicle formation may also be used.
Formation of giant liposomes from such mixtures is
conveniently achieved by mixing a chloroform solution of
these components with a sucrose solution to form an
emulsion, then mixing that with a similar ether water
emulsion to provide a water-in-oil-in-water emulsion, from
which are removed the organic solvents to generate
liposomes. Formation of DRVs may conveniently be achieved
by dissolving equimolar PC, or DSPC, and cholesterol in
chloroform and rotary evaporating the mixture to leave a
thin film of lipid on a flask wall. This film is then
disrupted at 4°C (for PC) or 60°C (for DSPC) with 2 ml
distilled water followed by probe sonication for 2 minutes
to yield small unilamellar vesicles (SWs). This suspension
is t:nen suitable for freeze-drying with material to be
encapsulated whereby the multilamellar DRVs of greater than
0.1 pm diameter form.
In yet a further aspect of the present invention
ther~_= is provided a method for separation of liposomes of
the :invention from non-entrapped microorganisms, cells or
waterr insoluble structures comprising the steps of: placing
a mi:~ture of the liposomes and the non-entrapped particulate
materials on a density gradient; centrifuging the mixture;
removing fractions of the gradient thus formed after
centrifuging; and separately collecting the liposomes and
the non-entrapped materials from their respective fractions
using conventional methods; the non-entrapped particulate
materials usually being collected in the lower fractions and
the =Liposomes in the upper fractions. Preferably the
gradient is a 0.4 M to 4 M sucrose gradient or gradient
including an equivalent density range or analogous sugar.
Where separation from soluble materials is also required the
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8a
liposomes are centrifuged at approximately 600xg in buffer,
eg. PBS, whereby they are collected as a pellet.
In yet another aspect the present invention
provides liposomes of the invention free from non-entrapped
form of the undissolved or insoluble particulates they
contain. Such forms are of course advantageous
determination of dosage given.
WO 95109610 _
PCT/GB9-1/02191
9
As stated in the introductory paragraphs above, it is sometimes
~ advantageous to present more than one agent to a target area of a
patient simultaneously, and the present invention provides such
advantage wherein the liposomes of the invention, the method of
preparing them and the method of separating them from non-entrapped
materials all incorporate or cater for handling of water soluble
agent. Thus the liposomes produced by the method of the second aspect
of the invention may contain living or attenuated microorganisms,
cells and/or water insoluble structures together with water soluble
agents such as vaccines, antibodies, antigens or enzymes.
Thus this method of preparing the liposomes of the invention will,
when soluble materials are also to be incorporated, include the
soluble erial with the insoluble material with the liposomes in the
rehydrat~on step, preferably in the freeze drying step, and the method
for separating non-entrapped material from the liposomes will utilise
both the density gradient and buffer centrifugation methods.
A further aspect of the present invention, by virtue of the aforesaid
aspects unique advantages, provides novel liposomes characterised in
that they contain whole live or attenuated microorganisms, plant or
animal cells, or water insoluble non-living structures having organic
solvent labile biochemical or immunological activity. The latter will
include killed organisms that retain a desired activity that is labile
to organic solvent treatment. The liposomes of the present invention
can readily be identified in that their content can be released and
demonstrated to have retained the ability to be cultured and/or to
illicit biochemical or immunological responses. In addition to
bacteria, protozoans, cells or viruses, the liposomes of the present
invention may comprise inanimate structures such as cytokine, enzyme,
antigen or antibody bearing support materials, such as latex beads or
other polymeric support bodies.
In a preferred form of this aspect the liposomes, and thus the
WO 95109610 PCT/GB94/02191
approximate size of their vesicles, are from O.lum to 50um in
diameter, preferably from 1um to 30um, and conveniently lum to l4um,
with a convenient mean diameter being 5.5um + 2.2; but vesicle size
required will necessarily be dictated by the amount or size of -
solution, microorganism, cell or water insoluble structure that is
intended to be encapsulated. To this end the means by which the
liposomes are initially formed is not important, and thus variation in
vesicle size is potentially unlimited as a method is provided for
incorporating the labile materials, particularly microorganisms or
insoluble structures, into already formed liposomes without killing or
inactivating them or destroying Iiposome integrity.
