U.S. patent application number 12/553086 was filed with the patent office on 2011-03-03 for multi-phase emulsions based on amphiphilic block copolymers.
This patent application is currently assigned to NATIONAL HEALTH RESEARCH INSTITUTES. Invention is credited to Hsin-Wei Chen, Pele Choi-Sing Chong, Ming-Hsi HUANG, Chih-Hsiang Leng, Shih-Jen Liu.
Application Number | 20110052633 12/553086 |
Document ID | / |
Family ID | 43625270 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110052633 |
Kind Code |
A1 |
HUANG; Ming-Hsi ; et
al. |
March 3, 2011 |
MULTI-PHASE EMULSIONS BASED ON AMPHIPHILIC BLOCK COPOLYMERS
Abstract
An composition comprises (a) a continuous aqueous phase; (b) an
oily phase; and (c) an amphiphilic emulsifying system comprising a
block copolymer having the formula (A)p-(B)q-(C)r, in which (B)q is
a hydrophilic block and (C)r is a hydrophobic block; wherein p, q
and r are integers with the proviso that: if p is 0, then A is
other than hydroxyl or reactive functional groups and the block
copolymer is a diblock copolymer; B and C are each individual
repeating) units; if p is >1, then the block copolymer is a
triblock copolymer, in which (A)p is a hydrophobic block; the
hydrophobic blocks (A)p and (C)r are each homopolymers or
heteropolymers; the repeating units A and C are the same or
different; and wherein the ratio q/(p+r) is sufficient high so that
the hydrophilic-lipophilic balance value of the block copolymer is
>10.
Inventors: |
HUANG; Ming-Hsi; (Miaoli
County, TW) ; Chong; Pele Choi-Sing; (Miaoli County,
TW) ; Leng; Chih-Hsiang; (Miaoli County, TW) ;
Liu; Shih-Jen; (Miaoli County, TW) ; Chen;
Hsin-Wei; (Miaoli County, TW) |
Assignee: |
NATIONAL HEALTH RESEARCH
INSTITUTES
Miaoli County
TW
|
Family ID: |
43625270 |
Appl. No.: |
12/553086 |
Filed: |
September 2, 2009 |
Current U.S.
Class: |
424/209.1 ;
523/105 |
Current CPC
Class: |
A61P 37/04 20180101;
C12N 2760/16134 20130101; Y10S 514/937 20130101; C08G 63/664
20130101; A61K 2039/55555 20130101; A61K 39/12 20130101; A61K
2039/5252 20130101; Y10S 514/938 20130101; A61K 39/39 20130101;
A61K 39/145 20130101; A61K 2039/55566 20130101; A61P 31/16
20180101; C08G 63/06 20130101 |
Class at
Publication: |
424/209.1 ;
523/105 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61K 8/86 20060101 A61K008/86 |
Claims
1. A composition comprising: (d) a continuous aqueous phase
comprising H, O, (e) an oily phase comprising oil; and (f) an
amphiphilic emulsifying system stabilizing the interface between
the oily phase and the continuous aqueous phase, comprising a block
copolymer having the formula (A)p-(B)q-(C)r wherein: p, q and r are
integers with the proviso that: if p is 0, then A is other than
hydroxyl or reactive functional groups and the block copolymer is a
diblock copolymer, in which (B)q is a hydrophilic block and (C)r is
a hydrophobic block, B and C are each individual repeating units;
if p is >1, then the block copolymer is a triblock copolymer, in
which (B)q is a hydrophilic block and (A)p and (C)r are each
hydrophobic blocks; the hydrophobic blocks (A)p and (C)r are each
homopolymers or heteropolymers; A, B and C are each individual
repeating units; the repeating units A and C are the same or
different; and wherein the ratio q/(p+r) is sufficient high so that
the hydrophilic-lipophilic balance (HLB) of the block copolymer is
>10.
2. The composition of claim 1, wherein p is 0 and A is methoxy.
3. The composition of claim 1, wherein the continuous aqueous phase
comprises an antigen and/or a bioactive substance.
4. The composition of claim 1, wherein the oily phase entraps or
encapsulates an antigen and/or a bioactive agent.
5. The composition of claim i, wherein the oily phase further
comprises an emulsion comprising: (c) an internal aqueous phase
comprising H.sub.2O, being dispersed in the oily phase; and (d) a
lipophilic emulsifying system stabilizing the interface between the
inner aqueous phase and the oily phase to form a water-in-oil (W/O)
emulsion; wherein the composition is in the form of a W/O/W
emulsion.
6. The composition of claim 5, wherein the lipophilic emulsifying
system comprises at least one physiologically acceptable emulsifier
selected from the group consisting of mannide monooleat and
sorbitan esters.
7. The composition of claim 5, further comprising an antigen and/or
a bioactive agent dissolved in the internal aqueous phase and/or in
the continuous aqueous phase.
8. The composition of claim 1, wherein the block copolymer is
bioresorbable.
9. The composition of claim 1, wherein the hydrophilic block (B)q
is a liner polymer.
10. The composition of claim 1, wherein the repeating unit B is
selected from the group consisting of ethylene oxide,
vinylpyroolidone, and acrylamide.
11. The composition of claim 1, wherein the hydrophobic blocks (A)p
and (C)r are each polyester polymers.
12. The composition of claim 1, wherein the hydrophobic blocks (A)p
and (C)r are each aliphatic polyesters.
13. The composition of claim 12, wherein the aliphatic polyesters
are polymers of dicarboxylic acids and a diols.
14. The composition of claim 12, wherein the repeating units A and
C are each selected from the group consisting of hydroxyacids,
lactones, and combinations thereof.
15. The composition of claim 14, wherein the hydroxyacids are
selected from the group consisting of lactic acid, 6-hydroxycaproic
acid, glycolic acid, malic acid monoesters, and combinations
thereof.
16. The composition of claim 14, wherein the lactones are selected
from the group consisting of .epsilon.-caprolactone, lactide,
glycolide, para-dioxanones, and combinations thereof.
17. The composition of claim 1, wherein the hydrophobic blocks (A)p
and (C)/r are each comprises a polymer selected from the group
consisting of: (i) a hydroxyacid-containing polymer: poly(lactide),
poly(lactic acid), poly(lactide-co-glycolide), poly(glycolide),
poly(lactic acid-co-glycolic acid), poly(malic acid), poly(malic
acid ester), or poly(glycolic acid); (ii) a lactone-containing
polymer: poly(lactide-co-.epsilon.-caprolactone),
poly(.epsilon.-caprolactone), or
poly(glycolide-co-.epsilon.-caprolactone); and (iii) a
hydroxycaproic acid-containing polymer: poly(lactic
acid-co-6-hydroxycaproic acid), or poly(glycolic
acid-co-6-hydroxycaproic acid).
18. The composition of claim 1, wherein the block copolymer is
selected from the group consisting of poly(ethylene
glycol)-block-poly(lactide-co-.epsilon.-caprolactone),
poly(ethylene glycol)-block-polylactide, and poly(ethylene
glycol)-block-poly(.epsilon.-caprolactone).
19. A block copolymer comprising the formula (A)p-(B)q-(C)r
wherein: p, q and r are integers with the proviso that: if p is 0,
then A is other than hydroxyl or reactive functional groups and the
block copolymer is a diblock copolymer, in which (B)q is a
hydrophilic block and (C)r is a hydrophobic block; B and C are each
individual repeating units; if p is >1, then the block copolymer
is a triblock copolymer, in which (B)q is a hydrophilic block and
(A)p and (C)r are each hydrophobic blocks; the hydrophobic blocks
(A)p and (C)r are each homopolymers or heteropolymers; A, B and C
are each individual repeating units; the repeating units A and C
are the same or different; and wherein the ratio q/(p+r) is
sufficient high so that the hydrophilic-lipophilic balance (1-1.03)
of the block copolymer is >10.
20. The block copolymer of claim 19, wherein the hydrophilic block
(B)q constitutes at least 50% by weight of the block copolymer.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to emulsion
formulations, and more specifically to a multi-phase emulsion
formulations.
BACKGROUND OF THE INVENTION
[0002] Among the vaccine adjuvants evaluated in human trials, the
emulsion-type adjuvants have the advantages of ease of manufacture
and low costs. Freund's adjuvants and Montanide.RTM. ISA 51
(ISA51), containing mineral oil and lipophilic emulsifier named
mannide monooleate, are defined as water-in-oil (W/O) emulsions
with dispersed antigenic media and continuous oily phases. Although
the mechanisms of adjuvant's action are poorly understood, the W/O
types of adjuvant products have been evaluated to improve the
innocuity of the vaccine and to achieve long-term protective immune
responses. It is difficult to give injections with syringe needle
having a small diameter. There are also local reactions at the
injection site, which limit their applications to humans. To
improve the injectability of such vaccines, a method for
re-dispersing them in an aqueous phase containing a hydrophilic
emulsifier Tween.RTM.80 (polyoxyethylene sorbitan monooleate) has
been described. Nevertheless, Tween.RTM.80 is a lipid dispersant,
can attack cell walls and hence is potentially toxic. Preclinical
experience has found that Tween.RTM.80-stabilized emulsions were
generally more immunogenic than non-adjuvanted vaccines but also
increased the reactogenicity.
[0003] To increase the number of safe emulsifiers for vaccine
adjuvants, synthetic polymer is regarded as an interesting
alternative to low-molecular weight surfactants (LMWS) because the
size and the relative positions of hydrophilic and lipophilic
blocks can be easily tailored by the order of the monomer addition
and by the amounts of monomer used to produce a broad range of
surfactant characteristics. One of the examples is TiterMax.RTM.,
in which a squalene-based water-in-oil (W/O) emulsion is stabilized
by microparticulate silica and the non-ionic block copolymer
polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE,
known as Pluronic.RTM. or Poloxamer.RTM.). TiterMax.RTM. elicits
more potent immune responses than the LMWS-emulsified formulation
but its application in human vaccine delivery is still dubious
because the stabilizers used are toxic and non-biodegradable.
[0004] A heretofore unaddressed need exists in the art to address
the aforementioned deficiencies and inadequacies, especially in
connection with enhancement of the water affinity of W/O
emulsion-adjuvanted vaccines.
SUMMARY OF THE INVENTION
[0005] One aspect of the invention relates to a composition
comprising: [0006] (a) a continuous aqueous phase comprising
H.sub.2O; [0007] (b) an oily phase comprising oil; and [0008] (c)
an amphiphilic emulsifying system stabilizing the interface between
the oily phase and the continuous aqueous phase, comprising a block
copolymer having the formula
[0008] (A)p-(B)q-(C)r [0009] wherein: [0010] p, q and r are
integers with the proviso that: [0011] if, is 0, then A is other
than hydroxyl or reactive functional groups and the block copolymer
is a diblock copolymer, in which (B)q is a hydrophilic block and
(C)r is a hydrophobic block; B and C are each individual repeating
units; if p is >1, then the block copolymer is a triblock
copolymer, in which (B)q is a hydrophilic block and (A)p and (C)r
are each hydrophobic blocks; the hydrophobic blocks (A)p and (C)r
are each homopolymers or heteropolymers; A, B and C are each
individual repeating units; the repeating units A and C are the
same or different; [0012] and wherein the ratio q/(p+r) is
sufficient high so that the hydrophilic-lipophilic balance (HLB)
oldie block copolymer is >10.
[0013] In one embodiment of the invention, the aforementioned
composition is dispersed in a phosphate-buffered saline solution
(PBS), wherein the composition is in the form of an oil-in-water
(O/W) emulsion, and wherein the aqueous phase comprises PBS.
[0014] The oily phase may entrap or encapsulate an antigen and/or a
bioactive agent. Alternatively, the continuous aqueous phase may
comprise an antigen and/or a bioactive substance.
[0015] In another embodiment of the invention, the aforementioned
composition is free of an organic solvent.
[0016] In another embodiment of the invention, the oily phase
further comprises an emulsion comprising: [0017] (a) an internal
aqueous phase comprising H.sub.2O, being dispersed in the oily
phase; and [0018] (b) a lipophilic emulsifying system stabilizing
the interface between the inner aqueous phase and the oily phase to
form a water-in-oil (W/O) emulsion; [0019] wherein the composition
is in the form of a W/O/W emulsion.
[0020] In another embodiment of the invention, the lipophilic
emulsifying system comprises at least one physiologically
acceptable emulsifier selected from the group consisting of mannide
monooleat and sorbitan esters.
[0021] The W/O/W emulsion formulation may further comprise an
antigen and/or a bioactive agent dissolved in the internal aqueous
phase and/or in the continuous aqueous phase. The antigen may be an
inactivated virus, e.g., H5N1 virus, or a bacterium and/or an
antigenic protein or an antigenic fusion protein.
[0022] In another embodiment of the invention, the inner aqueous
phase further comprises an antigen and/or a bioactive agent.
[0023] Another aspect of the invention relates to a block copolymer
comprising the formula:
(A)p-(B)q-(C)r
[0024] wherein: [0025] p, q and r are integers with the proviso
that: [0026] if p is 0, then A is other than hydroxyl or reactive
functional groups and the block copolymer is a diblock copolymer,
in which (B)q is a hydrophilic block and (C)r is a hydrophobic
block; B and C are each individual repeating units; if p is >1,
then the block copolymer is a triblock copolymer, in which (B)q is
a hydrophilic block and (A)p and (C)r are each hydrophobic blocks;
the hydrophobic blocks (A)p and (C)r are each homopolymers or
heteropolymers; A, B and C are each individual repeating units; the
repeating units A and C are the same or different; [0027] and
wherein the ratio q/(p+r) is sufficient high so that the
hydrophilic-lipophilic balance (HLB) value of the block copolymer
is >10.
[0028] In one embodiment of the invention, the hydrophilic block
(B)q constitutes at least 50% by weight of the block copolymer.
[0029] In one embodiment of the invention, p is 0 and A is
methoxy.
[0030] In another embodiment of the invention, the block copolymer
is bioresorbable.
[0031] In another embodiment of the invention, the hydrophilic
block (B)q is a liner polymer.
[0032] In another embodiment of the invention, the repeating unit B
is selected from the group consisting of ethylene oxide,
vinylpyroolidone, and acrylamide.
[0033] In another embodiment of the invention, the hydrophobic
blocks (A)p and (C)r are each polyester polymers.
[0034] Further in another embodiment of the invention, the
hydrophobic blocks (A)p and (C)r are each aliphatic polyesters.
[0035] In another embodiment of the invention, the repeating units
A and C are each selected from the group consisting of
hydroxyacids, lactones, and combinations thereof.
[0036] The hydroxyacids may be selected from the group consisting
of lactic acid, 6-hydroxycaproic acid, glycolic acid, malic acid
monoesters, and combinations thereof.
[0037] The lactones may be selected from the group consisting of
.epsilon.-caprolactone, lactide, glycolide, para-dioxanones, and
combinations thereof.
[0038] In another embodiment of the invention, the aliphatic
polyesters are polymers of dicarboxylic acids and a diols.
[0039] In another embodiment of the invention, the hydrophobic
blocks (A)p and (C)r are each comprises a polymer selected from the
group consisting of the following: [0040] (i) a
hydroxyacid-containing polymer: poly(lactide), poly(lactic acid),
poly(lactide-co-glycolide), poly(glycolide), poly(lactic
acid-co-glycolic acid), poly(malic acid), poly(malic acid ester),
or poly(glycolic acid): [0041] (ii) a lactone-containine, polymer:
poly(lactide-co-.epsilon.-caprolactone),
poly(.epsilon.-caprolactone), or
poly(glycolide-co-.epsilon.-caprolactone), and [0042] (iii) a
hydroxycaproic acid-containing polymer: poly(lactic
acid-co-6-hydroxycaproic acid), or poly(glycolic
acid-co-6-hydroxycaproic acid).
[0043] Yet in another embodiment of the invention, the block
copolymer is selected from the group consisting of poly(ethylene
glycol)-block-poly(lactide-co-c-Caprolactone), poly(ethylene
glycol)-block-polylactide, and poly(ethylene
glycol)-block-poly(.epsilon.-caprolactone).
[0044] These and other aspects will become apparent from the
following description of the preferred embodiment taken in
conjunction with the following drawings, although variations and
modifications therein may be affected without departing from the
spirit and scope of the novel concepts of the disclosure.
[0045] The accompanying drawings illustrate one or more embodiments
of the invention and, together with the written description, serve
to explain the principles of the invention. Wherever possible, the
same reference numbers are used throughout the drawings to refer to
the same or like elements of an embodiment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 shows (a) the visual aspects of emulsions stored at
37.degree. C. and (b) the cumulative release of OVA from the
emulsion formulations based on bioresorbable polymers and the oily
adjuvant ISA5 I (-x-) Non-formulation, (open circle)
PEG-b-PLA/ISA51, (open square) PEG-b-PCUISA51, (open triangle)
PEG-b-PLACL/ISA51, (filled circle) PBS/ISA51. The OVA-containing
formulations (3 mg per 0.3 mL) were placed in a dialysis chamber in
a centrifuge tube containing 2 ml PBS and stood at 37.degree. C.
