U.S. patent application number 09/963601 was filed with the patent office on 2002-04-11 for liposomes containing particulate materials.
This patent application is currently assigned to Unite Kingdom Defence Evaluation and Research Agency.. Invention is credited to Antimisiaris, Sophia George, Gregoriadis, Gregory, Gursel, Ishan.
Application Number | 20020041895 09/963601 |
Document ID | / |
Family ID | 10743157 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020041895 |
Kind Code |
A1 |
Gregoriadis, Gregory ; et
al. |
April 11, 2002 |
Liposomes containing particulate materials
Abstract
A method is provided for the formation of liposomes of 0.1 .mu.m
to 50 .mu.m in diameter having unilamella or multilamella structure
and containing water insoluble or undissolved particulate materials
comprising (a) forming liposomes and removing substantially all of
any organic solvent used in their preparation, (b) freeze drying
the liposomes so formed and then (c) rehydrating them in intimate
admixture with the particulate material. Preferred encapsulated
materials are particulate materials, most preferably
microorganisms, plant or animal cells or water insoluble structures
having organic solvent labile biochemical or immunological
activity, but any water insoluble particulate may be encapsulated
using the method. For example catalysts or drugs that are sparingly
soluble may also be so incorporated such that slow release into the
a patients body may be provided while release of detergents
included in the many liposome preparation protocols may be
avoided.
Inventors: |
Gregoriadis, Gregory;
(Northwood, GB) ; Antimisiaris, Sophia George;
(Patras, GR) ; Gursel, Ishan; (Ankara,
TR) |
Correspondence
Address: |
NIXON & VANDERHYE P.C.
8th Floor
1100 North Glebe Road
Arlington
VA
22201-4714
US
|
Assignee: |
Unite Kingdom Defence Evaluation
and Research Agency.
|
Family ID: |
10743157 |
Appl. No.: |
09/963601 |
Filed: |
September 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09963601 |
Sep 27, 2001 |
|
|
|
08624556 |
Sep 20, 1996 |
|
|
|
Current U.S.
Class: |
424/450 |
Current CPC
Class: |
Y10S 977/907 20130101;
A61K 9/127 20130101; A61K 9/1277 20130101; Y10S 977/918
20130101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 009/127 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 7, 1993 |
GB |
9320668.8 |
Oct 7, 1994 |
GB |
PCT/GB94/02191 |
Claims
1. A method for forming liposomes of greater than 0.1 .mu.m
diameter containing undissolved or water insoluble particulate
biologically, chemically or physically active material comprising
(a) forming liposomes (b) freeze drying the liposomes so formed and
then (c) rehydrating them in intimate admixture with the material
to be contained therein.
2. A method for the formation of unilamella liposomes containing
undissolved or water insoluble particulate material as claimed in
claim 1 comprising (a) forming unilamella liposomes of size
sufficiently large to accommodate the particulate material to be
included therein, (b) freeze drying the liposomes so formed and
then (c) rehydrating them in intimate admixture with the
particulate material.
3. A method for the formation of multilamella liposomes containing
undissolved or water insoluble particulate material as claimed in
claim 1 comprising (a) forming unilamella liposomes of diameter
less than that of the multilamella liposome to be produced (b)
freeze drying the liposomes so formed in the presence of material
to be contained therein and then (c) rehydrating the freeze dried
liposomes and material.
4. A method as claimed in any one of the preceding claims wherein
substantially all of any organic solvent used in the preparation of
the liposomes in step (a) is removed before the rehydration.
5. A method as claimed in any one of the preceding claims wherein
substantially all of any organic solvent used in the preparation of
the liposomes in step (a) is removed prior to step of freeze drying
(b).
6. A method as claimed in any one of the preceding claims wherein
the substantially water insoluble or undissolved particulate
material comprises a microorganism, a plant or animal cell or a
water insoluble structure having organic solvent labile biochemical
or immunological activity.