Use of the liposomes of the present invention allows targeting of the
macrophages, phagocytes and/or antibody producing cells of the body
specifically, by virtue of the fact that the preferred liposomes, as
stabilised with cholesterol, PG or equivalent materials, do not
substantially release their particulate content spontaneously. Thus
the fate of the particulate material tends to be in processing by
macrophages or phagocytes whereby the immune response and related
effects, eg, of cytokines, are enhanced. Furthermore, the fact that
the particulate is protected from circulating antibodies by the lipid,
until such encounter with the macrophages or phagocytes, ensures
maximal presentation to the immune system and antibody producing cells.
The liposomes and methods of the present invention will now be
illustrated by reference to the following Figures, non-limiting
Examples, and Comparative Example. Msny other suitable liposomes and
methods for their preparation falling within the scope of the
invention being readily evident to those skilled in the art in the
light of these.
FORMATION OF GIANT UNILAMET.1.A LIPOSOMES
In these examples the giant liposomes are preformed, have a mean
diameter of 5.5~0.2 um and are mixed with the particulate or soluble
WO 95/09610 s~-~ PCT/GB94/02191
11
materials and subsequently subjected to controlled rehydration.
Generated liposomes were found to maintain their original mean
diameter and diameter range and to contain up to 26.7 (mean value) of
the added materials. Particulate-containing liposomes could be
freeze-dried in the presence of trehalose with most (up to 87x) of the
entrapped material recovered within the vesicles formed on
reconstitution with saline.
The sources and grades of egg phosphU~.idylcholine (PC), distearoyl
phosphatidylcholine (DSPC), cholesterol, immunopurified tetanus toxoid
and trehalose have been described elsewhere (Davis and Gregoriadis,
1987, Immunology, 61, 229-234). Phosphatidyl glycerol (PG) and
triolein {TO) were from Lipid Products (Nuthill, Surrey) and Sigma
Chemical Company (London) respectively. :billed Bac~ » u~ s~bti is
(B.subt~l;s) and Bacille Calmette-Guerin -{BCG) were gifts from Dr
Bruce Jones (Public Health Laboratories Service, Porton Down,
Salisbury, Wilts) and Dr J.L. Stanford (Dept of Medical Microbiology,
UCL Medical School, London) respectively. Radiolabelling of tetanus
toxoid, B.s,_,b.;iis, and BCG with 125I was carried out as described
previously (Kirby and Gregoriadis, 1984 as above). Labelling of
B.subtilis with fluorescein isothiocyanate (FITC) {Sigma) was carried
out by incubating the bacteria in lml O.1M sodium carbonate buffer
(pH9.0) containing lmg FITC for 24 h at 4°C (Mann and Fish,(1972) Meth.
Enzymology, 26, 28-42). All other reagents were of analytical grade.
FIGURES
Figure 1: shows ~ added radioactivity or lipid v gradient fractions
for a sucrose gradient centrifugation of giant liposomes containing
i25I labelled $, subt;l;~. Separation of liposome-entrapped from
non-entrapped B. s~ht;i;s (A) and of empty liposomes from added free
$. s,h~t;~;~ (B) was carried out by sucrose gradient separation as
described below. Patterns of 1251 radioactivity {o) and lipid (~)
shown are typical for PC or DSCP liposomes prepared by either of the
WO 95/09610 PCT/GB94/02191
12
techniques described in the Comparative Example and Example 1 below.
Values are y of radioactivity or lipid used for fractionation.
Figure 2: shows x B. subtilis retention in liposomes of the
invention v incubation time in plasma. PC or DSPC liposomes
containing lzSI labelled B.subtilis were incubated with mouse plasma
(o) or PBS (~) at 37°C. At time intervals samples were fractionated
on a sucrose gradient to separate freed from entrapped B. subtilis
and values for triplicate experiments are x+SD of radioactivity
recovered with liposomes.