The OVA release was regularly monitored by the BCA method (read by
an UV-vis instrument at 562 nm, using calibration curves obtained
from standard BSA solutions). The data are presented as the mean
with standard deviation of three samples.
[0047] FIGS. 2(a)-2(d) are schematic presentations showing an
ISA51-adjuvanted vaccine (a) before and (b) after stabilization
with (c) a bioresorbable polymer. The ISA51 contains lipophilic
mannide monooleate, which renders a W/O emulsion because of its
high affinity for the oily phase. After incorporating the
hydrophilic emulsifier PEG-b-PLA, PEG-b-PCL, or PEG-b-PLACL in the
antigen medium, the water affinity of the oily ISA51-adjuvanted
vaccine was greatly enhanced so that the antigen-encapsulated stock
emulsion was able to be re-dispersed in the PBS and resulted in an
emulsion with homogeneous fine particles having (d) a particle size
distribution of <1 .mu.m.
[0048] FIG. 3 is a graph showing specific antibody responses in
mice following immunization with OVA in different formulations.
BALB/c mice were subcutaneously vaccinated twice at weeks 0 and 2
with 0.5 .mu.g of OVA. Sera were collected from blood and the
antibody titers were measured by ELISA. The data are presented as
geometric mean titers with standard errors (5 mice per group).
*P<0.005: A comparison to the non-formulated OVA group was made
at the same time point.
[0049] FIGS. 4(a)-4(b) show the schemes for synthesizing (a) the
diblock copolymer PLA-PEG and (b) the triblock copolymer
PLA-PEG-PLA.
[0050] FIGS. 5(a)-5(b) show the MALD1-TOF mass spectra of (a)
MePEG.sub.2000 and (b) PLA-PEG. FIGS. 6(a)-6(b) show the MALDI-TOF
mass spectra of (a) di0H-PEG.sub.2000 and (b) PLA-PEG-PLA.
[0051] FIG. 7 is a graph showing the in vitro OVA release from the
squalene emulsions based on the PLA-PEG and PLA-PEG-PLA. The
OVA-containing formulations (3 mg per 0.3 mL) were placed in a
dialysis chamber in a centrifuge tube containing 2 ml of PBS and
stood at 37.degree. C. The release was regularly monitored by the
RCA method and read by an UV-vis instrument at 562 nm using
calibration curves obtained from standard BSA solutions. Data are
presented as the mean with standard errors of three samples. (-x-)
Non-formulation, (open circle) PLA-PEG/squalene, (open triangle)
PLA-PEG-PLA/squalene.
[0052] FIGS. 8(a)-8(c) are photographs showing polymer/oil
emulsions (a) before and (b) after homogenization at 6000 rpm for 5
min, and (c) the visual aspects of the emulsions stored at
4.degree. C. for two months. The polymer concentration in the
antigen medium aqueous solution was 13 wt % and the aqueous/oily
solution was 5/5 w/w. (i) PEG-b-PLACL/squalene, (ii)
PEG-b-PLACL/squalene/Span.RTM.85, (iii)
PBS/squalene/Span.RTM.085.
[0053] FIGS. 9(a)-9(b) are photographs showing the results of
droplet tests of the emulsion formulations (a)
PBS/squalene/Span.RTM.85 and (b) PEG-b-PLACL/squalene.
[0054] FIGS. 10(a)-10(b) show microscopic aspects and laser light
scattering analysis of polymer-emulsified formulations (a)
PEG-b-PLACL/squalene and (b) of
PEG-b-PLACL/squalene/Span.RTM.85.
[0055] FIG. 11(a) is a graph showing the cumulative release of OVA
from various formulations. (-x-) No adjuvant, (open circle) the
aqueous solution PEG-b-PLACL, (open square) the O/W emulsion
PEG-b-PLACL/Squalene, (open triangle) the W/O/W emulsion
PEG-b-PLACL/squalene/Span.RTM.85, (filled circle) the W/O emulsion
PBS/squalene/Span.RTM.85. The OVA-containing formulations (3 mg per
0.3 mL) were placed in a dialysis chamber in a centrifuge tube
containing 2 mL of PBS and stood at 37.degree. C. The OVA release
was monitored by the BCA method and read by an UV--vis instrument
at 562 nm using calibration curves obtained from the standard BSA
solutions (2, 1, 0.5, 0.25, 0.125 mg/mL). The data are presented as
the mean with standard errors of three samples.
[0056] FIG. 11(b) is photograph showing the recovered formulations
after the experiments in FIG. 11(a).
[0057] FIGS. 12(a)-12(b) show (a) T-cell proliferation and (b)
cytokine release responses in spleen cells from mice immunized with
OVA in different formulations with or without adjuvants. (i)
control; (ii) no adjuvant; (iii) PEG-b-PLACL/squalene; (iv)
PEG-b-PLACL/squalene/Span.RTM.85; (v) aluminum phosphate. The
BALB/c mice were vaccinated subcutaneously at week 0 with 0.5 .mu.g
of OVA and boosted at weeks 2 and 4. One week after the final
boost, splenocyte suspensions from a pool of two mice per group
were prepared for cytokines assay and incubated over five days with
or without 10 .mu.g/mL of antigen OVA. The stimulation index (SI)
is the ratio of the mean counts per minute (c.p.m.) in the presence
of the antigen to the c.p.m. in the absence of the antigen. Results
are expressed as the mean with standard errors (n=3). Proliferation
was read as positive when the SI values were >2. Supernatants
collected from triplicate cultures were measured by IFN-.gamma. and
IL-4 cytokine ELISA paired antibodies. Data are presented as
cytokine release in the presence of OVA minus that with medium only
(n=3).
[0058] FIG. 13 is a schematic representation of a W/O/W emulsion.
The emulsion PEG-b-PLACL/squalene/Span.RTM.85 comprises two
surfactants, PEG-b-PLACL and Span.RTM.85, rendering a W/O/W
multi-phase emulsion, in which the oil droplets were dispersed in
the continuous water, but the core oil also traps an internal
aqueous phase.
[0059] FIG. 14 shows a PELC-formulated influenza vaccine having
homogeneous fine particles with diameters ranging from 200 to 400
nm. The inset is a histogram showing the particles' size
distribution.
[0060] FIG. 15A is a graph showing T-cell proliferation in the
splenocytes of mice immunized with inactivated H5N1 virus alone or
with PELC. The BALB/c mice were vaccinated i.m. with a single-dose
of 0.5 .mu.g of viral HA. Twelve days after immunization,
splenocyte suspensions were pooled from three mice per group and
incubated for four clays with or without 0.25 .mu.g HA/mL of
antigen. The Stimulation Index (SI) is the ratio of the mean counts
per minute (cpm) with antigen to the cpm without antigen. The data
are expressed as the mean plus the standard deviation (n=3).
Proliferation was deemed positive when the SI value was >3.
[0061] FIG. 15B is a graph showing cytokines release from the
splenocytes. The supernatants collected from the triplicate
cultures in FIG. 15A were assessed with IFN-.gamma. and IL-4 ELISA.
The data are expressed as the mean plus the standard errors
(n=3).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0062] The terms used in this specification generally have their
ordinary meanings in the art, within the context of the invention,
and in the specific context where each term is used. Certain terms
that are used to describe the invention are discussed below, or
elsewhere in the specification, to provide additional guidance to
the practitioner regarding the description of the invention. For
convenience, certain terms may be highlighted, for example using
italics and/or quotation marks. The use of highlighting has no
influence on the scope and meaning of a term: the scope and meaning
of a term is the same, in the same context, whether or not it is
highlighted. It will be appreciated that same thing can be said in
more than one way. Consequently, alternative language and synonyms
may be used for any one or more of the terms discussed herein, nor
is any special significance to be placed upon whether or not a term
is elaborated or discussed herein. Synonyms for certain terms are
provided. A recital of one or more synonyms does not exclude the
use of other synonyms. The use of examples anywhere in this
specification including examples of any terms discussed herein is
illustrative only, and in no way limits the scope and meaning of
the invention or of any exemplified term. Likewise, the invention
is not limited to various embodiments given in this
specification.
[0063] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains. In the
case of conflict, the present document, including definitions will
control.
[0064] As used herein, "around", "about" or "approximately" shall
generally mean within 20 percent, preferably within 10 percent, and
more preferably within 5 percent of a given value or range.
Numerical quantities given herein are approximate, meaning that the
term "around", "about" or "approximately" can be inferred if not
expressly stated.
[0065] As used herein, "immunity adjuvants" shall generally mean
products which increase the reactions of the immunity system when
they are administered in the presence of antigen of virus,
bacterial or synthetic origin.
[0066] As used herein, the term "biodegradable" shall generally
mean solid polymeric materials which break down due to
macromolecular degradation with dispersion in vivo but no proof for
the elimination from the body (this definition excludes
environmental, fungi or bacterial degradation). Biodegradable
polymeric systems can be attacked by biological elements so that
the integrity of the system, and in some cases but not necessarily,
of the macromolecules themselves is affected and gives fragments or
other degradation by-products. Such fragments can move away from
their site of action but not necessarily from the body.
[0067] As used herein, the term "bioresorbable" shall generally
means solid polymeric materials which show bulk degradation and
further resorb in vivo; i.e. polymers which are eliminated through
natural pathways either because of simple filtration of degradation
by-products or after their metabolization. Bioresorption is thus a
concept which reflects a total elimination of the initial foreign
material and of bulk degradation by-products (low molecular weight
compounds) with no residual side effects. The use of the word
`bioresorbable` assumes that the elimination is shown conclusively
(Dietmar W. Hutmacher (2000) "Scaffolds in tissue engineering bone
and cartilage" Biomaterials 21:2529-2543, which is herein
incorporated by reference in its entirety).
[0068] As used herein, a "lactone" is a cyclic ester which can be
seen as the condensation product of an alcohol group --OH and a
carboxylic acid group --COOH in the same molecule. It is
characterized by a closed ring consisting of two or more carbon
atoms and a single oxygen atom, with a ketone group=O in one of the
carbons adjacent to the latter. A "lactide" is a cyclic diester of
lactic acid, i.e., a di-lactone. A "glycolide" is a cyclic diester
of glycolic acid, which is also a di-lactone.
[0069] As used herein, "dicarboxylic acids" are organic compounds
that are substituted with two carboxylic acid functional groups,
such as succinic acid.
[0070] As used herein, a "diol" or "glycol" is a chemical compound
containing two hydroxyl groups (--OH groups), such as ethylene
glycol.
[0071] As used herein, a "hydroxy acid" is an organic compound
which contains a carboxylic acid functional group and hydroxy
functional group.
[0072] As used herein, the term "amphiphile" means any organic
compounds composed of hydrophilic and hydrophobic portions.
[0073] As used herein, the term "the ratio q/(p+r) is sufficient
high" means that the block copolymer of the formula of
(A)p-(13)q-(C)r has a hydrophilic-lipophilic balance (HLB) greater
than 10 so that it promotes an oily phase to disperse in an aqueous
phase and result in an oil-in-water (O/W) emulsion. For example,
the HLB of the amphiphilc block copolymer may be greater than 10,
11, 12, 13, 14, 15, 16, 17, 18, or near 20.
[0074] As used herein, the term "A is other than a reactive
functional group or a reactive group" refers to any group that does
not react under conditions where the non-protected group reacts. In
this case, the group "A" protects reactive functional groups, such
as hydroxyl or amino groups, from their reaction with growing
species in polymerization.
[0075] As used herein, the term "hydroxy-protecting group" refers
to any group commonly used for the protection of hydroxy functions
during subsequent reactions, including, for example, alkoxyl, acyl
or alkylsilyl groups such as trintethylsilyl, triethylsilyl,
t-butyldimethylsilyl and analogous alkyl or arylsilyl radicals, or
alkoxyalkyl groups such as methoxymethyl, ethoxymethyl,
methoxyethoxymethyl, tetrahydrofuranyl or tetrahydropyranyl. A
"protected-hydroxy" is a hydroxy function derivatized by one of the
above hydroxy-protecting groupings. "Alkyl" represents a
straight-chain or branched hydrocarbon radical or 1 to 10 carbons
in all its isomeric forms, such as methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, pentyl, etc., and the terms
"hydroxyalkyl," "fluoroalkyl" and "deuteroalkyl" refer to such an
alkyl radical substituted by one or more hydroxy, fluoro or
deuterium groups, respectively. An "acyl" group is an alkanoyl
group of 1 to 6 carbons in all its'isomeric forms, or an aroyl
group, such as benzoyl, or halo-, nitro-, or alkyl-substituted
benzoyl groups, or an alkoxycarbonyl group of the type
Alkyl--O--CO--, such as methoxycarbonyl, ethoxycarbonyl,
propyloxycarbonyl, etc., or a dicarboxylic acyl group such as
oxalyl, malonyl, succinoyl, glutaroyl, or adiopoyl. The term "aryl"
signifies a phenyl-, or an -alkyl-, nitro-, or halo-substituted
phenyl group. The term alkoxy signifies the group, alkyl-O--.
[0076] The full names for abbreviations used herein are as follows:
.sup.1H NMR, .sup.1H nuclear magnetic resonance; APCs,
antigen-presenting cells; BCA, bicinchinonic acid; ELISA,
enzyme-linked immunosorbent assay; FDA; Food and Drug
Administration; GPC, gel permeation chromatography; HLB,
hydrophilic-lipophilic balance; IgG, Immunoglobulin G; ISA51,
Montanide.RTM. ISA 51 oily adjuvant; LMWS, low-molecular weight
surfactants; MALDI-TOF MS, matrix-assisted laser
desorption/ionization time of flight mass spectrometry;
MePEG.sub.5000, Poly(ethylene glycol) 5,000 monomethyl ether; Mn,
number average molecular weight; Mw/ Mn, molecular weight
distribution; OVA, ovalbumin; O/W, oil-in-water; PBS, phosphate
buffered saline; PBS/ISA51, PBS dispersed in ISA51 emulsion;
PEG-b-PCL, poly(ethylene glycol)-Nock-poly(.epsilon.-caprolactone);
PEG-b-PLA, poly(ethylene glycol)-block-polylactide; PEG-b-PLACL,
poly(ethylene
glycol)-block-poly(lactide-co-.epsilon.-caprolactone);
PEG-b-PLACL/ISA51/PBS, PEG-b-PLACL-stabilized ISA51 emulsion
following dispersed in PBS; POE-POP-POE,
polyoxyethylene-polyoxypropylene-polyoxyethylene, s.c.,
subcutaneous; SnOct.sub.2, Tin(II) 2-ethylhexanoate; THF,
tetrahydrofuran; TMB, tetramethylbenzidine; Tween.RTM.80,
polyoxyethylene sorbitan monooleate; W/O, water-in-oil; W/O/W,
water-in-oil-in-water.
[0077] Emulsifiers may be defined by their hydrophilic-lipophilic
balance (HLB) values, which give information on their relative
affinity for aqueous and oily phases. An emulsifying system which
contains an emulsifier of low HLB value renders a W/O emulsion with
a high affinity for an oily phase. In contrast, an emulsifying
system which contains a high HLB value affords an O/W emulsion with
a high affinity for an aqueous phase. A W/O/W multi-phase emulsion
may be achieved when an emulsifying system contains an intermediate
HLB value. These parameters, however, are strongly influenced by
the optimization of a surfactant system and the manufacture
process.
[0078] For an emulsifier system comprising one or more emulsifiers,
the HLB is calculated based on the following equation:
HLB.sub.mix=.SIGMA.X.sub.i.times.HLB.sub.i
[0079] where X is the weight fraction of surfactant i.
[0080] For non-ionic surfactants, HLB may be calculated with the
Griffin's method:
HLB=20.times.M.sub.h/M
where M.sub.h is the molecular mass of the hydrophilic portion of
the molecule, and M is the molecular mass of the whole molecule.
The HLB of the most lipophilic molecule is close to 0, while the
HLB of the most hydrophilic molecule is about 20.
[0081] For a non-ionic copolymer, the equation can be represented
as follows:
HLB.sub.copolymer=20.times.W.sub.h/W.sub.copolymer
where W.sub.h/W.sub.copolymer is the weight ratio of the
hydrophilic portion of the main chain polymer and is obtained from
their number average molecular weight ratio Mn.sub.h/
Mn.sub.copolymer.
[0082] A W/O emulsion based on lipophilic mannide monooleate and
water-immiscible oil has been available. The oil used is the
mineral oil Markol (Freund's adjuvants), metabolizable mineral oil
Drakeol (Montanide.RTM. ISA 51) or metabolizable nonmineral
squalene (Montanide.RTM. ISA 720). TiterMax.RTM. is a
squalene-based W/O emulsion stabilized by microparticulate silica
and the nonionic block copolymer
polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE,
known as Pluronic.RTM. or Poloxamer.RTM.). These W/O emulsions are
difficult for injection with a syringe having a needle of small
diameters, and cause local reactions at the injection site of
animals, which considerably restrained the potential of this type
of emulsions for human use.