7. A method as claimed in any one of claims 1, 2 or 4 to 6 wherein
the step (a) of forming liposomes comprises the formation of
liposomes of diameter 0.1 .mu.m to 50 .mu.m.
8. A method as claimed in claim 7 wherein the liposomes are giant
liposomes of diameter of 1 .mu.m to 30 .mu.m.
9. A method as claimed in any one of the preceding claims wherein
step (b) is carried out with the material to be encapsulated
already intimately mixed with the liposomes.
10. A method as claimed in any one of the preceding claims wherein
a protectant against the effects of water loss is added to the
liposome product after rehydration.
11. A method as claimed in claim 10 wherein the protectant is
trehalose.
12. A method as claimed in any one of the preceding claims wherein
step (c) is carried out by controlled addition of water in a small
quantity just sufficient to produce a suspension, followed after a
first period of several minutes by a similar amount of a suitable
buffer biologically acceptable to the material to be encapsulated
such as to retain its desired activity; and the suspension so
obtained mixed with a larger volume of buffer after a second period
of several minutes.
13. A method as claimed in claim 12 wherein the first and second
periods are of from 20 to 40 minutes each.
14. A method as claimed in claim 12 or 13 wherein the buffer added
is phosphate buffered saline of about pH 7.4.
15. A method as claimed in any one of the preceding claims wherein
the freeze-drying step (b) is carried out by freeze drying a
suspension of liposomes and material to be encapsulated, and the
total volume of water and saline added in rehydration step (c) is
sufficient to provide from 1 to 10 times that of the volume of the
suspension.
16. A method as claimed in any one of the preceding claims wherein
the liposomes formed in step (a) comprise a lipid composition
comprising phosphatidylcholine (PC) and/or distearoylphosphatidyl
choline (DSPC).
17. A method as claimed in claim 16 wherein the lipid composition
further comprises one or more of cholesterol, phosphatidyl glycerol
(PG) and triolein (TO).
18. A method as claimed in any one of the preceding claims wherein
liposome formation step (a) is carried out by mixing a chloroform
solution of a lipid composition with sucrose solution to form an
emulsion, and that is mixed with a similar ether water emulsion to
provide a water in oil in water emulsion, whereupon substantially
all of the organic solvent is removed from that, resulting in
generation of liposomes.
19. A method for the separation of liposomes from non-entrapped
water insoluble or undissolved particulate materials characterised
in that it places a mixture of the two on a density gradient and
centrifuges it, the fractions of the gradient are removed, those
containing the separated liposomes collected, and the liposomes
separated from these with the free materials being collected in the
lower fractions.
20. A method as claimed in claim 19 wherein the density gradient is
a sucrose gradient from 0.4 to 4M in strength, or a gradient
including an equivalent density range and/or sugar.
21. A method as claimed in claim 1 to 20 wherein the material to be
contained within the liposome comprises a living microorganism or
plant or animal cell, characterised in that after the rehydration
step (c) the microorganism or cell is provided with nutrients
through the liposome wall such that it is cultured and
multiplies.
22. A method as claimed in claim 21 wherein the nutrients are
provided with an inhibitor of the microorganism or cell's ability
to metabolise the lipid of the liposome.
23. A method as claimed in claim 21 or 22 wherein the cell is in
the form of a spore.
24. A liposome obtained by a method as claimed in any one of claims
1 to 21.
25. A liposome characterised in that it contains one or more live
or attenuated microorganisms, plant or animal cells, or water
insoluble structures having organic solvent labile biochemical or
immunological activity.
26. A liposome characterised in that it contains live or attenuated
virus, bacteria and/or protozoa.
27. A liposome as claimed in any one of claims 23 to 26 wherein the
live or attenuated microorganisms are selected from measles, polio
virus, Bordetella pertussis, Bacille Calmette-Guerin and Salmonella
typhi.