Figure 3: shows retention of tetanus toxoid by liposomes of the
invention in the presence of plasma. PC or DSPC liposmes containing
izSI labelled tetanus toxoid were incubated in the presence of plasma
(o) or PBS (s) at 37°C. At time intervals samples were centrifuged at
600xg to separate freed from entrapped toxoid. values from
triplicate experiments are %+SD of radioactivity recovered with
liposomes.
Figure 4: shows clearance of tetanus toxoid and B, subtilis after
intramuscular injection into mice. Mice were injected into their hind
legs with free (o) or liposome entrapped (~) izSI lebelled tetanus
toxoid (A) or B. subtilis (B). Animals were killed at time intervals
and radioactivity measured in the amputated leg. Each value
represents three animals and is expressed as x+SD of the radioactivity
recovered in the leg immediately after injection.
COMPARATIVE EXAMPLE
Prenarat~on of vaccin -containine~ eiant liposomes in the presence of
organic solvents.
Giant liposomes with entrapped materials were prepared by the solvent
spherule evaporation method (Kim and Martin, 1981) with modifications.
WO 95/0961 _ PCT/GB94/02191
13
In brief, 1mL of a 0.15M sucrose solution containing the 125I-labelled
material to be encapsulated (2x103-104 cpm; 1-2 mg tetanus toxoid and
105 B.smb ;~; bacteria as spores) was mixed by vortexing for 45s with
. 1mL of a chloroform solution containing PC or DSPC, Chol, PG and TO
(4:4:2:1 molar ratio; 9 umoles total lipid). The resulting water-in
-chloroform emulsion was mixed by vortexing for 15s with a diethyl
ether-in-water emulsion prepared from 0.5m1 of a solution of the
lipids as above in diethyl ether and 2.5mL of a 0.2M sucrose solution.
The water-in-oil-in-water emulsion thus formed was placed in a 250mL
conical flask and the organic solvents were evaporated by flushing
nitrogen at 37°C while the sample was gently agitated in a shaking
incubator. Generated liposomes were centrifuged twice at 600xg for 5
min over a 5y glucose solution and the pellet was resuspended in 0.1 M
sodium phosphate buffer supplemented with 0.9x NaCl, pH '7.4 (PBS).
This last step enabled the separation of the entrapped from the
non-entrapped toxoid. For the separation of non-entrapped B.subt;~;s
(which sedimented together with liposomes) sucrose gradient
fractionation was used (see below). In some experiments, FITC
-labelled polystyrene particles (0.5 and 1um diameter; Polysciences)
or FITC-labelled B.st,b ;i; were entrapped as above in liposomes which
were used in fluorescence microscopy studies.
EXAMPLE 1
flf the 1nV .ni-i nn
In order to entrap potentially labile particulate materials in giant
liposomes in the absence of organic solvents, "empty" giant liposomes
containing only sucrose were preformed as described in the Comparative
Example above (with two preparations the total amount of lipid was 36
umoles) and centrifuged over a 5~ glucose solution in a bench
centrifuge at 600xg for 5 min. The liposomal pellet was resuspended
in lml 0.1 M sodium phosphate buffer supplemented with 0.9$ NaCl, pH
'7.4 (PBS), mixed with 1 mL of a solution or suspension of the
materials to be encapsulated (B.s~b ;~i as spores, tetanus toxoid and
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14
BCG as spores) and freeze-dried as described previously
(Gregoriadis et al, 1987; Kirby and Gregoriadis, 1984) by
treatment under vacuum. Typically 2 ml of liposome
suspension in distilled water were mixed with 2 ml of
suspension or solution of material to be entrapped in a
50 ml round bottomed flask or 21 mm diameter glass tube, and
the mixture flash frozen as a thin shell by swirling in a
freezing mixture of cardice and isopropanol. After freezing
the preparations were lyophilised, eg. in a HetosiccTM freeze
drier, at a vacuum of 0.1 torr for 4 to 5 hours, or 13 Pa
overnight.