[0083] The invention relates to formulations for enhancing the
water affinity oil-in-water emulsions, such as oily ajuvanted
vaccines. We designed different types of emulsion formulations
using block copolymers having the formula
(A)p-(B)q-(C)r
[0084] Wherein: [0085] p, q and r are integers with the proviso
that: [0086] if p is 0, then A is other than hydroxyl or reactive
functional groups and the block copolymer is a diblock copolymer,
in which (B)q is a hydrophilic block and (C)r is a hydrophobic
block; B and C are each individual repeating units; [0087] if p is
>1, then the block copolymer is a triblock copolymer, in which
(B)q is a hydrophilic block and (A)p and (C)r are each hydrophobic
blocks; the hydrophobic blocks (A)p and (C)r are each homopolymer
or heteropolymer; A, B and C are each individual repeating units;
the repeating units A and C are the same or different.
[0088] The HLB is sufficient high so that the block copolymer is
capable of stabilizing the interface between an oil phase and an
aqueous phase and promotes the dispersion of the oil phase into the
aqueous phase and forms a stable O/W emulsion. This is particular
useful in redispersing an ISA51-adjuvanted oily vaccine, a W/O
emulsion, into a PBS to form a W/O/W multiphase emulsion.
[0089] In one embodiment of the invention, q is an integer and the
hydrophilic portion (B)q has a molecular weight of from about 550
to about 10,000 daltons, preferably from about 2,000 to about 8,000
daltons, and the ratio q/(p+r) is sufficient high so that the
hydrophilic portion (B)q constitutes from about 50 to about 95% by
weight of the main chain polymer (A)p-(B)q-(C)r, which renders the
hydrophilic-lipophilic balance (HLB) of the block copolymer ranging
from about 10 to about 19. Preferably the hydrophilic portion (B)q
constitutes from about 70 to about 95% by weight of the main chain
polymer so that the HLB value ranges from about 14 to about 19.
[0090] The ratio of q versus p+r is important for the HLB of
amphiphilic macromolecules. If q/(p+r) is not high enough, then the
macromolecules do not dissolve in water. For illustration purpose
Tables A and B list the block copolymers' molar ratios, q/(p+r),
and HLB values (See Huang et al. (2009) "Development of Multi-Phase
Emulsions Based on Bioresorbable Polymers and Oily Adjuvant"
Pharmaceutical Research 26(8): 1856-1862; Huang et al., (2004)
Degradation and cell culture studies on block copolymers prepared
by ring opening polymerization of .epsilon.-caprolactone in the
presence of poly(ethylene glycol). J. Biomed Mater Res 69A:
417-427, which is herein incorporated by reference in its
entirety). In an extreme case where the HLB of a block copolymer is
20, the main chain polymer would consist of only a hydrophilic
block and the polymer would not possess amphiphilic property. (See
Siao et al., (2009) "Characterization and Emulsifying Properties of
Block Copolymers Prepared from Lactic Acid and Poly(ethylene
glycol)" Journal of Applied Polymer Science 114: 509-516, which is
herein incorporated by reference in its entirety).
TABLE-US-00001 TABLE A % of hydrophilic [LA]/[CL]/[OE] q/(p + r)
portion HLB PEG-b-PLA 0.243/--/1 4.1 72% 14.4 PEG-b-PCL --/0.166/1
6.0 70% 14.0 PEG-b-PLACL 0.100/0.068/1 6.0 75% 15.0
TABLE-US-00002 TABLE B % [CL]/[OE] q/(p + r) of hydrophilic portion
HLB PCL-PEG 3.9/1 0.256 9% 1.8 PCL-PEG-PCL 3.7/1 0.270 9.5% 1.9
OCL-PEG 0.03/1 33.3 93.5% 18.7 OCL-PEG-OCL 0.04/1 25.0 91.5% 18.2
PCL stands for poly(.epsilon.-caprolactone). OCL stands for
oligo(.epsilon.-caprolactone).
[0091] The oil must be non-toxic, metabolizable, physiologically
acceptable, and form fluid emulsions when stored at 4.degree. C.
Oils are selected from mineral oils, vegetable or animal oils known
for low toxicity. The selected mineral oils may be straight chain
mineral oils, e.g., Markol (Freund's adjuvants) or Drakeol
(Montanide.RTM. ISA 51). Synthetic hydrocarbons include
polyisobutene and polyisoprene.
[0092] Suitable vegetable oils include oleic type unsaturated oils
that are biodegradable and known for immunogenic power, such as
ground-nut oil, olive oil, sesame oil, soya bean oil, corn oil, and
jojoba oil, etc. Suitable animal oils require the same criteria of
tolerance and immunological efficiency, such as squalane and
squalene (MF59.RTM., AS03, Montanide.RTM. ISA 720, TiterMax).
[0093] The polymer blocks (A)p and (C)r are hydrophobic linear
polyester blocks, e.g., aliphatic polyesters. Aliphatic polyesters
may be obtained as follows: a) either by self-polycondensation of a
hydroxyacid (i.e., homopolymers), or by polycondensation of
different hydroxyacids (i.e., heteropolymers), b) by the
polymerization via ring-opening of lactones; and c) by
polycondensation of diacids and diols.
[0094] Hydroxyacid monomers may be chosen from lactic acid,
glycolic acid, 6-hydroxycaproic acid, malic acid monoesters, e.g.,
alkyl or aralkyl monoesters, or monoesters resulting from the
monoesterification of mane acid with a hydroxylated active
compound, in particular a hydrophobic active compound; lattides
(D-lactide, L-lactide, DL-lactide, and meso-lactide), glycolide,
.epsilon.-caprolactone, para-dioxanone, and the like. The
hydrophobic polymer blocks (A)p and (C)r may be copolymers formed
by the polymerization of different monomers.
[0095] The optimal length of (A)p and (C)r chains may be
determined. The polymer was added to PBS in the presence of the
water-insoluble dye diphenylhexatriene, which would dissolve in the
hydrophobic core of polymeric micelles or aggregates. After
sonication and centrifugation, we observed an abrupt enhancement in
the ultraviolet (356 nm) absorption of the dye, which indicated
micelle formation.
[0096] The polymeric emulsifiers of the invention exhibit
distinguishing features. They are biodegradable and bioresorbable.
Degradation studies on these copolymers have shown that the
polyester chains (A)p and (C)r are gradually degraded by
hydrolysis. The final products are corresponding hydroxyacids (or
diacids and diols), which are bioresorbable. Continuing hydrolysis
will eventually release the hydrophilic polyester chain (B)q, the
central block of the copolymer. Such polymers of relatively low
molecular mass (less than 10,000) are bioresorbable and may be
excreted from the kidney.
[0097] The preparation factors a two-step preparation process and
an emulsifier with djuvant (an O/W submicron emulsion), which
HLB.sub. =15) and 0.5% Span.RTM. 85 (HLB.sub.Span.RTM.85=1.8) as
single-step manufacture process through a microfluidizer at an
internal followed by filtration through a 0.22-.mu.m filter
membrane to achieve O/W submicron emulsion particles. The GSK ASO3
adjuvant, which contains is prepared via a two-step manufacture
process Tween.RTM.80, and alpha-tocopherol in a fluid O/W emulsion.
It however lacks an HLB value.
[0098] In another aspect, the invention relates to a process for
making a multi-phase W/O/W emulsion, which may trap and/or
encapsulate antigens and/or bioactive substances in the multi-phase
emulsion. The process comprises a homogenizing (or called
emulsifying) step and a diluting (or called dispersing) step. In
the homogeniziml (or emulsifying) step, a pre-emulsified stock
comprising an oil and emulsifiers is obtained, in which a designed
block copolymer serves as a hydrophilic emulsifier to stabilize an
oil-water interface, and a lipophilic emulsifier serves to
stabilize a water-oil interface and result in a stable and
isotropic W/O/W emulsion. The W/O/W multi-phase emulsion is
achieved with an emulsifying system containing the designed block
copolymer with an intermediate HLB value. The oil droplets are
dispersed in a continuous aqueous phase, in which the oil-water
interface is stabilized by the block copolymer (HLB>>10). In
addition, the core oil entraps an aqueous phase, in which the
entrapped water-oil interface is stabilized by a lipophilic
emulsifier (HLB<<10).
[0099] In the diluting (or dispersing) step, the above
pre-emulsified stock was diluted by redispersing it into an aqueous
solution. The aqueous solution may be an aqueous medium alone, such
as PBS, or an aqueous medium containing an antigen or a bioactive
substance such as peptides, anticancer agents, hormones, or other
active agents such as antibiotics or antiparasitics. The antigen or
the bioactive substance may help disolving in either the oily or
the aqueous phase. A either in the internal and/or external aqueous
phase of the release formulation, dissolving and encapsulating an
has the effect of protecting the antigen. Conversely has the effect
of facilitating the
Examples
[0100] Without intent to limit the scope of the invention,
exemplary instruments, apparatus, methods and their related results
according to the embodiments of the present invention are given
below. Note that titles or subtitles may be used in the examples
for convenience of a reader, which in no way should limit the scope
of the invention. Moreover, certain theories are proposed and
disclosed herein; however, in no way they, whether they are right
or wrong,.should limit the scope of the invention so long as the
invention is practiced according to the invention without regard
for any particular theory or scheme of action.
Example 1
[0101] This example illustrates the incorporation of hydrophilic
polymeric emulsifiers, namely, poly(ethylene
glycol)-block-polylactide (PEG-b-PLA), poly(ethylene
glycol)-block-poly(.epsilon.-caprolactone) (PEG-b-PCL), and
poly(ethylene glycol)-block-poly(lactide-co-.epsilon.-caprolactone)
(PEG-b-PLACL) in the antigen medium to alter the water affinity of
oily ISA51-adjuvanted vaccines. These amphiphilic block copolymers
were selected because of their biocompatibility and
bioresorbability. Various physiochemical properties of emulsions
have been characterized, namely, stability, the droplet test,
particle size distribution and in vitro release of a model protein
ovalbumin (OVA). Finally, a preliminary immunogenicity evaluation
of OVA after formulation with the PEG-b-PLACL-stabilized ISA51
adjuvant was determined in mice for induction of antibody responses
in comparison with non-formulated OVA and conventional ISA51 oily
adjuvant-formulated OVA (Huang et al. (2009) "Development of
Multi-Phase Emulsions Based on Bioresorbable Polymers and Oily
Adjuvant" Pharmaceutical Research 26(8): 1856-1862. which is herein
incorporated by reference in its entirety).
Materials and Methods
Polymer Synthesis and Characterization
[0102] Tin(II) 2-ethylhexanoate (stannous octoate, SnOct.sub.2) was
purchased from Sigma (St. Louis, Mo., USA). DL-lactide (a cyclic
di-ester of lactic acid) was purchased from Aldrich (Seelze,
Germany) and recrystallized from ethyl acetate.
.epsilon.-Caprolactone was purchased from Aldrich. Poly(ethylene
glycol) 5,000 monomethyl ether (MePEG.sub.5000) was purchased from
Fluka (Buchs, Switzerland). All solvents were of analytical
grade.
[0103] PEG-b-PLACL was synthesized by ring-opening polymerization
of lactide and .epsilon.-caprolactone, using SnOct.sub.2 as a
catalyst and MePEG.sub.5000 as an initiator. Briefly, a
predetermined amount of MePEG.sub.5000 (2.1 g), lactide (0.58 g),
and .epsilon.-caprolactone (0.47 g) were placed in a dried
round-bottomed bottle, and an appropriate amount of SnOct.sub.2 (30
mg) was added as a solution in dried toluene (10 mL).
Polymerization was performed at 140.degree. C. under reflux for 24
hr. The product was recovered by precipitation in an excessive
amount of ethanol. PEG-b-PLA or PEG-b-PCL was synthesized in the
same manner with MePEG/lactide or MePEG/.epsilon.-caprolactone at a
weight ratio of 2/1. The resulting polymers were characterized by
.sup.1H nuclear magnetic resonance (.sup.1H NMR) and gel permeation
chromatography (GPC). .sup.1H NMR spectra were recorded at room
temperature with a Varian VXR 300 MHz spectrometer (Varian, Palo
Alto, Calif., USA) using deuterated chloroform as the solvent. GPC
was performed by using a setting composed of a Waters 510 HPLC
pump, a Waters 410 differential refractometer, one PLgel mixed-C 5
.mu.m 100 .ANG. column (7.5.times.300 mm), and one PLgel 3 .mu.m
100 .ANG. column (7.5.times.300 mm), and the mobile phase being
tetrahydrofuran (THF) and the flow rate 0.8 mL/min. Data were
expressed with respect to polystyrene standards from
Polysciences.
Polymer-Stabilized Emulsions
[0104] The antigen medium was prepared with a particular
concentration of ovalbumin (OVA, Grade V, Sigma, St. Louis, Mo.,
USA) diluted in a phosphate-buffered saline (PBS). An aqueous
solution containing 120 mg of polymer and 0.8 mL of antigen medium
and an oil solution containing 1.1 mL of ISA51 (Montanide.RTM. ISA
51 F VG, SEPPIC, Paris, France) were emulsified using a
Polytron.RTM. PT 3100 homogeniser (Kinematica AG, Swiss) under
6,000 rpm for 5 min. A polymer-free PBS/ISA51 emulsion composed of
0.9 mL of antigen medium and 1.1 mL of ISA51 was also prepared at
8,000 rpm for 10 min. These emulsified formulations served as
stocks for further physicochemical characterizations namely
stability, the droplet test, particle size distribution and in
vitro release.
[0105] The stability test was performed by placing each sample at
4.degree. C. and 37.degree. C., and then noted the visual aspect at
a predetermined time. The droplet test was assessed by placing a
droplet (20 .mu.L) of an emulsion into the water in a beaker (200
mL). The particle size distribution was determined by using the
laser light scattering technique with a Brookhaven 90 plus particle
sizer (Brookhaven Instruments Limited, New York, USA). In vitro
release experiments were performed by using the inverted dialysis
tube method. OVA-containing formulations (3 mg per 0.3 mL) were
first placed in a dialysis chamber (cutoff 0.2 .mu.m) and then the
device was immersed in a 50 mL centrifuge tube containing 2 mL of
PBS at 37.degree. C. At different time intervals, 100 .mu.L of
sample were aspirated from the medium outside of the chamber and
replaced with 100 .mu.L of PBS buffer. The OVA release was
regularly determined by the bicinchinonic acid method (BCA.TM.
protein assay kit, Pierce, Rockford, Ill., USA).
Immunization and ELISA Immunoassay
[0106] Five-week old female BA Laic. mice were obtained from the
National Laboratory Animal Breeding and Research Center (Taipei,
Taiwan) and acclimatized for at least one week at the animal
facility of National Health Research Institutes (NHRI, Miaoli,
Taiwan) prior to use. All animal studies were approved by the
Animal Committee of NHRI. Mice were primed subcutaneously (s.c.;
100 .mu.L) with a syringe needle of 27G.times.1/2'' and 0.5 .mu.g
of OVA in PBS or formulated with PEG-b-PLACL/ISA51 or PBS/ISA51,
and boosted with the same formulation at week 2. To increase the
fluidity, the group of PEG-b-PLACL/ISA51 was investigated by
re-dispersing 100 .mu.L of stock emulsion (See "MATERIALS AND
METHODS" SECTION: Polymer-stabilized emulsions) into 900 .mu.L of
PBS before injection, resulting in a PEG-b-PLACL/ISA51/PBS emulsion
of only 5% oil solution.
[0107] To determine the antibody response, mice were bled at the
lateral tail vein and the collected sera were stored at -30.degree.
C. The presence of OVA-specific antibodies in the sera was
determined by enzyme-linked immunosorbent assay (ELISA). Briefly,
100 .mu.L of diluted OVA (10 .mu.g/mL) were coated onto 96-well
microtiter plates with 0.05 M carbonate buffer (pH 9.6). After the
overnight incubation at 4.degree. C., coated plates were washed
twice with PBS containing 0.05% Tween.RTM. 20 (Sigma, St. Louis,
USA) and then blocked with 5% non-fat milk in PBS at room
temperature for 2 hr. Diluted sera (starting dilution 1:50, serial
three-fold serum dilutions) from immunized animals were applied to
wells at room temperature for 2 hr. Following the addition of
HRP-conjugated goat anti-mouse IgG (ICN Cappel, Aurora, Ohio, USA),
the assay was developed with a substrate solution containing
tetramethylbenzidine (TMB, Sure Blue.TM., KPL, MD, USA), and the
reaction was stopped in 2 N H.sub.2SO.sub.4. Plates were read at
450 nm using an ELISA plate reader (Molecular Devices, Sunnyvale,
Calif., USA). The titers were determined based on the reciprocal of
the final dilution that gave 2-fold greater absorbance than the
pre-immune sera. For isotype determination, 100 .mu.L of an
appropriate dilution (1:2,000) of HRP-rabbit anti-mouse IgG1
(Zymed.RTM., Calif., USA) or HRP-rabbit anti-mouse IgG2a
(Zyme.RTM., Calif., USA) was added. Statistical significance
(p<0.005) was determined by performing two-tailed Student's
t-test on log-transformed values.