28. A liposome as claimed in claim 25 or 27 wherein the water
insoluble non-living structures comprise cytokine, enzyme, antigen
or antibody bearing support materials.
29. A liposome as claimed in any one of claims 23 to 28
characterised in that it further contain water soluble
biochemically or immunologically active materials.
28. A liposome preparation comprising liposomes as claimed in any
one of claims 22 to 27 being of 0.1 .mu.m to 50 .mu.m in diameter
and containing viable bacterial spores in excess of 4 spores per
vesicle per 6.2 .mu.m mean diameter sphere of liposome.
29. A liposome preparation as claimed in claim 28 wherein the
liposomes are multilamella and the number of viable bacterial
spores is 6 spores per 3.2 .mu.m mean diameter sphere of liposome
or more.
30. A liposome preparation as claimed in claim 28 wherein the
liposomes are unilamella and the number of viable bacterial spores
is 13 spores per 7.2 .mu.m mean diameter sphere of liposome or
more.
31. A composition comprising a physiologically acceptable carrier
and one or more liposomes or a liposome preparation as claimed in
any one of claims 23 to 30.
32. A method as claimed in any one of claims 1 to 22 substantially
as described in Example 1 or Example 2.
33. A liposome as claimed in any one of claims 23 to 32
substantially as claimed in Example 1 or Example 2.
Description
[0001] 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.
[0002] 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, 89-97 and
Alving C R (1991) J Immunol Meth, 140, p1-13.
[0003] 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 typhi (see Mimms C A et al (1993) Medical
Microbiology, Chapter 36, Mosby).
[0004] 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.
[0005] 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.
[0006] It is known to incorporate particulate materials into large
liposomes having average diameter up to 9.2 .mu.m 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 (Streptococcus
salivarius) 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.
[0007] 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,
Fla., pp19-28; 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 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 unilamella liposomes for the purpose of
encapsulating aqueous solutions (see EP 0171710).
[0008] 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.
[0009] 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.
[0010] The present inventors have now surprisingly found that
dehydration/rehydration 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. 0.1-50
.mu.m 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 vesicles of relatively high
capacity, being similar in size to their outer diameter in the case
of the unilamella giant liposomes.
[0011] It is particularly surprisingly that (i) when micrometer
sized liposomes are dehydrated then rehydrated in this manner,
unilamella 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 multilamella liposomes are formed containing insoluble or
undissolved material they are of micron size rather than the
previously obtained 40 to 80 nm in diameter.
[0012] Thus in a first aspect of the invention there is provided a
method for forming liposomes of greater than 0.1 .mu.m diameter,
preferably greater than 1 .mu.m diameter, containing undissolved or
insoluble particulate biologically, chemically or physically active
material comprising (a) forming unilamellar liposomes (b) freeze
drying the liposomes so formed and then (c) rehydrating them in
intimate admixture with the undissolved or insoluble material to be
contained therein.
[0013] Where unilamella liposomes are to be produced step (a) forms
liposomes of greater than 0.1 .mu.m in diameter and uses these in
step (b). Where multilamella 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 with in step (b) prior to rehydration in
step (c).
[0014] The freeze drying step is, in the case of both unilamella
and multilamella 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.
[0015] In a second 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 0.1 .mu.m, preferably over 1 .mu.m, in diameter
and contain biologically, chemically or physically active materials
that would have their activity damaged or destroyed by contact with
organic solvents.
[0016] It is particularly preferred that substantially all of any
organic solvent used in the step of liposome preparation (a) is
removed prior to the rehydration step (c), most conveniently before
the freeze drying step (b).
[0017] Preferred particulate materials are microorganisms,
including bacteria, protozoa and viruses, plant or animal cells or
water insoluble structures having organic solvent labile
biochemical or immunological activity. It should be noted however
that any water insoluble particulate may be encapsulated using the
method. For example catalysts or drugs that are sparingly soluble
may also be so incorporated such that slow release into the 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 main 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 yielding
increased capacity with multilamella liposomes.