The freeze-dried material was rehydrated initially by the
addition of 0.1 ml distilled water at 20°C (rehydration of
liposomes containing the "high melting" DSPC at 50-60°C did
not have a significant effect on percent entrapment of
materials). The suspension was vigourously swirled and
allowed to stand for 30 minutes. The process was repeated
after the successive addition of 0.1 ml PBS and of 0.8 ml
PBS 30 minutes later. All materials for entrapment were
lasl_labelled for assessing uptake. In one experiment,
B.subtilis-containing liposomes prepared as above, were
freeze-dried in the presence of 0.25 M (final concentration)
trehalose and subsequently reconstituted in PBS.
EXAMPLE 2.
Separation of liposomes containing entrapped material from
non-entrapped material.
Separation of entrapped from non-entrapped material was
carried out by sucrose gradient centrifugation (B.subtilis
and BCG; see below) or centrifugation at 600xg for
10 minutes followed by the suspension of the twice
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PBS-washed pellet in 1 ml PBS (tetanus toxoid). B.subtilis
retention by the re-formed vesicles was evaluated by sucrose
gradient fractionation. A discontinuous sucrose gradient
was prepared (Gregoriadis, (1970) J. Biol Chem 245,
5 5833-5837) by layering ten dilutions (sucrose content
ranging from 0.4 M to 4 M) of a 4 M sucrose solution in
swing out bucket centrifuge tubes. Preparations (1 ml) with
entrapped and non-entrapped B.subtilis or BCG from Example 1
were placed on the top of the gradient and then centrifuged
10 for 1.5 hours at 25,OOOrpm in a Dupont Combi PlusTM
ultracentrifuge using a swing-out bucket. After
centrifugation, 1 ml fractions were pippeted out from the
top of the gradient and assayed for 1251 radioactivity in a
Wallac MinigammaTM counter and phospholipid content.
15 (Stewart. (1979) Anal. Biochem. 104. 10-14). Separation of
liposomes from sucrose fractions was carried out by diluting
these with water or PBS, eg. using enough diluent to make up
the volume to that of the centrifugation bucket, then
centrifuging that at 600xg for 10 minutes as for removal of
non-entrapped tetanus toxoid.
Measurement of vesicle size The mean diameter in terms of
volume distribution of giant liposomes was measured in a
Malvern MastersizerTM.
Light Microscopy Light microscopy studies on liposomes
containing FITC-labelled B.subtilis or polystyrene particles
were carried out using a NikonTM microscope and a LeicaTM
confocal microscope, both equipped with fluorescence light
sources. To improve visualization of liposomes, these were
stained by the addition of oil-red-O in the chloroform
solution of lipids during liposome preparation.
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15a
Stability of giant liposomes in plasma PC or DSPC giant
liposomes (3-5 mg total lipid) containing radiolabelled
toxoid (103 cpm; 0.1 - 0.2 mg) or B.subtilis (3-5x103 cpm;
103 bacteria) and made by the method of the invention as in
Example 1 and 2 were mixed in triplicates with five volumes
of mouse (male, CDI strain) plasma or PBS and incubated at
37°C. At time intervals, samples were removed and retention
of entrapped materials by liposomes was ascertained by
sucrose gradient fractionation (B.subtilis) or
centrifugation at 600xg (toxoid). Vesicle stability was
expressed as percent of originally entrapped material
retained by the vesicles.
Clearance of liposome-entrapped toxoid and B.subtilis after
intramuscular injection into mice. Forty eight male CDI
mice. (body
WO 95/09610 PCT/GB9.~/02191
16
weight 20-25g) were randomly divided into four groups of twelve
animals each and injected intramuscularly (hind leg) with O.lml of (a)
free radiolabelled toxoid (6x103cpm; O.Olmg), (b) free radiolabelled
B.subtilis (3X103cpm; 103 bacteria as spores); (c) toxoid as in (a)
entrapped by the method of Example 1 into DSPC liposomes (lmg total
lipid); (d) B.subtilis as in (b) entrapped into DSPC liposomes as in
(c). Animals in groups of three, were killed and hind legs removed
immediately after injection (to establish zero time values) and at
time intervals thereafter. 1251 was measured in the isolated legs and
results were expected as percent of radioactivity recovered in the
tissue at zero time.