Results and Discussion
Polymer Design and Characterization
[0108] AB-type diblock copolymers consisting of a polyether block
(PEG) and a polyester block (PLA, PCL or PLACL) were synthesized by
ring-opening polymerization of lactide and/or
.epsilon.-caprolactone in the presence of MePEG, using SnOct.sub.2
as a catalyst. The molecular characteristics of the three
copolymers are summarized in Table 1. In this study, MePEG with a
molecular weight of 5,000 and an initial hydrophilic/lipophilic
ratio of 2/1 were selected as a compromise between the high
hydrophilicity of polymers and the bioresorbability of PEG-rich
degradation products. In fact, PEG is a water-soluble polymer,
particularly, low molecular weight PEG (<10,000 Daltons) can be
excreted through kidney filtration. The lipophilic block was
derived from the U.S. Food and Drug Administration (FDA)-approved
aliphatic polyesters, PLA and PCL. They show bulk degradation and
further resorb in vivo. PLA with a variable chain stereoregularity
provides a worthwhile means to adjust the rate of degradation. On
the other hand, the degradation products of PCL had a relatively
higher pKa than those of poly(lactide-co-glycolide) (4.8 for
.epsilon.-hydroxycaproic acid, and 3.8 for lactic acid and glycolic
acid at 25.degree. C.), and they may provide more conservation of
protein molecular integrity when being used for a long-term
controlled delivery of proteins. PLACL was chosen as the lipophilic
block for its fast degradation characteristics. In addition, its
amorphous nature provides a good affinity between the polymer
matrix and oil solutions. On the selection of catalyst, SnOct.sub.2
has been approved by the U.S. FDA for biomedical and therapeutic
applications.
TABLE-US-00003 TABLE 1 Product .sup.1H NMR GPC.sup.e) Feed
[LA]/[CL]/ Mw / Yield Size.sup.f) Polymer [LA]/[CL]/[OE]
[OE].sup.a) Mn.sup.b) W.sub.PFG:W.sub.PLA CL.sup.e) HLB.sup.d) Mn
Mn % nm MePEG.sub.5000 --/--/1 --/--/1 5,000 100:0 20.0 4,350 1.1
-- n.d. PEG-b- 0.306/--/1 0.243/--/1 7,000 72:28 14.4 9,250 1.2 75
360 .+-. 40 PLA PEG-b- --/0.193/1 --/0.166/1 7,150 70:30 14.0 8,900
1.3 85 470 .+-. 80 PCL PEG-b- 0.169/0.086/1 0.100/0.068/1 6,700
75:25 15.0 10,000 1.2 80 370 .+-. 20 PLACL n.d. not detected
.sup.aThe [LA]/[CL]/[OE] molar ratio was determined from the
integrations of the signals due to PLA blocks at 5.1 ppm, to PCL
blocks at 4.0 ppm, and to PEG blocks at 3.6 ppm on the 1H NMR
spectra .sup.b Mn = Mn.sub.PEG + Mn.sub.PLA CL = 5000 + 72 .times.
5000/44 .times. [LA]/[OE] + 114 .times. 5000/44 .times. [CL]/[OE]
.sup.cW.sub.PEG:W.sub.PLA/CL = Mn.sub.PEG: Mn.sub.PLA/CL
.sup.dHLB.sub.copolymer = 20 ( Mn.sub.PEG/ Mn.sub.copolymer)
.sup.eData obtained by GPC with respect to polystyrene standards
from Polysciences. .sup.fPolymer-stabilized ISA51 emulsion
particles were determined by the particle size analyzer. Each value
represents the mean of three experiments (mean .+-. s.d.)
[0109] The molar ratio of lactyl units to caproyl units to
oxyethylene or [LA]:[CL]:[OE] was determined from the integrations
of the proton resonances due to PLA blocks at 5.2 ppm, to PCL
blocks at 4.1 ppm, and to PEG blocks at 3.6 ppm on the .sup.1H NMR
spectra. The number average molecular weight ( Mn) were calculated
according to the following equation:
Mn= Mn.sub.PEG+
Mn.sub.PLA/CL=5000+72.times.5000/44.times.[LA]/[OE]+114.times.5000/44.tim-
es.[CL]/[OE]
where 44, 72 and 114 are the molecular weights of OE, LA and CL
repeat units, respectively. The HLB (hydrophilic-lipophilic
balance) values of non-ionic copolymers PEG-b-PLA, PEG-b-PCL, and
PEG-b-PLACL were expressed according to Griffin's method as
follows:
HLB copolymer = 20 ( W PEG W copolymer ) ##EQU00001##
where W.sub.PEG/W.sub.copolymer is the weight ratio of the
hydrophilic portion of the main chain polymer and is obtained from
Mn.sub.PEG/ Mn.sub.copolymer. The most lipophilic polymer has an
HLB number approaching 0, and the most hydrophilic polymer has a
HLB of about 20. According to the equation, a high HLB value
(HLB.sub.PEG-b-PLA of 14.4, HLB.sub.PEG-b-PCL of 14.0, and
HLB.sub.PEG-b-PLACL of 15.0) was obtained, which indicated that the
three copolymers had a high affinity to water.
[0110] The GPC traces of PEG-b-PLA, PEG-b-PCL, and PEG-b-PLACL
exhibited monomodal and reflected rather narrow molecular weight
distributions (Table 1), which indicated the absence of residual
low molecular weight species. The !Mn values calculated from GPC
were higher than those from .sup.1H-NMR. This finding could be
attributed to changes in the hydrodynamic volume of the hydrophilic
PEG and/or the lipophilic PLA, PCL, PLACL blocks as compared to the
polystyrene standards.
Preparation of Polymer-Stabilized Emulsions
[0111] With the aim of enhancing the potency of emulsion-adjuvanted
vaccines, we used hydrophilic PEG-b-PLA, PEG-b-PCL, and PEG-b-PLACL
as emulsifiers to stabilize the interface between the ISA51 oily
adjuvant and the antigen media. An aqueous phase of polymer
dissolved in antigen media and an oily phase of ISA51 were
emulsified using a homogenizer. The emulsifying formulation was
perfectly white and isotropic from the top to the bottom.
[0112] To mimic the usual storage conditions and the post-injection
stage, the stability test was investigated at 4.degree. C. and at
37.degree. C. During the storage at 4.degree. C., all emulsions
were stable for a few weeks without phase separation. In the case
of PEG-b-PLA/ISA5 I and PEG-b-PCL/ISA5I, 10% of water disassociated
after two weeks, but beyond this, no further water disassociation
from the emulsion occurred. An isotropic emulsion could be
re-formed by vortex mixing. On the other hand, approximately 10% of
free oil at the surface layer disassociated from the PBS/ISA51
emulsion after one month under the same storage conditions. The
PEG-b-PLACL/ISA51 emulsion was stable for at least six months
without phase separation. During 60 days' monitoring at 37.degree.
C., the PEG-b-PCL/ISA5I and PEG-b-PLACL/ISA51 emulsions were stable
without phase separation. On the other hand, approximately 10% of
free oil at the surface layer disassociated from the PBS/ISA51
emulsion after 3 days (FIG. 1a). After one week, 30% of free oil at
the surface and clear layers of water (30%) at the bottom
disassociated from the emulsion. Phase separation happened at day
60, indicating the emulsion breaks. In the case of PEG-b-PLA/ISA51,
the change in the visual aspect over time was similar to the
PBS/ISA51.
[0113] The water affinity of the emulsions was investigated by the
droplet test and laser light scattering. As shown in FIG. 2a, an
emulsified PBS/ISA51 droplet (arrow in FIG. 2a inset) kept on
floating on the water surface after 24 hr. The particle size was
not detected by using light scattering technology. However,
homogeneous particles with the size distribution of 1 .mu.m were
observed by optical microscope when re-dispersing the emulsion in
the ISA51 oil solution (data not shown). On the other hand, each
polymer-stabilized ISA51 droplet (arrow in FIG. 2b inset) could
stand only for seconds in the aqueous phase and then diffuses in
the water, which indicated its high affinity for water (FIG. 2b).
The dynamic light scattering pattern showed that PEG-b-PLA,
PEG-b-PCL or PEG-b-PLACL was a suitable emulsifier for the
ISA51/water interface, and yielded narrowly distributed
nanoparticles (FIG. 2d and Table 1). Typically, a bimodal
distribution with two different sizes was observed, the relatively
large particles of 500 nm and smaller ones of 100 nm (FIG. 2d).
This dimension is appropriate for uptake by antigen-presenting
cells (APCs) to facilitate the induction of potent immune responses
due to the pseudo-natural targeting of antigens. Homogenization
using MePEG failed to improve the water affinity of ISA51.
emulsion. The droplet floated on the surface instead o f diffusion
in the water, which indicated that only PEG bearing short
lipophilic units in the main chain polymer exhibited emulsifier
property (FIG. 2c).
[0114] In addition to the water affinity, PEG-b-PLA-, PEG-b-PCL-,
or PEG-b-PLACL-stabilized ISA51 emulsion also provided different
controlled-release profiles to hydrophilic OVA protein with respect
to free OVA or PBS/ISA51 -formulated OVA, as shown in FIG. 1b.
Initially, a fast release was observed in the case of OVA without
formulation from which more than 50% of loaded OVA was released
into the outside PBS medium within the first 30 hr. The
PEG-b-PLA-modified ISA51 emulsion has similar release profiles to
free OVA, in which less than 30% of OVA were released during the
same period of time. Furthermore, the protein release increased
continuously until it reached the equilibrium concentration in the
inside and outside of the dialysis device. Conversely, the oily
PBS/ISA51 emulsion presented well depot effect to OVA so that
hydrophilic OVA was slowly released over 500 hr. Following the
intermediate controlled-release mechanisms, the hydrophilic
bioactive agents (or antigens) trapped within the PEG-b-PCL- or
PEG-b-PLACL-stabilized emulsion will be released mostly by
diffusion from the core oil to the surface, but also to a lesser
extent by degradation mechanisms and emulsion breaks.
[0115] As shown in FIG. 2a, an ISA51 oily adjuvant contains only
emulsifier of low HLB value (2.6 to mannide monooleate 202, in
which the hydrophobic end 204 facing toward the oil phase, and the
hydrophilic end 206 facing toward the aqueous phase). The water
affinity test and in vitro release showed that the resulting
PBS/ISA51 emulsion has a continuous phase of oil and the dispersed
phase being water (FIG. 2a). On the other hand, a
polymer-stabilized ISA51 system composes of two surfactants,
hydrophilic polymer 208 and mannide monooleate 202, rendered a
water-in-oil-in-water (W/O/W) multi-phase emulsion (FIG. 2b). In
this case, oil droplets dispersed into the continuous water
(stabilized by the polymeric emulsifier 208), and the core oil also
entrapped an aqueous phase (stabilized by mannide monooleate 202;
FIG. 2b). The polymer-emulsified particles could serve as either
carriers or vehicles to deliver antigens to APCs in a targeted and
prolonged manner.
[0116] From a viewpoint of emulsion stability, the vaccine
emulsions met the requirements for in vitro storage and the
post-injection depot. It is generally recognized that oil droplets
with small particle size and homogeneous distribution are more
stable. These parameters are however strongly influenced by the
optimization of the emulsification process and the surfactant
system. To this end, addition of excipients like glycine or
glycylglycine in Montanide.RTM. ISA 720, an oily adjuvant
containing squalene and mannide monoolete, provided a potential way
of stabilizing the emulsions during storage and post-injection. On
the other hand, two O/W emulsion-type adjuvants that possess
significant potential for the development of human vaccines are
MF59.RTM. (developed from Novartis) and AS03 (developed from
GlaxoSmithKline). MF59.RTM. is accomplished by using a combination
of a hydrophilic Tween.RTM.80 emulsifier and a lipophilic
Span.RTM.85 (sorbitan trioleate) while AS03 is stabilized by
Tween.RTM.80 and alpha-tocopherol. In the present study.
PEG-b-PLA-stabilized ISA51 emulsion remained the same or reduced
the stability intrinsic to ISA51 oily adjuvant. However,
homogenization of PEG-b-PCL- or PEG-b-PLACL- containing aqueous
solution and the mannide monooleate-contained oily phase (e.g.
ISA51) provides a potential way of stabilizing the emulsified
particles both at storage and at post-injection stage
conditions.
Immunological Evaluation in Mice
[0117] To further evaluate the potential applications of
polymer-stabilized emulsions in vaccine adjuvants, the antibody
assays were performed by subcutaneous vaccination in BALB/c mice
with OVA, alone or formulated with PBS/ISA5I or
PEG-b-PLACL/ISA51/PBS. The latter contained only 5% of ISA51 oily
adjuvant in the formulation (See "MATERIALS AND METHODS" section:
Immunization and ELISA immunoassay).
[0118] As shown in FIG. 3, the serum antibody IgG, IgG1 and IgG2a
titers were significantly enhanced for the groups of PBS/ISA51 -
and PEG-b-PLACL/ISA51/PBS- formulated OVA in comparison with the
group of OVA alone (p<0.005). Moreover,
PEG-b-PLACIASA51/PBS-formulated OVA induced the same level of
antibody titers as those induced by PBS/ISA51-formulated OVA. Thus,
the PEG-b-PLACL/ISA51/PBS reserved the adjuvant effects of
PBS/ISA51. We have attempted to study the effect of polymeric
aqueous solutions on the enhancement of the OVA immunity. Our
findings indicated that polymeric aqueous solutions had no adjuvant
effect, the induced antigen (OVA)-specific antibodies were still at
the same level as those without formulation (data not shown). We
also observed that the PEG-b-PLACL/ISA51/PBS emulsion could be
absorbed after 5 weeks of s.c. inoculation, while PBS/ISA5I
emulsion still remained at the injection site. Furthermore, there
was no adverse side effect in the animal.
[0119] After the injection, vaccine antigens may be directly taken
up by APCs, bind to the surface antibody on B cells, or undergo
degradation. Only those antigens that are taken by APCs can
integrate into the immune responses. The pathway is largely
dependent on the characteristics of the antigen, but may also be
influenced by the presence of adjuvants. Although emulsion-type
adjuvants have been widely used for several decades, their
immunogenicity-enhancing effects are still controversial due to the
lack of understanding about the complexity of colloidal
dispersions, the emulsion stability of post-injection and the
mechanism of the immune response. In the present study, the
adjuvant effect of the W/O emulsion (PBS/ISA51 as an example) could
be explained by the depot of emulsion that is capable of slowly
releasing antigen over a long period of time. In the case of the
W/O/W emulsion (PEG-b-PLACL/ISA51/PBS emulsion as an example), even
with only 5% of ISA51 oily adjuvant, it induced significantly
higher responses than non-formulated OVA. It is probable that W/O/W
not only reserved the depot effects intrinsic to ISA5 I oil, but
also combined the antigen presentation effects. Moreover, the
ameliorated W/O/W emulsion increases injectability and conceptually
diminishes local reactions with respect to the W/O type vaccines
produced from the same oil.
[0120] Amphiphilic copolymers consisting of 70 wt-% hydrophilic PEG
block and 30 wt-% lipophilic PLA, PCL or PLACL block were
synthesized by the ring-opening polymerization of lactide and/or
.epsilon.-caprolactone on monomethoxy PEG.sub.5000. The resulting
polymers can serve as a hydrophilic emulsifier to alter the water
affinity of oily ISA51-adjuvanted vaccines so that the stock
antigen-encapsulated emulsion could be re-dispersed into PBS before
injection and thus resulting in stable and injectable W/O/W
emulsion nanoparticles. Preliminary immunological evaluation showed
that only 5% of oil within the PEG-b-PLACL/ISA51/PBS formulation
reserves the adjuvant effects of the ISA51 oily adjuvant. These
features are of great interest for further investigations of
single-dose prophylactic and therapeutic vaccine development and
via alternative immunization routes, such as intramuscular,
transdermal or mucosal administration.
Example 2
[0121] The example here illustrates the synthesis and
characterization of PEG-bearing PLA di- and tri-block copolymers,
PLA-PEG and PLA-PEG-PLA, prepared by the direct polycondensation of
an aqueous lactic acid solution on monomethoxy or dihydroxyl PEG.