[0018] 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 liposome production,
the step (a) will comprise a method for the formation of so called
`giant liposomes` of 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
.mu.m to 50 .mu.m in diameter, more preferably 1 .mu.m to 30 .mu.m.
For multilamella liposome production standard
dehydration/rehydration vesicles (DRVs) may be formed.
[0019] 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 acse where solutions are being incorporated as in the prior
art.
[0020] 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 quantity sufficient just to produce a suspension,
followed after several minutes, preferably 20 to 40 minutes, eg. 30
minutes, by a similar amount of a buffer which is suitable for
allowing the material to be encapsulated to retain its desired
activity; one such suitable buffer being phosphate buffered saline
(PBS) pH7.4. Again, the buffer is preferred at this stage in order
to balance the high osomotic pressure of the solution forming in
the vesicles of the liposomes as the materials present before the
drying step are slowly rehydrated.
[0021] The suspension so obtained is preferably mixed with a larger
volume of buffer, eg. PBS, after a further period, again preferably
20 to 40 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 provide from 1 to 10 times that of the
volume of the suspension, although no particular limits are placed
here.
[0022] 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.degree. C. to 60.degree. 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.degree. C. to
40.degree. C. would be more usual, preferably 10.degree. C. to
30.degree. C. It will be realised however that certain organisms
and proteins will be capable of treatment at much higher
temperatures.
[0023] 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.
[0024] 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 vesicle formation may also be
used.
[0025] Formation of giant liposomes from such mixtures is
conveniently achieved by mixing a chloroform solution of these
components with a sucrose solution to form and 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.degree.
C. (for PC) or 60.degree. C. (for DSPC) with 2 ml distilled water
followed by probe sonication for 2 minutes to yield small
unilamella vesicles (SUVs). This suspension is then suitable for
freeze drying with material to be encapsulated whereby the
multilamella DRVs of greater than 0.1 .mu.m diameter form.
[0026] In a third aspect of the present invention there is provided
a method for separation of liposomes of the invention from
non-entrapped microorganisms, cells or water insoluble structures
characterised in that it places a mixture of the two on a density
gradient and centrifuges it, the fractions of the gradient are
removed, those containing the separated liposomes collected, and
the liposomes separated from these by conventional methods; the
free materials usually being collected in the lower fractions and
the liposomes in the upper fractions. Preferably the gradient is a
0.4M to 4M sucrose gradient or gradient including an equivalent
density range or analogous sugar. Where separation from soluble
materials is also required the liposomes are centrifuged at
approximately 600 xg in buffer, eg. PBS, whereby they are collected
as a pellet.
[0027] A fourth aspect of the present invention is therefor
provided in the form of 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.
[0028] 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.
[0029] Thus this method of preparing the liposomes of the invention
will, when soluble materials are also to be incorporated, include
the soluble material with the insoluble material with the liposomes
in the rehydration 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.
[0030] 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.
[0031] In a preferred form of this aspect the liposomes, and thus
the approximate size of their vesicles, are from 0.1 .mu.m to 50
.mu.m in diameter, preferably from 1 .mu.m to 30 .mu.m, and
conveniently 1 .mu.m to 14 .mu.m, with a convenient mean diameter
being 5.5 .mu.m.+-.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 liposome integrity.
[0032] 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.
[0033] The liposomes and methods of the present invention will now
be illustrated by reference to the following Figures, non-limiting
Examples, and Comparative Example. Many 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.
[0034] Formation of Giant Unilamella Liposomes
[0035] In these examples the giant liposomes are preformed, have a
mean diameter of 5.5.+-.0.2 .mu.m and are mixed with the
particulate or soluble materials and subsequently subjected to
controlled rehydration.
[0036] 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
87%) of the entrapped material recovered within the vesicles formed
on reconstitution with saline.