RESULTS: FntraDmen s ~die~ Evaluation of B.subtilis entrapment into
giant liposomes could not be achieved by centrifugation as both
entities sedimented at low speed. Complete separation was however,
obtained by sucrose gradient fractionation as already described. The
1251 radioactivity (B.subtilis) and phospholipid (liposomes) values
measured in the fractions after centrifugation indicate that
bacteria-containing PC or DSPC liposomes were recovered in the upper
ten 0.5m1 fractions of the gradient, with free B.subtilis sedimenting
to the bottom fraction.
The mean (+SD) diameter of these vesicles in eight different PC and
DSPC preparations made according to the protocol of the examples was
found to vary significantly (5.5 ~ 2.2um) with a lower and upper
diameter range (all preparations) of 1-14 um. There was no
statistically significant difference in mean diameter between PC and
DSPC liposomes. The possibility that bacteria had adsorbed to the
surface of empty vesicles was discounted since incubation of such
liposomes with B.subtilis for 22 hours prior to gradient fractionation
resulted in the quantitative recovery of the latter in the bottom
fraction. On the basis of B.sub ~1;~ presence in the top ten
fractions of the sucrose gradient (which coincided with the presence
of liposomal phospholipid in the same fractions), B.subtilis
WO 95109610
PCT/GB94/02191
17
entrapment in twelve separate experiments by the method of Kim and
Martin was variable with a mean value of 31.6x of the bacteria used.
However, attempts to entrap tetanus toxoid by the same method (in the
presence of organic solvents) failed presumably because of protein
denaturation at the water-chloroform interface and subsequent
inability of the altered protein to remain in the water phase.
This finding and the prospect of similar damage or otherwise
inactivation of other protein antigens and attenuated or live microbes
destined for entrapment demonstrates the advance of the method and
product liposomes of the invention. Controlled rehydration of the
powder obtained by freeze drying the preformed liposomes with
B.subtilis, BCG and tetanus toxoid resulted in the formation of
vesicles with a mean diameter (4.6~1.3 um and a lower and upper range
of 1-18 um; all preparations) similar to those of the parent vesicles
(see above), containing variable but substantial proportions of
B s»bt;~;s, BCG and toxoid (8.4 to 27.8; of the material used). There
was no significant difference in values of B.subtilis entrapment
obtained with the present procedure and that of Kim and Martin, but
unlike in that method, ability to produce bacterial growth when
liposome content was inoculated onto culture plates was retained.
Morpholo~ica? St~r7;A~ Light microscopy revealed that nearly all giant
vesicles stained with oil red-0 contained a varying number of
FITC-labelled B.sLbt;~; bacteria, which in many instances appeared to
adhere to (or precipitate towards) the inner wall of the vesicles.
Experiments using polystyrene particles and latex particles (0.5um and
lum in diameter) as particulate material for entrapment in l8um
diameter liposomes gave similar results. That localization of
particles within liposomes had been achieved was further evidenced by
confocal microscopy which enabled their visualization in different
"sections" of space within the vesicles.
S ab;7; ~~.i~nt lwn~nma in D~p~m~ An important prerequisite for
WO 95/09610 ~ ' y . PCT/GB9-1/02191
is
the successful use of liposomes as carriers of live or attenuated
microbial vaccines (alone or in combination with soluble antigens) in
pre-immunized animals, animals with maternal antibodies to the
vaccines, or with antibodies to vaccine impurities, would be
avoidance of interaction between antibodies and vaccines prior to
their delivery to antigen presenting cells. Previous work
(Gregoriadis and Allison, 1974) demonstrated that such interaction was
indeed avoided when protein containing multilamellar liposomes were
injected intravenously into mice pre-immunized with the protein:
whereas all animals injected with the free protein died of
anaphylactic shock, those treated with the entrapped protein survived,
presumably because of antigen confinement within the bilayers.