Unlike the block copolymers reported in the literature, which were
prepared in the presence of cytotoxic catalyst-containing heavy
metals, in this study, no catalyst was added during polymerization,
which increased the confident biocompatibility in the final
material. The resulting copolymers were characterized by MALDI-TOF
MS, GPC, and .sup.1H-NMR. In contrast to micelles or nanospheres in
which the polymer provides a matrix or a vehicle to encapsulate the
bioactive agents, here we wanted to demonstrate whether the
amphiphilic polymer could play an auxiliary role (as a surfactant)
to stabilize the oil/water interface so that the bioactive
candidates could be either surface attached or encapsulated within
the core oil. The emulsifying properties were investigated by the
homogenization of a polymer aqueous solution and oily squalene.
Stability, size distribution, and in vitro release with ovalbumin
(OVA) as model protein were performed to identify the resulting
emulsion. For the sake of biocompatibility, squalene was selected
as the core oil because it has a low toxicity and is used in the
clinical trial (Siao et al., (2009) "Characterization and
Emulsifying Properties of Block Copolymers Prepared from Lactic
Acid and Poly(ethylene glycol)" Journal of Applied Polymer Science
114: 509-516, which is incorporated by reference in its
entirety).
Polymer Synthesis
[0122] Lactic acid was purchased as a 85-90% aqueous solution from
TEDIA (Fairfield, Ohio). Poly(ethylene glycol) 2000 monomethyl
ether (MePEG.sub.2000) and polyethylene glycol) 2000
(diOH-PEG.sub.2000) were purchased from Fluka (Buchs, Switzerland).
These materials were used without further purification. All
solvents were analytical grade.
[0123] The PLA-PEG diblock copolymer was synthesized through the
polycondensation of lactic acid on MePEG2000, in the absence of any
catalyst. Briefly, 10 g of MePEG.sub.2000 and 10 g of an aqueous
lactic acid solution were placed in a round-bottomed bottle. We
performed the polymerization by distilling out water from lactic
acid at 140.degree. C. for 24 hr using a system composed of a
Rotavapor.RTM. R-210 (Buchi Labortechnik AG, Switzerland) and
vacuum pump V-700 (Buchi Labortechnik AG). The products were
recovered by precipitation in an excessive amount of ethanol. They
were further purified twice by successive dissolution/precipitation
cycles with acetone as a solvent and ethanol as a nonsolvent to
eliminate low-molecular-weight byproducts. The yield was about 25
wt-%. The PLA-PEG-PLA triblock copolymer was synthesized according
to the same procedure with diOH-PEG.sub.2000 used instead of
MePEG.sub.2000. The product was recovered by precipitation in cold
ethanol (<10.degree. C.), and the yield was about 30 wt %.
Measurements
[0124] MALDI-TOF MS was recorded on a Waters.RTM. MALDI micro
MX.TM. mass spectrometer (Milford, Mass.) equipped with a nitrogen
laser (337 nm). All spectra were recorded in the reflection mode
with an acceleration voltage of 12 kV. The irradiation targets were
prepared from 0.1% trifluoroacetic acid (Riedel-de Haen, Seelze,
Germany) in an acetonitrile/water mixture at a ratio of 50/50 (v/v)
with .alpha.-cyano-4-hydroxy cinnamic acid (Sigma, Steinheim,
Germany) as the matrix and sodium trifluoroacetate (Na-TFA, Fluka)
as the dopant. The sample solutions were then spotted on a MALDI
sample plate and air-dried before analysis. GPC was performed with
a setup composed of an isocratic pump (Waters.RTM. high-performance
liquid chromatography (HPLC) Model 510), a refractive index
detector (Waters.RTM.410 differential refractometer), and two
columns connected in series, one PLgel 5-.mu.m mixed-C column
(100-.ANG. pore size, 7.5.times.300 mm, Polymer Laboratories, Ltd.,
Shropshire, United Kingdom), and one PLgel 3-.mu.m column
(100-.ANG. pore size, 7.5.times.300 mm). The mobile phase was
tetrahydrofuran and the flow rate was 0.8 mL/min, Data were
expressed with respect to polystyrene standards (Polysciences,
Inc., Warrington, Pa.). .sup.1H-NMR spectra were recorded at room
temperature with a Varian VXR 300-MHz spectrometer (Varian, Palo
Alto) with dimethyl sulfoxide-4 (Aldrich, Steinheim, Germany) and
tetramethylsilane as the solvent and shift reference,
respectively.
Polymer-Stabilized Emulsions
[0125] The polymer aqueous solution [120 mg of polymer dissolved in
0.8 mL of phosphate-buffered saline (PBS)] and 1.1 mL of squalene
oil (Sigma, Steinheim, Germany) were emulsified with a
Polytron.RTM. PT 3100 homogeniser (Kinematica AG, Lucerne,
Switzerland) under 6000 rpm for 5 min. The emulsified formulations
served as stocks for further physicochemical characterizations.
[0126] To mimic the usual storage conditions and the
post-administration stage, the stability test was performed by
placing each formulation at 4 and 37.degree. C. and observed the
visual aspects. To investigate the size distribution of the
emulsions, the stock emulsion was redispersed in the PBS before the
measurement by using the laser light-scattering technique with a
Brookhaven 90 plus particle size analyzer (Brookhaven Instruments
Limited, New York). In vitro release experiments were performed
with the inverted dialysis tube method. Formulations containing OVA
(albumin from chicken egg white, Grade V, Sigma; 3 mg/0.3 mL) were
placed in a dialysis chamber (cutoff=0.2 .mu.m, Pall Life Sciences,
Ann Arbor, Mich.). The device was immersed in a 50-mL centrifuge
tube containing 2 mL of PBS and left to stand at 37.degree. C. At
different time intervals, 100 .mu.L of sample were aspirated from
the medium outside of the chamber and replaced with 100 .mu.L of
fresh PBS buffer. The OVA release was regularly determined by the
bicinchinonic acid method (BCA.TM. protein assay kit, Pierce,
Rockford, Ill.).
Results and Discussion
[0127] FIG. 4 shows the synthesis and the chemical structure of the
block copolymers. The PLA-PEG diblock copolymer was synthesized by
the polycondensation of lactic acid in the presence of monomethoxy
PEG, which resulted in a copolymer composed of a hydrophilic block
PEG and a lipophilic block PLA. Similarly, the triblock copolymer
PLA-PEG-PLA was obtained from the polymerization of lactic acid in
the presence of dihydroxyl PEG. In general, PLA compounds are
synthesized by the ring-opening polymerization of lactide (a cyclic
diester of lactic acid) or the polycondensation of lactic acid.
Although the latter is a reasonably low-cost and straightforward
method for synthesizing polymers bearing PLA segments this route
generally leads to oligomers with low-molar-mass chains. The
molecular characteristics of the resulting copolymers are
summarized in Table 2.
Characterization of the PLA/PEG Block Copolymers by MALIDI-TOF
MS
[0128] Mass spectrometry is used to measure the real MW of
synthetic polymers. With this technique, the molecular structure
and chemical composition of copolymers can be accurately studied.
FIGS. 5a-5b show the MALDI-TOF MS spectra of PLA-PEG and the
corresponding MePEG.sub.2000. The MePEG.sub.2000 spectrum was well
resolved [FIG. 5(a)], and the peaks were separated by 44 mass
units, which corresponded to the MW of the PEG monomer (oxyethylene
(OE) units=44.03 g/mol). The subsidiary peaks were assigned to the
isotopes of elements. The MW of MePEG.sub.2000 ranged from 1200 to
2800 g/mol with Mn=1970 and Mw/Mn=1.05. After the condensation
(140.degree. C., 24 h, no catalyst) of the lactic acid aqueous
solution in the presence of MePEG.sub.2000, the MW distribution of
the resulting polymer shifted to 1600-3200 g/mol with Mn of 2370
and Mw/Mn=1.03 [FIG. 5b], which indicated the chain extension of
the lactyl (LA) monomer onto the macroinitiator MePEG. No signals
characteristic of MePEG were detected on the MALDI-TOF MS spectra
of PLA-PEG, which indicated that PLA-free MePEG.sub.2000 species
were removed during purification. On the spectra of MePEG2000 and
PLA-PEG, the number of oxyethylene units (OE) and lactyl (LA) units
could be uniquely determined (x and y, respectively) from the MW of
the major peaks. Each major peak in the mass spectrum corresponded
to a polymer species (molecular structure proposed in FIG. 4) that
has OE units and LA units (MW=72.06) in addition to the end groups
(one methyl and one hydroxyl, MW=32.03) and a Na.sup.+ ion
(MW=22.99, due to Na-TFA).
MW.sub.MePEG=x(44.03)+32.03+22.99
MW.sub.PLA-PEG=x(44.03)+y(72.06)+32.03+22.99
[0129] For example, the major five polymer species between 2030 and
2100 m/z in the spectra of PLA-PEG (FIG. 5b) are represented as
follows:
MW.sub.PLA-PEG=2032, x=40, y=3
MW.sub.PLA-PEG=2048, x=42, y=2
MW.sub.PLA-PEG=2064, x=44, y=1
MW.sub.PLA-PEG=2076, x=41, y=3
MW.sub.PLA-PEG=2092, x=43, y=2
[0130] To substantiate the speculation of correlation between the
MW and molecular architecture of copolymers, the same method can be
employed for the analysis of the triblock copolymer PLA-PEG-PLA.
Each major peak in the mass spectrum, as shown in FIG. 6,
corresponded to a polymer species that had OE units, LA units, the
end groups of one hydrogen and one hydroxyl, and Na.sup.+ ion:
MW.sub.diOH-PEG2000=x(44.03)+18.02+22.99
MW.sub.PLA-PEG-PLA=x(44.03)+y(72.06)+18.02+22.99
[0131] For example, the major five polymer species between 2010 and
2080 m/z in the spectra of PLA-PEG-PLA (FIG. 6b) were represented
as follows:
MW.sub.PLA-PEG-PLA=2018, x=40, y=3
MW.sub.PLA-PEG-PLA=2035, x=42, y=2
MW.sub.PLA-PEG-PLA=2050, x=44, y=1 or x=26, y=12
MW.sub.PLA-PEG-PLA=2062, x=41, y=3
MW.sub.PLA-PEG-PLA=2078, x=43, y=2 or x=25, y=13
[0132] After calculating the repeat unit masses and the end group
masses through the MALDI spectra, we could distinguish the
molecular structure between the diblock and triblock copolymers.
The hydrophilic-lipophilic balance (HLB) value of the nonionic
PLA/PEG diblock or triblock copolymer was expressed according to
Griffin's method as follows:
[0133] HLB.sub.PLA/PEG=20 x(W.sub.PEG/W.sub.PLA-PEG)
where W.sub.PEG/W.sub.PLA/PEG is the weight ratio of the
hydrophilic portion of the main-chain polymer and was obtained from
Mn.sub.PEG/Mn.sub.PLA-PEG. The most lipophilic portion has an HLB
number approaching 0, and the most hydrophilic portion has a number
of about 20. According to this equation; high HLB values
(HLB.sub.PLA-PEG=16.6 and HLB.sub.PLA-PEG-PLA=16.4) were obtained,
which indicated that the two copolymers had high affinities for
water. There was however no significant difference between the
copolymers initiated by MePEG.sub.2000 and the copolymers initiated
by diOH-PEG.sub.2000.
TABLE-US-00004 TABLE 2 MALDI- TOF MS.sup.a GPC.sup.b .sup.1H
NMR.sup.c Polymer Mn Mw/Mn Mn Mw/Mn Mn MePEG.sub.2000 1970 1.05
2650 1.10 2000 PLA-PEG 2370 1.03 3360 1.08 2150 diOH-PEG.sub.2000
1840 1.04 2700 1.08 2000 PLA-PEG-PLA 2240 1.04 3520 1.07 2200
.sup.aData obtained by MALDI-TOF MS with .alpha.-cyano-4-hydroxy
cinnamic acid as the matrix and Na-TFA as a dopant. .sup.bData
obtained by GPC with respect to polystyrene standards from
Polysciences. .sup.cMn = Mn.sub.PEG + Mn.sub.PLA = 2000 + 72
.times. 2000/44 .times. ([LA]/[OE]), where [LA]/[OE] was determined
from the integrations of the signals due to the PEG blocks at 3.6
ppm and to the PLA blocks at 1.5 ppm on the .sup.1H NMR
spectra.
MW Determined by GPC and .sup.1H-NMR
[0134] GPC is a separation technique based on the molecular
hydrodynamic volume. By comparing with a standard curve of a known
MW species, the relative MW of the samples could be easily
calculated. Table 2 shows molecular characteristics of the block
copolymers of PEG and lactic acid initiated by PEG. The average MW
increased after the introduction of lactic acid chains onto the
prepolymer PEG. The GPC traces of PLA-PEG and PLA-PEG-ALA exhibited
monomodal distributions and reflected rather narrow MW
distributions, which indicated the absence of residual
low-molecular-weight species. .sup.1H-NMR data revealed that these
low-molecular-weight species consisted of unreacted lactic acid
and/or LA-rich species. The Mn values calculated from GPC were
higher than those calculated from MALDI-TOF MS and .sup.1H-NMR
(Table 2). This finding could be assigned to changes in the
hydrodynamic volume of the hydrophilic PEG and/or PLA blocks as
compared with that of the polystyrene standards.
[0135] The LA units/OE units molar ratio or [LA]/[OE] was
determined from the integrations of the proton resonances due to
PEG blocks at 3.6 ppm and to PLA blocks at 1.5 ppm on the
.sup.1H-NMR spectra. The single peak at 3.3 ppm assigned to the
hydrogens of methyl groups was also detected on the NMR spectra of
MePEG.sub.2000 and PLA-PEG. The MW of the copolymers was determined
according to the following relationship:
M _ n ( NMR ) = M _ n PEG + M _ n PLA = 2000 + 72 .times. 2000 / 44
.times. ( [ LA ] / [ OE ] ) ##EQU00002##
where 44 and 72 were the MWs of the OE and LA repeat units,
respectively, and 2000 was the average
[0136] MW of PEG indicated by the supplier.
Emulsifying Properties of Amphiphilic Block Copolymers
[0137] To demonstrate whether PLA-PEG and PLA-PEG-PLA could be used
as emulsifiers, the polymer aqueous solution was homogenized with
squalene oil, which resulted in an isotropic emulsified
formulation. The emulsions remained stable for a few weeks when
they were stored at 4.degree. C. After 2 weeks, 5% of water
disassociated, but beyond this no further water disassociation from
the emulsion occurred. The isotropic emulsion could be reformed by
vortex mixing. Little difference was observed between the PLA-PEG-
and PLA-PEG-PLA-stabilized emulsions. Homogenization of squalene
oil with MePEG.sub.2000 or diOH-PEG.sub.2000 failed to stabilize
the squalene/water interface. This indicated that even bearing only
short PLA units in the main-chain polymer PLA-PEG or PLA-PEG-PLA,
the block copolymer could display an amphiphilic behavior.
[0138] The size distribution of the emulsions and in vitro OVA
release were measured to identify the dispersion characteristics of
the resulted emulsions and to understand the effect of the
copolymer in the emulsification process. The emulsion was
redispersed in the PBS and the size distribution was measured with
a particle size analyser. Typically, a water-in-oil (W/O) emulsion
droplet remains floating on a water surface, and the particle size
is undetectable with a light-scattering technology. Conversely, an
O/W emulsion droplet can stand only for seconds in an aqueous phase
and then diffuses into the water. The dynamic light-scattering
pattern showed that PLA-PEG or PLA-PEG-PLA was a suitable
emulsifier for squalene/water emulsions and yielded narrowly
distributed nanoparticles in the PBS. Table 3 shows physicochemical
characteristics of the squalene emulsions based on PLA-PEG and
PLA-PEG-PLA. 7 shows the cumulative release of OVA from different
formulations. Initially, a fast release was observed in the
nonformulated OVA, from which more than 80% of loaded OVA were
released into the outside PBS medium within the first 50 hr. The
PLA-PEG/squalene or PLA-PEG-PLA/squalene emulsion allowed a slight
delay, then the protein was quickly released. The visual aspect
showed that the emulsions remained stable only 5% of water
disassociated at the bottom over 200 h at 37.degree. C. Surfactants
as emulsifiers can be defined by their HLB values, which give
infbrmation about the relative affinity to aqueous and oily phases.
A lipophilic emulsifier renders a W/O emulsion with a high affinity
to an oily phase, whereas a hydrophilic emulsifier renders an O/W
emulsion with a high affinity to an aqueous phase. These are
however strongly influenced by the optimization of the surfactant
system and the emulsification process. Here, light-scattering and
in vitro release data indicated that polymers with high HLB values
rendered a stable O/W emulsion. Moreover, no significant difference
was found between PLA-PEG- and PLA-PEG-PLA- stabilized
emulsions.