[0037] The sources and grades of egg phosphatidylcholine (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. Killed
Bacillus subtilis (B.subtilis) 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.subtilis, and BCG with
.sup.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 1 ml 0.1M sodium carbonate buffer
(pH9.0) containing 1 mg FITC for 24 h at 4.degree. C. (Mann and
Fish,(1972) Meth. Enzymology, 26, 28-42). All other reagents were
of analytical grade.
[0038] Figures
[0039] FIG. 1: shows % added radioactivity or lipid v gradient
fractions for a sucrose gradient centrifugation of giant liposomes
containing .sup.125I labelled B. subtilis. Separation of
liposome-entrapped from non-entrapped B. subtilis (A) and of empty
liposomes from added free B. substilis (B) was carried out by
sucrose gradient separation as described below. Patterns of
.sup.125I radioactivity (.smallcircle.) and lipid (.circle-solid.)
shown are typical for PC or DSCP liposomes prepared by either of
the techniques described in the Comparative Example and Example 1
below. Values are % of radioactivity or lipid used for
fractionation.
[0040] FIG. 2: shows % B. subtilis retention in liposomes of the
invention v incubation time in plasma. PC or DSPC liposomes
containing .sup.125I labelled B.subtilis were incubated with mouse
plasma (.smallcircle.) or PBS (.circle-solid.) at 37.degree. C. At
time intervals samples were fractionated on a sucrose gradient to
separate freed from entrapped B. subtilis and values for triplicate
experiments are %+SD of radioactivity recovered with liposomes.
[0041] FIG. 3: shows retention of tetanus toxoid by liposomes of
the invention in the presence of plasma. PC or DSPC liposomes
containing .sup.125I labelled tetanus toxoid were incubated in the
presence of plasma (.smallcircle.) or PBS (.circle-solid.) at
37.degree. C. At time intervals samples were centrifuged at 600 xg
to separate freed from entrapped toxoid. values from triplicate
experiments are %+SD of radioactivity recovered with liposomes.
[0042] FIG. 4: shows clearance of tetanus toxoid and B. subtilis
after intramuscular injection into mice. Mice were injected into
their hind legs with free (.smallcircle.) or liposome entrapped
(.circle-solid.) .sup.125I 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 %+SD of the radioactivity
recovered in the leg immediately after injection.
Comparative Example
[0043] Preparation of Vaccine-containing Giant Liposomes in the
Presence of Organic Solvents
[0044] Giant liposomes with entrapped materials were prepared by
the solvent spherule evaporation method (Kim and Martin, 1981) with
modifications.
[0045] In brief, 1 mL of a 0.15M sucrose solution containing the
.sup.125I-labelled material to be encapsulated
(2.times.10.sup.3-10.sup.4 cpm; 1-2 mg tetanus toxoid and 10.sup.5
B.subtilis bacteria as spores) was mixed by vortexing for 45 s with
1 mL of a chloroform solution containing PC or DSPC, Chol, PG and
TO (4:4:2:1 molar ratio; 9 pmoles total lipid). The resulting
water-in-chloroform emulsion was mixed by vortexing for 15 s with a
diethyl ether-in-water emulsion prepared from 0.5 ml of a solution
of the lipids as above in diethyl ether and 2.5 mL of a 0.2M
sucrose solution. The water-in-oil-in-water emulsion thus formed
was placed in a 250 mL conical flask and the organic solvents were
evaporated by flushing nitrogen at 37.degree. C. while the sample
was gently agitated in a shaking incubator. Generated liposomes
were centrifuged twice at 600 xg for 5 min over a 5% glucose
solution and the pellet was resuspended in 0.1 M sodium phosphate
buffer supplemented with 0.9% 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.subtilis (which
sedimented together with liposomes) sucrose gradient fractionation
was used (see below). In some experiments, FITC-labelled
polystyrene particles (0.5 and 1 .mu.m diameter; Polysciences) or
FITC-labelled B.subtilis were entrapped as above in liposomes which
were used in fluorescence microscopy studies.