Similarly, there was no Arthus reaction in such mice injected
subcutaneously (foot pad) with the liposomal antigen (Gregoriadis and
Allison, 1974). It was, therefore, of interest to see whether giant
liposomes prepared by dehydration-rehydration would still retain their
bacteria or tetanus toxoid content entrapped in the presence of mouse
blood plasma at 37°C.
In the event B.subtilis-containing PC giant liposomes retained 80-90x
of their bacteria content for at least 24 hours in the presence of
plasma. As about lOx of bacteria were freed soon after mixing with
plasma it is possible that these represent bacteria adsorbed to the
liposome surface during entrapment and subsequently removed by plasma
components. This is supported by the-fact that liposomes exposed to
PBS retained all their content B.subtilis. Retention of B.subtilis by
DSPC liposomes in the presence of plasma was even greater (>95y) for
at least 7 hours. Further, retention values were not significantly
different than those seen in the presence of PBS, probably because a
liposomal bilayer containing DSPC in its structure would be more rigid
and therefore more resistant to particle adsorption. In contrast to
the nearly quantitative retention of B.subtilis (dia 0.8um), giant
liposomes appeared to gradually lose significant amounts or their
toxoid content in the presence of plasma with the loss being more
W O 95/09610
PCT/GB94/02191
19
pronounced for PC (48:) than for DSPC liposomes (38x; 24 hour
values).
The
extent of tetanus toxoid and B.mb ;~;~ retention by giant liposomes
after intramuscular injection into mice could not be estimated
directly, for instance by measuring released and liposome-entrapped
materials in the muscle tissue. However, it was reasoned that
comparison of the clearance rates of free and liposome-entrapped
. tetanus toxoid and B.subt?~;s from the tissue could provide an
indication of the extent to which liposomes retained their content 'fir
situ. Much more (67;~ of the content measured in the tissue
immediately after injection) of the tetanus toxoid administered via
liposomes was recovered in the tissue 10 hours after injection than
toxoid given as such (27x). This substantial difference in clearance
rates between the two toxoid formulations (entrapped and free)
suggests that much of the liposomal toxoid is retained within the
vesicles in situ.
It appears from the slow rate of liposomal toxoid clearance that
liposomes are gradually destabilized to release toxoid which would
then be cleared from the tissue as free. It is possible however that
some of the vesicles, especially those of smaller size, may also
migrate to the lymphatics. The rates of clearance of free and
liposomal B.s b ;~; on the other hand, were initially similar. and
assuming that liposomes containing B.subtilis or toxoid are
destabilized (and release their contents) to the same extent in the
local milieu, the slower clearance of .;iposomal B.subtilis (as
compared to that of liposomal toxoid) during the first 5 hour is
probably due to the similarly slow clearance of free bacteria.
Nevertheless, the substantial difference in tissue levels between the
two B.~ub ;~; formulations 24 hours after injection indicates the
considerable extent to which liposomes remain stable in the injected
tissue.
WO 95/09610 PCT/GB94/02191
Thus use of solvents detrimental to live or attenuated microbes and
the presence of detergent in formulations rendering them toxic for '
in-vivo use has been demonstrated to be avoidable by use of the
present invention. Liposomes prepared by the present procedure retain
(in isolation from the external milieu) most of their bacteria or
soluble antigen content in the presence of blood plasma and a
considerable proportion of it in vivo. Thus, liposomes could not only
serve as immunoadjuvants for microbial vaccines, in connection with
co-entrapped soluble antigens or cytokines if required, but also as
carriers of live or attenuated microbial vaccines in cases where there
is a need to prevent interaction of the latter with maternal
antibodies or preformed antibodies to vaccine impurities.
Furthermore, the present invention allows for the entrapment of
microorganisms or cells within liposomes, followed by culturing of
these in the entrapped state to increase the 'dose' of active material
without the necessity to optimize the entrapment step. For example,
only a few cells need be entrapped in the rehydration step, then
these could be supplied with nutrients through the liposome wall,
preferably with an inhibitor of lipid metabolism, such that they
multiply thus making the liposome vesicle more heavily loaded with
desired material. Once a suitable loading had been achieved, the
liposomes would be freeze dried for storage, with rehydration being
used just prior to use.