TABLE-US-00005 TABLE 3 Component Emulsion Particle Aqueous phase
Oily phase HLB.sup.a type size (nm).sup.b PLA-PEG/PBS Squalene 16.6
O/W 343 .+-. 67 PLA-PEG-PLA/PBS Squalene 16.4 O/W 331 .+-. 68
.sup.aHLB.sub.PLA/PEG = 20 ( M.sub.n PEG/ M.sub.n PLA/PEG).
.sup.bEach value represents the mean of three experiments (Mean
.+-. Standard deviation).
[0139] Mostly, degradable aliphatic polyesters used for vaccine or
protein delivery have been in the form of injectable microspheres
or implant systems. Such systems require complicated fabrication
processes using organic solvents, which may cause denaturation when
antigens (virus or proteins) are to be encapsulated. Moreover, the
systems require polymers with high MW (generally >50,000 Da),
which require severe polymerization conditions (extreme temperature
and pressure and toxic catalysts). In this study, the stable
squalene; water emulsions were obtained with PEG-containing PLA
oligomers as emulsifiers without the addition of any other
stabilizer. The bioactive candidates could be either surface
attached to or encapsulated within a core oil. The obtained
emulsions had a high affinity to water so that nanoparticles were
obtained after they were redispersed into the PBS. Moreover, no
catalyst was required for the preparation of the designed polymers.
The emulsified formulation developed here was free of organic
solvents. These features are of great interest for a local delivery
of bioactive agents, especially for applications in candidate
vaccines delivery and anti-cancer treatments.
CONCLUSION
[0140] PLA/PEG diblock and triblock copolymers with high HLB values
were synthesized by the direct polycondensation of an aqueous
lactic acid solution on monomethoxy PEG or dihydroxyl PEG in the
absence of a catalyst. MALDI-TOF MS data allowed us to calculate
the repeat unit masses and end-group masses so that the molecular
structure between the diblock and triblock copolymers could be
distinguished. The obtained copolymers could serve as hydrophilic
emulsifiers and rendered stable O/W emulsified nanoparticles when
the polymer aqueous solution was homogenized with squalene oil.
Little difference was found in the physiochemical characteristics,
such as the stability, particle size, and emulsion type between the
PLA-PEG- and PLA-PEG-PLA-stabilized emulsions. These formulations
have potential to be used in a delivery system for prophylactic and
therapeutic vaccine candidates and for anticancer drugs.
Example 3
[0141] This example illustrates the use of an amphiphilic polymer,
namely, poly(ethylene
glycol)-block-poly(lactide-co-.epsilon.-caprolactone)
(PEG-b-PLACL), as an emulsification agent to render different types
of vaccine formulations. The hydrophilic block was made of PEG
because of its availability, water-solubility, and high
biocompatibility. Degradable aliphatic polyesters, in particular
polylactides (PLA) and poly(.epsilon.-caprolactone) (PCL), have
been widely used as medical and drug delivery devices with FDA
approval. PLA with variable chain stereoregularity provides a
worthwhile means to adjust the rate of degradation, in addition to
its physical and mechanical properties. The degradation products of
PCL have a relatively higher pKa than those of
poly(lactide-co-glycolide) (PLG) (4.8 for .epsilon.-hydroxycaproic
acid, and 3.8 for lactic acid and glycolic acid at 25.degree. C.),
and they may provide more conservation of protein molecular
integrity when being used for a long-term controlled delivery of
proteins. PLACL was thus chosen as a lipophilic block for its fast
degradation characteristics. In addition, its amorphous nature
provides good affinity between the polymer matrix and oil
solutions. Squalene was selected as the core oil because unlike
mineral oil., it is natural and metabolizable. The excipient use of
Span.RTM.85 in the oily phase is also positively indicated, as it
is an emulsifying agent in licensed human vaccines. Various
properties of emulsions have been characterized, including
stability, the droplet test, microscopic aspects, and the in vitro
release profile of a model antigen ovalbumin (OVA). The 13-cell and
T-cell responses in mice after immunization were characterized to
evaluate the immunogenicity-enhancing effect of novel emulsion-type
vaccine formulations (Huang et al., (2009) "Formulation and
Immunological Evaluation of Novel Vaccine Delivery Systems Based on
Bioresorbable Poly(ethylene
glycol)-block-poly(lactide-co-.epsilon.-caprolactone" J Biomed
Mater Res Part B: Appl Biomaler 90B: 832-841,which is herein
incorporated by reference in its entirety).
Materials and Methods
Polymer Synthesis and Characterization
[0142] PEG-b-PLACL was synthesized by the ring-opening
polymerization of lactide (Aldrich) and .epsilon.-caprolactone (CL.
Aldrich), using SnOct.sub.2 (stannous octoate, Sigma) as a catalyst
and MePEG (polyethylene glycol 5000 monomethyl ether, Fluka) as an
initiator. Briefly, predetermined amounts of MePEG (2.1 g), lactide
(0.58 g), and .epsilon.-caprolactone (0.47 g) were placed in a
dried round-bottomed bottle, and the appropriate amount of
SnOct.sub.2 (30 mg) was added as a solution in dried toluene (10
mL). The polymerization was performed at 140.degree. C. under
reflux for 24 h. The product was recovered by precipitation in an
excessive amount of ethanol. The yield was .about.75 wt %. The
resulted polymer was characterized by .sup.1H nuclear magnetic
resonance (.sup.1H NMR) and gel permeation chromatography (GPC).
.sup.1H NMR spectra were recorded at room temperature with a Varian
VXR 300 MHz spectrometer (Varian, Palo Alto) using deuterated
chloroform as a solvent. The molar ratio of oxyethylene to lactyl
units to caproyl units or [OE]:[LA]:[CL] was determined from the
integrations of the proton resonances due to PEG blocks at 3.6 ppm,
to PLA blocks at 5.2 ppm and to PCL blocks at 4.1 ppm on the NMR
spectra. The average molecular weights (M.) were calculated on the
basis of the M.sub.n of PEG ( M.sub.n=5000 daltons) according to
the following equations:
M.sub.nPEG-b-PLACL=5000+72.times.50000/44.times.[LA]/[OE]+114.times.500-
0/44.times.[CL]/[OE]
where 44, 72, and 114 are the molecular weights of OE, LA, and CL
repeat units, respectively. The hydrophilic-lipophilic balance
(HLB) value of nonionic PEG-b-PLACL is expressed according to the
Griffin's Method as follows:
HLB.sub.PEG-b-PLACL=20 (W.sub.PEG/W.sub.PEG-PLACL)
where W.sub.PEG/W.sub.PEG-b-PLACL is the weight ratio of the
hydrophilic portion of the main chain polymer and is obtained from
M.sub.n PEG/ M.sub.n PEG-b-PLACL.
[0143] GPC was performed by using a setting composed of a Waters
510 HPLC pump, a Waters 410 differential refractometer, one PLgel
mixed-C 5 .mu.m 100 .ANG. column (7.5.times.300 mm), and one PLgel
3 .mu.m 100 .ANG. column (7.5.times.300 mm), and the mobile phase
being THF and the flow rate being 0.8 mL/min. Data were expressed
with respect to polystyrene standards from Polysciences. A unimodal
and narrow molecular weight distribution (polydispersity index
being 1.1) was observed in GPC chromatograms of PEG-b-PLACL and the
corresponding MePEG.sub.5000, which indicated a full initiation of
the macroinitiator.
[0144] The fact that the polymer PEG-b-PLACL aqueous solution forms
micelles is an indication that the polymer possesses an amphiphilic
nature. This was confirmed by dye solubility experiments and light
scattering analysis. Briefly, five milligrams of polymer were added
to 1 mL of PBS in the presence of the water-insoluble dye
diphenylhexatriene (DPH, Sigma), which is known to dissolve in the
hydrophobic core of polymeric micelles or aggregates. After
sonication and centrifugation, an abrupt enhancement in the
ultraviolet (356 nm) absorption of the dye was observed, which was
an indication, of micelle formation. The particle size distribution
was determined by using the laser light scattering technique with a
Brookhaven 90 plus particle sizer (Brookhaven Instruments
Limited).
Polymer-Based Emulsions
[0145] An aqueous solution containing PFG-b-PLACL (120 mg)
dissolving in an antigen medium (OVA in 0.8 mL of PBS) and 1.1 mL
of an oily phase containing squalene only or squalene/Span.RTM.85
mixture (85/15 v/v) were emulsified with a Polytron.RTM. PT 3100
homogenizer (Kinematica AG, Swiss) at 6000 rpm for 5 min. A
polymeric surfactant-free emulsion composed
PBS/squalene/Span.RTM.85 was also prepared at 8000 rpm for 10 min.
These emulsified formulations serving as stocks for further
physicochemical characterizations, including stability, the droplet
test, microscopic aspects, and in vitro release. The stability test
was performed by placing each formulation at 4.degree. C. and
observed the emulsion at predetermined time points (2 weeks, 1
month, 3 months, 6 months, 1 year). The droplet test was assessed
by placing a droplet (20 .mu.L) of emulsion into a water-containing
beaker (200 mL). The microscopic aspects of the emulsions were
investigated by redispersing them (100 .rho.L) into a continuous
phase (900 .mu.L) and monitoring with an Olympus DP70 microscope.
Particle size distribution was determined by using the laser light
scattering technique. The In vitro release experiments were
performed by using the inverted dialysis tube method.
OVA-containing formulations (3 mg per 0.3 mL) were placed in a
dialysis chamber (cutoff 0.2 .mu.m) and the device was then
immersed in a 50 mL centrifuge tube containing 2-mL of `PBS at
37.degree. C. At different time points, 100 .mu.L of sample were
aspirated from the medium outside of the chamber and replaced with
100 .mu.L of fresh PBS buffer. The OVA release was regularly
determined by the bicinchinonic acid method (BCA.TM. protein assay
kit, Pierce).
Mice and Immunizations
[0146] Five-week old female BALBk mice were obtained from the
National Laboratory Animal Breeding and Research Center (Taipei,
Taiwan) and acclimatized for at least 1 week at the animal facility
of the National Health Research Institutes (NHRI, Miaoli, Taiwan)
before use. All animal studies were approved by the Animal
Committee of the NHRI. Mice were immunized subcutaneously with
syringe needles of 27G.times.1/2'' at weeks 0, 2, and 4 by 0.5
.mu.g of OVA in PBS or formulated with PEG-b-PLACL/squalene or
PEG-b-PLACL/squalene/Span.RTM.85 or aluminum phosphate suspension
(alum, 150 .mu.g per dose). To increase the fluidity, the
PEG-b-PLACL/squalene and PEG-b-PLACL/squalene/Span.RTM.85
formulations were prepared by redispersing 100 .mu.L of a stock
emulsion (see MATERIALS AND METHODS: Polymer-Based Emulsions) into
900 .mu.L of PBS before the injections, which resulted in the
formulations with .about.5% oil. Sera and spleen collections were
performed to determine B- and T-cell responses, respectively.
IgG and its Isotypes in Immunity
[0147] To determine the B-cell response, mice were bled at the
lateral tail vein and the collected sera were stored at -30.degree.
C. until assaying. The presence of OVA-specific antibodies in the
sera was determined by enzyme-linked immunosorbent assay (ELISA).
In brief, 100 .mu.L of diluted OVA (10 .mu.g/mL) were coated onto
96-well microtiter plates with 0.05M carbonate buffer (pH 9.6) for
overnight incubation at 4.degree. C. The coated plates were washed
twice with PBS containing 0.05% Tween.RTM.20 (Sigma) and then
blocked with 5% nonfat milk in PBS at room temperature for 2 h.
Diluted sera (starting dilution 1:50, serial threefold serum
dilutions) from immunized animals were applied to wells at room
temperature for 2 h. After the addition of HRP-conjugated goat
anti-mouse IgG (ICN Cappel), the assay was developed with the
substrate solution tetramethylbenzidine (TMB, Sure BIue.TM., KPL)
and the reaction was stopped with 2NH.sub.2SO.sub.1. Plates were
read at 450 nm using an ELISA plate reader (Molecular Devices,
Sunnyvale, Calif.). The data for each sample were fit using a
curve-fitting method to an exponential function, and the titers
were expressed as the reciprocal dilution that gave an optical
density of twofold absorbance of preimmune sera. For isotype
determination, 100 .mu.L of an appropriate dilution (1:2000) of
HRP-rabbit anti-mouse IgG1 (Zymed.RTM., CA) or HRP-rabbit
anti-mouse IgG2a (Zymed.RTM., CA) was added. The statistical
significance (p<0.05) was determined by performing a two-tailed
Student's t-test on log-transformed values.
T-Cell Immune Responses
[0148] To determine T-cell responses, 1 week after the boost, the
mouse spleen was removed aseptically and placed in an eppendorf
containing 1 mL of culture medium (cRPMI) consisting of RPMI 1640
(SAFC, Kansas) with 2 mM L-glutamine, and supplemented with 25 mM
HEPES (Gibco, Invitrogen, NY), 0.05 mM 2-mercaptoethanol, 10%
heatinactivated fetal bovine serum (FBS, HyClone, Perbio) and 1%
antibiotics. The single cell suspension was prepared using the end
of a syringe and grinding the spleen through a cell strainer (BD,
Biosciences). The cell suspension was collected in a 50 mL
centrifuge tube and then centrifuged at 1000 rpm for 5 min. To
remove the erythrocytes, the cell pellet was resuspended in 5 mL of
the ACK lysis buffer placed to room temperature for 1 min,
terminated the reaction with 20 mL RPMI 1640, followed by
centrifugation for 5 min. The pellet was washed twice with cRPMI
and resuspended in 5-mL of cRPMI. After cell counting with a
hemocytometer using the trypan blue dye exclusion technique,
U-bottomed 96-well plates were seeded with 2.times.10.sup.5 cells
in cRPMI at a total volume of 200 .mu.L per well. Cells were
stimulated in triplicate in the presence or absence of 10 .mu.g/mL
of OVA. Concanavalin A (Con A, 5 .mu.g/mL, Sigma) was used as a
positive control, and plates were then incubated for 5 days at
37.degree. C. in 5% CO.sub.2 in humidified air. Cellular
proliferation was assessed by the addition of 1 .mu.Ci of tritiated
methylthymidine (Perkin Elmer, Mass.) to the cell suspension for
the final 16.h of culture. Interferon-.gamma. (IFN-.gamma.) and
interleukin-4 (IL-4) concentrations in supernatants were measured
by ELISA using paired antibodies according to the manufacturer's
instructions (R&D Systems, Abingdom).
Results
Polymer Design and Polymer-Based Emulsions
[0149] A diblock copolymer consisting of 75 wt % of the hydrophilic
block PEG and 25 wt % of the lipophilic block PLACL with molecular
weight of 7000 daltons, polydispersity index of 1.1 and a
calculated HLB of 15 was synthesized via the ring-opening
polymerization of lactide and .epsilon.-caprolactone on monomethoxy
PEG with the catalyst SnOct.sub.2. The amphiphilic nature of
PEG-b-PLACL was confirmed by self-association of micelles in the
polymer aqueous solution. Dynamic light scattering displayed
polymeric micelles with a unimodal size distribution with an
average diameter of 18.2.+-.0.4 nm. Preliminary immunogenicity
studies however showed that the polymeric aqueous solution had no
adjuvant effect because they induced the same level of antigen
(OVA)-specific antibodies as those formulations without the
adjuvant (data not shown).
[0150] With the aim of enhancing the vaccine potency, the
amphiphilic polymer was used as an emulsifier to make different
emulsion-type formulations by homogenizing a mixture of the polymer
aqueous solution and squalene or the squalene/Span.RTM.85 oil (FIG.
8a). Table 4 lists the physicochemical characteristics of various
formulations based on the bioresorbable polymer PEG-b-PLACL and
selected oils. The emulsified formulation was white and isotropic
from the top to the bottom (FIG. 8b). FIG. 8c shows the stability
of the emulsions stored at 4''C. The emulsion
PEG-b-PLACL/squalene/Span.RTM.85 was stable for at least 1 year
without occurrence of phase separation. Conversely, .about.10% of
free oil at the surface layer disassociated from the emulsion
PBS/squalene/Span.RTM.85 after I month. In the case of the emulsion
PEG-b-PLACL/squalene with aqueous/oily being 5/5 w/w, 20% of water
disassociated under the same storage conditions after 2 weeks, but
beyond this no further water disassociation from the emulsion
occurred. An isotropic emulsion could be reformed by homogenization
in the same condition or simply by vortex mixing. An increase in
the squalene content could significantly enhance the stability of
the emulsion PEG-b-PLACL/squalene and result in a stable emulsion
without phase separation for at least 1. year when the aqueous/oily
was 3/7 w/w.