EXAMPLE 1
[0046] Entrapment of Live Vaccines in Preformed Giant Liposomes by
Method of the Invention
[0047] 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 .mu.moles) and centrifuged over a 5% glucose
solution in a bench centrifuge at 600 xg for 5 min. The liposomal
pellet was resuspended in 1 ml 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.subtilis as spores, tetanus toxoid and 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 distiled 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 Hetosicc freeze drier, at a vacuum of 0.1 torr for 4 to 5
hours, or 13 Pa overnight.
[0048] The freeze-dried material was rehydrated initially by the
addition of 0.1 mL distilled water at 20.degree. C. (rehydration of
liposomes containing the "high melting" DSPC at 50-60.degree. C.
did not have a significant effect on percent entrapment of
materials). The suspension was vigourously swirled and allowed to
stand for 30 min. The process was repeated after the successive
addition of 0.1 mL PBS and of 0.8 mL PBS 30 min later. All
materials for entrapment were .sup.125I-labelled for assessing
uptake. In one experiment, B.subtilis--containing liposomes
prepared as above, were freeze-dried in the presence of 0.25M
(final concentration) trehalose and subsequently reconstituted in
PBS.
EXAMPLE 2
[0049] Separation of Liposomes Containing Entrapped Material from
Non-entrapped Material
[0050] Separation of entrapped from non-entrapped material was
carried out by sucrose gradient centrifugation (B.Subtilis and BCG;
see below) or centrifugation at 600 xg for 10 min followed by the
suspension of the twice 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, 5833-5837) by layering ten dilutions (sucrose content ranging
from 0.4M to 4M) of a 4M 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 for 1.5 hours at 25,000
rpm in a Dupont Combi Plus ultracentrifuge using a swing-out
bucket. After centrifugation, 1 mL fractions were pippeted out from
the top of the gradient and assayed for .sup.125I radioactivity in
a Wallac Minigamma counter and phospholipid content.(Stewart,
(1979) Anal. Biochem, 104, 10-14). Separation of liposomes form
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 600 xg for 10
minutes as for removal of non-entrapped tetanus toxoid.
[0051] Measurement of Vesicle Size
[0052] The mean diameter in terms of volume distribution of giant
liposomes was measured in a Malvern Mastersizer.
[0053] Light Microscopy
[0054] Light microscopy studies on liposomes containing
FITC-labelled B.subtilis or polystyrene particles were carried out
using a Nikon microscope and a Leica confocal microscope, both
equipped with fluoresence 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.
[0055] Stability of Giant Liposomes in Plasma
[0056] PC or DSPC giant liposomes (3-5 mg total lipid) containing
radiolabelled toxoid (10.sup.3 cpm; 0.1-0.2 mg) or B.subtilis
(3-5.times.10.sup.3 cpm; 10.sup.3 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.degree. 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
600 xg (toxoid). Vesicle stability was expressed as percent of
originally entrapped material retained by the vesicles.
[0057] Clearance of Liposome-entrapped Toxoid and B.subtilis after
Intramuscular Injection into Mice
[0058] Forty eight male CDI mice.(body weight 20-25 g) were
randomly divided into four groups of twelve animals each and
injected intramuscularly (hind leg) with 0.1 ml of (a) free
radiolabelled toxoid (6.times.10.sup.3 cpm; 0.01 mg), (b) free
radiolabelled B.subtilis (3.times.10.sup.3 cpm; 10.sup.3 bacteria
as spores); (c) toxoid as in (a) entrapped by the method of Example
1 into DSPC liposomes (1 mg 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. .sup.125I was measured in the isolated legs and results
were expected as percent of radioactivity recovered in the tissue
at zero time.
[0059] Results
[0060] Entrapment Studies
[0061] 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 .sup.125I
radioactivity (B.subtilis) and phospholipid (liposomes) values
measured in the fractions after centrifugation indicate that
bacteria-containing PC or DSPC liposoines were recovered in the
upper ten 0.5 ml fractions of the gradient, with free B.subtilis
sedimenting to the bottom fraction.