RMATION
AMPLE-'~
Entrapment of live spores and other materials in multilamella DRV
liposomes.
Small unilamella vesicles composed of PC or DSPC and equimolar
cholesterol (l8umoles phospholipid) were prepared as described in
Kirby and Gregoriadis, 1984) whereby phospholipid and cholesterol
were dissolved in chloroform which was then rotary evaporated to
CA 02173601 2004-04-08
28472-98
21
leave a thin lipid film on the walls of the flask. The film
was disrupted at 4°C (PC) or 60°C (DSPC) with 2 ml of
distilled water and subsequently probe-sonicated for about
2 minutes to produce SUVs in suspension. The suspension was
mixed with 2 ml of l2sl-labelled live spores (10'), 100-150 ~g
l2sl-labelled toxoid or a mixture of radiolabelled spores and
unlabelled toxoid and freeze-dried overnight. The
freeze-dried material was mixed vigorously with 0.1 ml water
at the appropriate temperatures (as Example 2) and allowed
to stand for 30 minutes. This step was repeated with 0.1 ml
PBS and after 30 minutes the suspension supplemented with
0.8 ml PBS. The final suspension was subjected to sucrose
gradient fractionation to separate the entrapped from
non-entrapped spores or to centrifugation at 90,OOOxg twice
for 20 minutes to separate the free from entrapped toxoid.
Unlabelled toxoid was measured by the fluorescamine method
(Anderson and Desnick (1979) J. Biol. Chem., 254, 6924).
Results: Spore entrapment was unexpectedly high (63.6 - 78%)
in spite of previous findings of relatively small vesicle
size for these type of vesicles. The mean size for PC-DRVs
was 3.6 ~,m and for DSPC-DRVs was 3.2 ~m diameter.
SPORE VIABILITY:
Giant or DRV liposomes containing B.subtilis spores were
tested for spore viability by treating them with
TritonT"" X-100 to liberate spores were serially diluted in
nutrient broth and then spread on nutrient agar plates to
estimate the total colony count, the viability count and the
average number of viable spores per individual vesicle.
Results are shown in Table 1 below wherein the number of
colonies are given.
CA 02173601 2004-04-08
28472-98
21a
The number of colonies produced for each treated preparation
tested was much greater than that observed with intact
liposomes; spores within an intact liposome being liable to
only form a single colony. The table indicates that up to
20 spores could be found within
WO 95109610 PCT/GB9.1/02191
~:~~~~a~~
22
individual vesicles with no apparent relationship between spore
numbers and the phospholipid used. There was a positive relationship
between the number of spores entrapped and vesicle size.
Among the six different giant liposome preparations used the lowest
spore number was obtained with the smallest diameter of vesicle and
the highest with the largest diameter vesicle. However, with DRV
liposomes their smaller size was not reflected in lower spore numbers.
TABLE
1.
Estimated
average
number
of
viable
B.
subtilis
spores
per
vesicle.
Type Number of colonies Spores per vesicleVesicle
Method Triton Control size um
Giant PC A96 29 3 6,3
Giant DSPCA162 38 4 6.5
Gisnt PC B203 10 20 8.4
Giant DSPCB250 21 12 '7.2
Giant PC B160 12 13 8.0
Giant DSPCB109 15 '7 6.4
DRV PC 320 44 7 3.6
DRV DSPC 189 31 6 3.2
B. mb ~1;~ spores were entrapped in giant liposomes by method of the
Comparative example (A), Example 1 (B), or DRV liposomes; both PC and
DSPC lipids being used to make respective types. Estimation of spores
per vesicle is carried out by dividing colonies number after triton
treatment with the control number; the Triton number being equal to
the number of viable spores liberated from vesicles. The control
number is equivalent to the number of vesicles as entrapped spores
produce only one colony. The toxic effect of the solvent {method A) on
spores is demonstrated; this effect is increased on vegetative bacteria.