[0151] The type of dispersion of the emulsion was examined by a
droplet test, microscopic aspects, and in vitro release. The
droplet test allowed the identification of a continuous phase of
emulsion. A droplet of PBS/squalene/Span.RTM.85 emulsion remained
floating on the water surface after 24 h (FIG. 9a) even after
gentle hand stirring of the beaker. Conversely, the
PEG-b-PLACL/squalene emulsion droplet could stand for only one
half-hour in an aqueous phase and then diffused into the water
(FIG. 9b), a feature similar to the
PEG-b-PLACL/squalene/Span.RTM.85 system. This result revealed that
the PBS/squalene/Span.RTM.85 emulsion contained only the lipophilic
emulsifier Span.RTM.85 and rendered an emulsion with a high
affinity to the oily phase, whereas the emulsion
PEG-b-PLACL/squalene or PEG-b-PLACL/squalene/Span.RTM.85 had a
hydrophilic polymeric emulsifier and rendered an emulsion with a
high affinity to the aqueous phase.
TABLE-US-00006 TABLE 4 Component Particle Aqueous phase Oily phase
HLB Emulsion type size PEG-b-PLACL/PBS Squalene 15.0 O/W emulsion
~1-10 .mu.m PEG-b-PLACL/PBS Squalene/ 7.5.sup.a W/O/W emulsion
<1 .mu.m Span .RTM. 85 --/PBS Squalene/ 1.8 W/O emulsion -- Span
.RTM. 85 .sup.aHLB.sub.mix = X.sub.PEG-b-PLACL .times.
HLB.sub.PEG-b-PLACL + X.sub.Span85 .times. HLB.sub.Span85, where X
is the weight fraction of each surfactant.
[0152] The emulsion drops were invisible under an optical
microscope since they were crowded in the dispersed phase. The size
distribution of the emulsion was investigated by redispersing the
emulsion in the continuous phase and measuring the size with a
microscope and particle sizer. As shown in FIG. 1.0a, the emulsion
PEG-b-PLACL/squalene was composed of nonhomogeneous particles with
a bimodal distribution. Two different sizes of particles were
observed, relatively large particles of about 10 .mu.m and smaller
particles of about 1 .mu.m. By contrast, homogeneous fine particles
less than 1 .mu.m were observed with an optical microscope when
redispersing the PEG-b-PLACL/squalene/Span.RTM.85 in the PBS (FIG.
10b). The dynamic light scattering pattern showed a unimodal
distribution with an average diameter of 457.7.+-.25.8 nm.
Stabilized particles of .about.1-10 .mu.m in diameter are
appropriate for uptake by antigen-presenting cells (APCs) to
facilitate the induction of potent immune responses due to the
pseudo-natural targeting of antigens.
In vitro Protein Release from Various Formulations
[0153] FIG. 11 shows the cumulative release of OVA from various
formulations: (-x-) no adjuvant, (open circle) the aqueous solution
PEG-b-PLACL, (open square) the O/W emulsion PEG-b-PLACL/Squalene,
(open triangle) the W/O/W emulsion
PEG-b-PLACIL/squalene/Span.RTM.5, (filled circle) the W/O emulsion
PBS/squalene/Span.RTM.85. Initially, a fast release was observed in
the case of OVA without adjuvant from which more than 80% of loaded
OVA were released into the outside PBS medium within the first 50
h. The polymer aqueous solution or PEG-b-PLACL/squalene emulsion
allowed a slight delay but the protein was quickly released. The
emulsion PEG-b-PLACL/squalene/Span.RTM.85 released less than 50% of
OVA during the same period of time. Afterwards, the protein release
increased continuously until it reached an equilibrium
concentration in the inside and outside of the dialysis device. The
PBS/squalene/Span.RTM.85 emulsion presented a well depot effect on
OVA so that hydrophilic OVA was slowly released over 200 h.
[0154] FIG. 11b shows the recovered formulations after 200 h
experiments. Phase separation occurred in the formulation
PBS/squalene/Span.RTM.85. By contrast, the PEG-b-PLACL-based
emulsion remained stable with clear layers' of water at the bottom.
The protein (ovalbumin as an example) initially encapsulated within
the emulsion was almost released at this time, which indicated that
the hydrophilic bioactive agents (or antigens) trapped within the
polymer-emulsified oily emulsion were released from the core oil to
the surface mostly by diffusion, but also to a lesser extent by
degradation mechanisms and emulsion breaks.
Immunogenicity Studies in Mice
[0155] Physicochemical characterization showed that various
emulsions provide different size ranges of particles and different
antigen controlled-release mechanisms so that the emulsified
particles could serve as either carriers or vehicles to deliver
antigens to APCs in a targeted and prolonged manner. To evaluate
the potential application of these polymer-based emulsions as
adjuvant, BALB/c mice were vaccinated subcutaneously with OVA using
various formulations. Table 5 shows the B-cell response to
OVA-formulations with different adjuvants. Following immunization,
the antigen-specific antibody IgG response in the group of OVA
atone was undetectable in an initial serum dilution of 1:50 at week
2, and less than 10.sup.4 serum titers were detected at week 4.
However, the serum antibody IgG titers as well as isotypes IgG1 and
IgG2a were significantly enhanced for the group of
PEG-b-PLACL/squalene or PEG-b-PLACL/squalene/Span.RTM.85- or
alum-formulated OVA in comparison to the free OVA group
(p<0.05). For IgG responses, the ratio between the geometric
mean titer (GMT) obtained with PEG-b-
PLACL/squalene/Span.RTM.85-formulated OVA vaccine and the GMT
obtained with vaccine alone was found to be 19.9 at week 4, in
comparison with the group of PEG-b-PLACL/squalene being 2.4 and the
group of alum being 6.3. PEG-b-PLACL/squalene-formulated OVA
induced comparable levels of serum antibody titers as
alum-formulated OVA within 10 weeks. The highest antibody responses
were elicited in the group of
PEG-b-PLACL/squalene/Span.RTM.85-formulated OVA, in which
statistical significance with respect to alum was detected at weeks
2 and 4, that is, in the early stages after immunization. The
immunogenicity increase was probably due to appreciable particle
size and/or carrier/depot activity.
TABLE-US-00007 TABLE 5 [OK] IgG Formulation Week 2 Week 4 Week 6 No
adjuvant <50 5,400 79,200 (2,200-13,300) (62,000-101,100)
PEG-b-PLACL/Squalene 400.sup.a 12,700 101,800 (50-2,800)
(4,000-40,400) (69,200-149,700) PEG-b- 3800.sup.a, b 107,500*.sup.a
163,700.sup.a PLACL/squalene/Span .RTM. 85 (3,200-4,500)
(63,300-182,500) (85,600-312,900) Alum 500.sup.a 34,000.sup.a
124,000.sup.a (100-2,600) (20,300-57,000) (79,800-192,700) IgG IgG
1 IgG 2a Formulation Week 8 Week 10 Week 4 Week 4 No adjuvant
90,400 74,700 18,100 100 (55,100-148,300) (50,300-111,000)
(9,500-34,600) (80-200) PEG-b-PLACL/Squalene 124,800 67,100 49,600
500 (69,100-225,500) (28,500-157,700) (15,200-161,600) (100-2,750)
PEG-b- 182,000.sup.a 130,100.sup.a 274,600.sup.a,b 5,500.sup.a
PLACL/squalene/Span .RTM. 85 (129,600-255,600) (99,100-170,700)
(183,200-411,700) (2,000-14,900) Alum 138,600 99,100 112,400.sup.a
2,600.sup.a (74,900-256,500) (70,000-140,300) (83,200-152,000)
(1,120-6,200) BALB/c mice were vaccinated three times
subcutaneously (week 0, 2, 4) with dose of 0.5 .mu.g OVA. Sera were
collected from blood and the antibody titers were measured by
ELISA. The data are presented as geometric mean titers with 95%
confidence intervals of five mice per group. .sup.aP < 0.05:
Comparison with free OVA group at the same time point. .sup.bP <
0.05: Comparison with alum-formulated OVA group at the same time
point. <50 means undetectable in an initial dilution of
1:50.
[0156] T-cell proliferation and cytokine responses were measured in
the splenocytes following restimulation of the cells in vitro with
OVA antigen. FIG. 12a showed that following one immunization, OVA
alone did not induce antigen-specific proliferative response well
due to the low dosage of antigen so that the stimulation index of
the OVA/PBS group was only slight higher than the threshold value.
Once OVA was formulated with the polymer-based emulsion or adsorbed
to alum, positive T-cell proliferative response was induced. FIG.
12b shows that sufficiently elevated IFN-.gamma. secretion, a
predominant T helper type 1 cytokine, was detected in the
supernatants of splenocyte collected from mice treated with
PEG-b-PLACL/squalene- and PEG-b-PLACL/squalene/Span.RTM.85-
formulated OVA, following in vitro restimulation of splenocytes
with OVA. By contrast, T helper type 2 cytokine IL-4 was measured
at the same or reduced level as OVA alone.
Discussion
[0157] Delivery of antigens in a targeted or prolonged manner can
be performed with different emulsion-type adjuvants and can be
achieved it specific surfactant systems. Surfactants as emulsifiers
can be defined by their hydrophilic-lipophilic balance (HLB) value,
which gives information on their relative affinity to both aqueous
and oily phases. In this study, the droplet test and in vitro
release data showed that the squalene/Span.RTM.85 oil solution
contained an emulsifier of low HLB value (HLB.sub.Span.RTM.85=1.8),
which rendered a W/O emulsion. By contrast, the
PEG-b-PLACL/squalene system with a high HLB value emulsifier
rendered an O/W emulsion. Finally, the emulsion
PEG-b-PLACL/squalene/Span.RTM.85 is composed of two emulsifiers and
renders a water-in-oil-in-water (W/O/W) multiphase emulsion (FIG.
13). From a viewpoint of emulsion stability, oil droplets with a
small particle size and homogeneous distribution are more stable,
which are however strongly influenced by the optimization of the
surfactant system and the emulsification process. Homogenization of
PEG-b-PLACL-containing aqueous solution and the
Span.RTM.85-contained oily phase provides a potential way of
stabilizing emulsified particles both at storage and at
postinjection stage conditions.
[0158] A polymer can play different roles in a particulate delivery
system. The most direct role is to provide a matrix or a vehicle
that builds the microparticle. Degradable PLG, PLA, and PCL used
for vaccine or protein delivery have mostly been in the form of
injectable microspheres or implant systems. Such systems require
complicated fabrication processes using organic solvents and may
cause denaturation when antigens (virus or proteins) are to be
encapsulated. Moreover, the systems require polymers with high
molecular weight (generally >100,000 Da), which require severe
polymerization conditions (extreme temperature and pressure and
toxic catalysts). By contrast, amphiphilic copolymers such as poly
(ethylene glycol)-block-poly(propylene sulphide)-blockpoly(ethylene
glycol) and poly(ethylene
glycol)-block-polyoxypropylene-block-poly(ethylene glycol) (known
as Pluronic.RTM. or Poloxamers) could be formed as polymersomes, as
oil free thermosensitive hydrogels or as a surfactant to render an
emulsion (known as TiterMax.RTM.) in vaccine delivery systems.
These polymeric emulsifiers render stable emulsions and elicit both
potent humoral and cellular immune responses with respect to other
formulations, using OVA as model. Their use in human vaccine
delivery is however problematic because they are rather toxic and
nonbiodegradable. We have attempted to study the effects of polymer
aqueous solution or polymer-based emulsions on the activation and
antigen-presenting functions of bone marrow-derived dendritic cells
to understand the biological interactions and immunological
mechanisms of action. Our findings indicated that PEG-b-PLACL-based
formulations were biologically inert in dendritic cells (data not
shown). These formulations probably could not act as
immunostimulatory adjuvants for dendritic cells of the innate
system but could he used as vaccine delivery systems instead.
[0159] To our knowledge, we are the first group to investigate the
synthetic, amphiphilic, bioresorbable polymer as an emulsification
agent to stabilize aqueous/oily interfaces. The emulsified vaccine
delivery systems have several advantages over traditional vaccine
adjuvants. Firstly, synthetic polymeric emulsifier is reproducible
from batch to batch, and the relative hydrophobic/hydrophilic
balance can be easily manipulated by the amounts of monomer used,
thus producing a broad range of emulsifier characteristics.
Secondly, unlike antigen adsorption onto alum, the system allows
either the surface attachment or encapsulation of antigens, and the
emulsified formulation can be stored at or below room temperature
as a stock before injection. Moreover, the acidic condition is not
required in the preparation of PEG-b-PLACL and/or Span.RTM.85
emulsified delivery systems because both are nonionic emulsifiers.
Thirdly, bioresorbable polymeric emulsifiers, with a hydrophobic
block that is degradable, show bulk degradation and further resorb
in vivo. Fourthly, the raw materials for polymer synthesis
described here are commercially available and frequently used for
temporary therapeutic applications. Furthermore, the formulation is
easy for preparation, that is, no complicating processes or
supplemental equipment are required and thus the cost is reduced.
Moreover, the polymer-emulsified delivery system is free of organic
solvents, in contrast to the common polymeric microspheres.
Fifthly, with only 5% of oil included in the vaccines (see
MATERIALS AND METHODS: Mice and Immunizations), the W/O/W emulsion
increases injectability and conceptually diminishes local
reactions, which is encountered by the W/O type vaccines produced
from the same oil. Finally, from the immunity viewpoint, antigen:
specific antibody titers and T-cell proliferative responses as well
as IFN-.gamma. responses were significantly enhanced (p <0.05)
to ovalbumin after being formulated with the PEG-b-PLACL-based
emulsions. These features are of great interest for local delivery
of bioactive agents, especially for application in candidate
vaccine delivery systems.
CONCLUSION
[0160] With the aim of enhancing vaccine potency, we used a
bioresorbable diblock tri-component copolymer PEG-b-PLACL as an
emulsifier and rendered an O/W or W/O/W multiphase emulsion when
the polymer aqueous solution was homogenized with squalene or the
squalene/Span.RTM.85 mixture. Novel polymer-emulsified formulations
have high affinity to water so that the stock OVA-containing
emulsion could be redispersed into PBS before injection, thus
resulting in fluid emulsion (only 5% of oil within the emulsion)
with homogeneous particles rangimi between 1 and 10 .mu.m. The
emulsified particles could serve as either carriers or vehicles to
deliver biologically active agents (ovalbumin as an example) to
APCs in a targeted and prolonged manner, thus effectively enhancing
immunity. These formulation have potential to be used in adjuvants
for prophylactic and therapeutic vaccine candidates. Such
applications include single-dose multivalent vaccine development
and via alternative immunization routes, such as intramuscular or
transdermal administration.
Example 4
[0161] Previously, based on the bioresorbable diblock tri-component
co-polymer polyethylene
glycol)-block-poly(lactide-co-.epsilon.-caprolactone)
(PEG-b-PLACL), we developed a water-in-oil-in-water multiphase
emulsion-type vaccine delivery system called PELC. In this
emulsion, PEG-b-PLACL served as a hydrophilic emulsifier and
Span.RTM.85 acted as a hydrophobic emulsifier to stabilize the
water/squalene interface, resulting in a stable and homogeneous
nanoemulsion. Preliminary immunogenicity studies in mice using
ovalbumin as a model antigen showed that antigen-specific antibody
titers, T-cell proliferative response, and interferon-.gamma.
(IFN-.gamma.) secretion increased significantly after formulation
with PELC. Thus, this approach is of great interest for
applications in prophylactic and therapeutic vaccination. In
preparation for a potential shortage of pandemic influenza vaccine,
we aimed to increase the efficacy of vaccine candidates via
formulation with PELC. In the work described here, a single-dose
immunization was performed using inactivated H5N1 virus adjuvanted
with PELC. The PELC-formulated hemagglutinin (HA; 0.5 .mu.g) of
inactivated virus induced more potent antigen-specific antibodies,
hemagglutination inhibition, and virus neutralization than HA (5
.mu.g) of non-adjuvanted virus, demonstrating the antigen
economization of the PELC-based vaccine. Moreover, T-cell
proliferative responses as well as IFN-.gamma. and interleukin-4
(IL-4) secretion were significantly enhanced after formulation with
the PELC emulsion. These results demonstrate that PELC could play
an important role in the influenza pandemic vaccine development
(Huang et al. (2009) "Enhancement of potent antibody and T-cell
responses by a single-dose, novel nanoemulsion-formulated pandemic
influenza vaccine" Microbes and Infenction 11: 654-660, which is
herein incorporated by reference in its entirety).
Materials and Methods
Vaccine Preparation
[0162] The vaccine used in this study was the formalin-inactivated
whole-virus vaccine NIBRG-14, which was kindly supplied by the UK
National Institute of Biological Standard and Control, NIBSC. The
vaccine was derived from a reassorted H5N1 vaccine strain
containing modified HA and neuraminidase (NA) from a highly
pathogenic avian influenza strain A/Vietnam/1194/2004 and
propagated in Madine-Darby canine kidney (MDCK) cells.