[0062] 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.+-.2.2 .mu.m) with a
lower and upper diameter range (all preparations) of 1-14 .mu.m.
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.subtilis presence
in the top ten fractions of the sucrose gradient (which coincided
with the presence of liposomal phospholipid in the same fractions),
B.subtilis entrapment in twelve separate experiments by the method
of Kim and Martin was variable with a mean value of 31.6% 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.
[0063] 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 .mu.m and a
lower and upper range of 1-18 .mu.m; all preparations) similar to
those of the parent vesicles (see above), containing variable but
substantial proportions of B.subtilis, 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.
[0064] Morphological Studies
[0065] Light microscopy revealed that nearly all giant vesicles
stained with oil red-O contained a varying number of FITC-labelled
B.subtilis 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 (0.5
.mu.m and 1 .mu.m in diameter) as particulate material for
entrapment in 18 .mu.m 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.
[0066] Stability of Giant Liposomes in Plasma
[0067] An important prerequisite for 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.degree. C.
[0068] In the event B.subtilis-containing PC giant liposomes
retained 80-90% of their bacteria content for at least 24 hours in
the presence of plasma. As about 10% 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 (>95%) 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 0.8 .mu.m), giant
liposomes appeared to gradually lose significant amounts or their
toxoid content in the presence of plasma with the loss being more
pronounced for PC (48%) than for DSPC liposomes (38%; 24 hour
values).
[0069] Stability of Giant Liposomes in Vivo after Intramuscular
Injection
[0070] The extent of tetanus toxoid and B.subtilis 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.subtilis from the tissue
could provide an indication of the extent to which liposomes
retained their content in 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 (27%). 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.
[0071] 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.subtilis 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 liposomal
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.subtilis formulations 24 hours after
injection indicates the considerable extent to which liposomes
remain stable in the injected tissue.
[0072] 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.
[0073] 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.
[0074] Formation of Multilamella Liposomes of the Invention
EXAMPLE 3
[0075] Entrapment of Live Spores and Other Materials in
Multilamella DRV Liposomes
[0076] Small unilamella vesicles composed of PC or DSPC and
equimolar cholesterol (18 .mu.moles phospholipid) were prepared as
described in Kirby and Gregoriadis, 1984) whereby phospholipid and
cholesterol were dissolved in chloroform which was then rotary
evaporated to leave a thin lipid film on the walls of the flask.
The film was disrupted at 4.degree. C. (PC) or 60.degree. 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 .sup.125I-labelled live spores (10.sup.7),
100-150 .mu.g .sup.125I-labelled toxoid or a mixture of
radiolabelled spores and unlabelled toxoid and freez-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 90,000 xg 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)
[0077] Results
[0078] Spore entrapment was unexpectedly high (63.6-78%) inspite of
previous findings of relatively small vesicle size for these type
of vesicles. The mean size for PC-DRVs was 3.6 .mu.m and for
DSPC-DRVs was 3.2 .mu.m diameter.
[0079] Spore Viability
[0080] Giant or DRV liposomes containing B.subtilis spores were
tested for spore viability by treating them with Triton 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.
[0081] 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 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.
[0082] 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.
1TABLE 1 Estimated average number of viable B. subtilis spores per
vesicle. Type Number of colonies Spores Vesicle Method Triton
Control per vesicle size .mu.m Giant PC A 96 29 3 6.3 Giant DSPC A
162 38 4 6.5 Giant PC B 203 10 20 8.4 Giant DSPC B 250 21 12 7.2
Giant PC B 160 12 13 8.0 Giant DSPC B 109 15 7 6.4 DRV PC 320 44 7
3.6 DRV DSPC 189 31 6 3.2
[0083] B. subtilis 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.
* * * * *