Formalin-inactivated vaccines were prepared with 0.1% formalin at
37.degree. C. for 24 h. The HA content was determined by single
radial diffusion (SRD). Production details for the H5N 1 vaccine
candidate are reported elsewhere.
Adjuvant Preparation
[0163] The diblock co-polymer PEG-b-PLACL was synthesized by the
ring-opening polymerization of lactide and .epsilon.-caprolactone
on monomethoxy PEG as previously described. The PEG-b-PLACL
consisted of 75 wt % hydrophilic block PEG and 25 wt % lipophilic
block PLACL. The calculated hydrophilic lipophilic balance (HLB)
value was 15 with a molecular weight of 7000. PELC is an
emulsion-type vaccine delivery system based on PEG-b-PLACL,
Span.RTM.85, and squalene. Briefly, a PEG-b-PLACL-containing
aqueous solution (120 mg dissolved in 0.8 mL of phosphate-buffered
saline (PBS)) and 1.1 mL of an oily phase containing a
squalene/Span.RTM.85 mixture (85/15 v/v) were emulsified using a
Polytron.RTM. PT 3100 homogenizer (Kinematica AG, Swiss) at 6000
rpm for 5 min. The emulsified formulation was stored at 4.degree.
C. until use. The PELC-adjuvanted vaccine was formulated by
re-dispersing 200 .mu.L of the stock PELC emulsion in 1800 .mu.L of
bulk vaccine before injection. The size distribution of the
emulsion droplets was determined with a microscope (Olympus DP70)
and the laser light scattering technique (Brookhaven 90 plus
particle size analyzer, Brookhaven Instruments Limited).
Mice and Immunizations
[0164] Five-week-old female BALB/c mice were obtained from the
National Laboratory Animal Breeding and Research Center (Taipei,
Taiwan) and acclimatized for at least one week at the animal
facility of the National Health Research Institutes (NHRI, Miaoli,
Taiwan) prior to use. To investigate the potency of a single-dose
of H5N1 influenza vaccine, all mice were vaccinated intramuscularly
(i.m.) with one of two different closes (0.5 .mu.g or 5 .mu.g HA)
administrated with or without PELC. Serum and tissue collection
were performed to determine B- and T-cell responses. Serum samples
were collected from immunized mice and antibody titers were
determined by enzyme-linked immunosorbent assay (ELISA) as well as
by hemagglutination inhibition titration and viral neutralizing
assays.
ELISA Immunoassay
[0165] The presence of NIBRG-14-specific antibodies in the sera was
determined via ELISA. Briefly, 96-well microtiter plates were
coated with 100 mL of dilute inactivated virus (1 .mu.g/mL) and
incubated overnight at room temperature. The coated plates were
washed once with PBS containing 0.05% Tween.RTM.20 (Sigma) and
blocked with 1% bovine serum albumin (BSA, Sigma) in PBS at room
temperature for 2 h. Diluted sera (starting dilution 1:1000, serial
two-fold serum dilutions) from immunized mice were applied to the
blocked wells at room temperature for 2 h. After another wash,
HRP-conjugated goat anti-mouse IgG (ICN Cappel, 1:5000) was added
to all wells at room temperature for 30 min. The assay was
developed using the substrate solution 2,
20-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS.RTM.
Peroxidase, KPL) for 20 min at room temperature and shielded from
light. Plates were read at 405 nm with an ELISA plate reader
(Thermo Multiskan.RTM. spectrophotometer, Vantaa, Finland). For
isotype determination, 10 .mu.L of HRP-conjugated rabbit anti-mouse
IgG1 (AbD Serotec, Kidlington, UK, 1:5000) or HRP-conjugated rabbit
anti-mouse IgG2a (AbD Serotec, Kidlington, UK, 1:2000) was added
instead of anti-mouse IgG. The titers were expressed based on the
inverse of the final dilution that gave two-fold greater absorbance
than the pre-immune sera.
Hemagglutination Inhibition (HI) Titration
[0166] The HI test was based on the ability of the specific
anti-influenza antibodies to inhibit hemagglutination of turkey red
blood cells (RBCs) by influenza virus HA. Non-specific inhibitors
of agglutination were removed by heat treatment and addition of
receptor-destroying enzymes. After pretreatment, serum samples
(two-fold dilutions starting at an initial dilution of 1:10) were
incubated with four hemagglutination units of influenza strain.
Turkey RBCs were then added and agglutination inhibition was
scored. The serum titer was expressed as the reciprocal of the
highest dilution that demonstrated complete HI. The seroprotection
rate (SPR, %) was calculated from the proportion of mice achieving
a post-vaccination titer .gtoreq.40.
Virus Neutralization (VN) Assay
[0167] The NIBRG-14 virus in 200 TCID50 (50% tissue culture
infective dose) per well was incubated with two-fold-diluted mice
sera at a starting dilution of 1:40. The mixtures of virus and
serum were transferred to monolayers of MDCK cells and incubated at
37.degree. C. and 5% CO.sub.2 for 4 days. The neutralizing titer
was expressed as the reciprocal of the highest serum dilution at
which the infectivity of the H5N1 virus 200 TOD50 for MDCK cells
was completely neutralized in 50% of the wells. Infectivity was
identified by the presence of cytopathy on Day 4 and the titer was
calculated using the ReedeMuench method.
Statistical Analysis
[0168] The statistical significance (P<0.05) was determined by
performing a two-tailed Student's t-test on log-transformed
values.
T-Cell Immune Assay
[0169] It has been demonstrated that 7-14 days post-immunization is
a reasonable time point for detecting influenza-specific T-cell
proliferation We chose the twelfth day after immunization as an
endpoint. The mouse spleen was removed aseptically twelve days
after immunization and transferred to a tube containing 1 mL of
RPMI-1640 culture medium (cRPMI) (SAFC, Kansas, USA) with 2 Mm
L-glutamine and supplemented with 25 mM HEPES (Gibco, Invitrogen,
NY, USA), 0.05 mM 2-mercaptoethanol, 10% heat-inactivated fetal
bovine serum (FBS; HyClone/Perbio), 100 ng/ml streptomycin, and 100
U/ml penicillin. Cell suspensions were prepared by mashing the
spleen through a cell strainer with a syringe plunger. The resulted
suspension was collected in a 50-mL tube and centrifuged at 1000
rpm for 5 min. To remove erythrocytes, the cell pellet was
resuspended in 5 mL of RBC lysis buffer (150 mM NH.sub.4Cl, 1 mM
KHCO.sub.3 and 0.1 mM EDTA (pH 7.3), Biolegend, CA) and incubated
at room temperature for 1 min. The reaction was then terminated
with 20 mL of RPMI-1640 and the mixture was centrifuged for 5 min,
The pellet was washed twice with cRPMI and resuspended in 5 mL of
cRPMI. After cell counting with a hemacytometer by the trypan blue
dye exclusion, U-bottomed 96-well plates were seeded with
2.times.10.sup.5 cells in cRPMI at a total volume of 200 .mu.L per
well. Cells were stimulated in triplicate in the presence or
absence of 2.5 .mu.g HA/mL of inactivated NIBRG-14 virus.
Concanavalin A (Con A, 5 .mu.g/mL, Sigma) was used to induce a
maximal proliferative response. Plates were then incubated for 4
days at 37.degree. C. and 5% CO2 in air. Cellular proliferation was
assessed with the addition of 1 uCi of tritiated methylthyrnidine
(Perkin Elmer, MA) to the cell suspension for the final 16 h of
culture. IFN-.gamma. and IL-4 concentrations'in the supernatants
were measured by ELISA with paired antibodies according to the
manufacturer's instructions (R&D Systems, Abingdom).
Results
[0170] Formulation of Inactivated Influenza Virus with PELC
[0171] The PELC-formulated influenza candidate vaccine consisted of
an inactivated virion and a pre-emulsified PELC stock (see
Materials and methods:Adjuvant preparation). Prior to the
injection, the PELC stock and inactivated virions were mixed to
form homogeneous particles. The size distribution of the particles
ranged from 200 to 400 nm in diameter (FIG. 14), which was
consistent with the definition of nanoemulsion. Importantly, it has
been reported that this nanoseale dimension is conducive to uptake
by antigen-presenting cells, which facilitates the induction of
potent immune responses.
Determination of NIBRG-14-Specific Antibodies Elicited in BALB/c
Mice
[0172] To evaluate whether protective antibodies were induced by a
single immunization with the candidate vaccine, BALB/c mice were
intramuscularly immunized with 0.5 .mu.g or 5 .mu.g HA of
inactivated NIBRG-14 virus formulated with or without PELC. Table 6
shows NIBRG-14-specific IgG1, and IgG2a antibodies elicited in
BALB/c mice following a single intramuscular dose of H5N1
inactivated virus vaccine. The elicited antigen-specific antibodies
are shown in Table 6. The results demonstrated that the specific
anti-virus lgG, IgG1, and IgG2a titers induced by the
PELC-formulated inactivated virus were significantly higher than
those induced by non-adjuvanted inactivated virus in both the 0.5
.mu.g and 5 .mu.g HA groups (P<0.05). Another advantage of
vaccination with PELC-formulated inactivated virus was revealed at
the fourth week after administration: the IgG titers induced by
PELC formulated with 0.5.mu. HA of inactivated virus were one order
higher than those induced by 5 .mu.g HA of inactivated virus. The
titers induced by 5 .mu.g HA were still lower than those induced by
one-tenth of the same amount of PELC-formulated antigen (0.5 .mu.g
HA) at the same time point.
TABLE-US-00008 TABLE 6 GMT .+-. SE Vaccine No adjuvant Formulated
with PELC IgG 0.5 .mu.g 5 .mu.g 0.5 .mu.g 5 .mu.g Week 2 <1000
2000 .+-. 500* 1800 .+-. 900* 5900 .+-. 1700*.sup.,# Week 4 1400
.+-. 200 4000 .+-. 1100* 11,300 .+-. 4700* 29,000 .+-. 8000*.sup.,#
Week 8 7100 .+-. 700 16,000 .+-. 4500* 45,250 .+-. 12,600*.sup.,#
64,000 .+-. 15,000*.sup.,# Week 12 6050 .+-. 1000 10,800 .+-. 2000
39,000 .+-. 7600*.sup.,# 55,700 .+-. 6400*.sup.,# Week 18 5650 .+-.
2000 13,100 .+-. 4300 21,500 .+-. 10,800* 42,200 .+-. 7800*.sup.,#
Week 26 7100 .+-. 2400 14,500 .+-. 4800 23,800 .+-. 9000* 48,500
.+-. 7800*.sup.,# IgG1 at week 8 3300 .+-. 800 7300 .+-. 3400
17,450 .+-. 6800* 29,000 .+-. 16,000*.sup.,# IgG2a at week 8.sup.a
7100 .+-. 2400 22,600 .+-. 4300 107,600 .+-. 45,250*.sup.,# 105,000
.+-. 25,900*.sup.,# The data are presented as geometric mean titers
(GMTs) with standard errors (SE) of eight mice per group. *P <
0.05: comparison with the group of 0.5 .mu.g HA without adjuvant at
the same time point. .sup.#P < 0.05: comparison with the group
of 5 .mu.g HA without adjuvant at the same time point. <1000
means undetectable in an initial dilution of 1:1000.
.sup.aTitrations do not reflect absolute concentrations as can be
seen in the IgG2a subtype assays that are more sensitive than the
total IgG assays.
Hemagglutination Inhibition (HI) Activities of Elicited
Antisera
[0173] The HI activity assay is the most common way to determine
the efficacy of an influenza vaccine. We determined HI activity
using turkey erythrocytes incubated with sera obtained from the
vaccinated groups. Table 7 shows that HI antibody responses were
elicited in BALB/c mice following a single intramuscular dose of
inactivated H5N1 virus vaccine formulated with or without PELC.
Following a single injection, sera from the mice vaccinated with
0.5 .mu.g HA of non-adjuvanted inactivated virus elicited an HI
geometric mean titer (GMT) of 6 and 9 at Weeks 2 and 4,
respectively. The highest GMT responses were 59 at Week 12 and 33
at Week 26. When the amount of non-adjuvanted virus administrated
was increased to 5 .mu.g HA, the HI titer was slightly enhanced at
Weeks 2, 4, and 8. There were however no statistically significant
differences (Pz) in HI titers between the 0.5 .mu.g and 5 .mu.g HA
groups. By contrast, the PELC-adjuvanted vaccines were capable of
inducing higher HI titers than those developed from the virus alone
(P<0.05). Even in the group of PELC-adjuvanted, 0.5 .mu.g HA of
inactivated virus, the HI titer was significantly higher than that
induced by non-adjuvanted, 5 .mu.g HA of inactivated virus.
Moreover, the seroprotection of those groups immunized with
inactivated virus alone never reached 100%. In contrast, after
forimilation with PELC, seroprotection easily reached 100% in both
0.5 .mu.g HA and 5 .mu.g HA groups, which indicated the merit of
PELC-adjuvanted inactivated influenza virus.
TABLE-US-00009 TABLE 7 GMT .+-. SE (SPR, %) No adjuvant Formulated
with PELC Vaccine 0.5 .mu.g 5 .mu.g 0.5 .mu.g 5 .mu.g Week 2 <10
(0%) 20 .+-. 13* (38%) 44 .+-. 9* (75%) 80 .+-. 24*.sup.,# (100%)
Week 4 <10 (14%) 22 .+-. 11 (50%) 62 .+-. 20*.sup.,# (100%) 108
.+-. 21*.sup.,# (100%) Week 8 24 .+-. 10 (57%) 26 .+-. 12 (63%) 95
.+-. 34*.sup.,# (100%) 238 .+-. 44*.sup.,# (100%) Week 12 59 .+-.
23 (86%) 44 .+-. 25 (63%) 108 .+-. 38 (100%) 422 .+-. 107*.sup.,#
(100%) Week 18 49 .+-. 39 (86%) 52 .+-. 26 (63%) 131 .+-. 36 (100%)
243 .+-. 58*.sup.,# (100%) Week 26 33 .+-. 13 (57%) 37 .+-. 25
(63%) 88 .+-. 37 (86%) 184 .+-. 45*.sup.,# (100%) The data are
presented as geometric mean titers (GMTs) with standard errors (SE)
of eight mice per group. The seroprotection rate (SPR, %) is the
percentage of mice achieving a post-vaccination titer .gtoreq.40.
*P < 0.05: comparison with the group of 0.5 .mu.g HA without
adjuvant at the same time point. .sup.#P < 0.05: comparison with
the group of 5 .mu.g HA without adjuvant at the same time point.
<10 means undetectable in an initial dilution of 1:10.
Virus Neutralization (VN) Activities of Elicited Antisera
[0174] VN assays were performed to provide a more functional
measure of vaccine-induced immunity. As shown in Table 8, the
neutralizing antibody titers were slightly enhanced When the amount
of non-adjuvanted antigen administered was increased. In contrast,
when the vaccine was formulated with PELC, the neutralizing
antibody titers were dramatically enhanced. The highest
neutralizing antibody titers were induced by the PELC-adjuvanted 5
.mu.g HA group. The VN capability of PELC-formulated, inactivated
virus was complementary to its adjuvanticity demonstrated by HI
titers. Thus, a combination of PELC and inactivated virus may
induce sufficient and sustainable protective antibodies.
Importantly, 0.5 .mu.g HA of PELC-formulated, inactivated virus
could induce higher antibody titers, higher HI activity, and higher
VN activity than 5 .mu.g HA of inactivated virus alone. These
results indicated that inactivated virus formulated with PELC could
not only facilitate a decrease in antigen dose, but also an
increase in the humoral protection.
T-Cell Proliferation and Cytokine Responses to PELC-Formulated
Antigens
[0175] We next sought to determine whether T-cell responses could
also be enhanced when antigen was formulated with PELC. Twelve days
after intramuscular immunization with 0.5 .mu.g HA of inactivated
virus in PBS or PELC single-cell suspensions were prepared from the
mouse spleen and restimulated in vitro in the presence of
inactivated virus for 4 days. FIG. 1SA shows that following one
vaccination, the virus alone did not induce a notable
antigen-specific proliferative response. The elicited Stimulation
Index (SI), which is the ratio of the mean counts per minute (cpm)
with antigen to the cpm without antigen, was only slightly higher
than the control value. However, once the PELC-formulated vaccine
candidate was administered, a positive T-cell proliferative
response was induced and the SI value was about two-fold higher
than that of the non-adjuvanted group. In addition, the IFN-.gamma.
and IL-4 concentrations detected in the splenocyte supernatants of
the PELC group were significantly higher than those of the
nonadjuvanted group (FIG. 158). IFN-.gamma. is a predominant T
helper type 1 (Th1) cytokine relevant to virus-specific cytotoxic T
lymphocyte (CTL) activity, while IL-4 is a common T helper type 2
(Th2) cytokine. Therefore, an antigen formulated with PELC may not
only increase the humoral protection but also enhance both Th1 and
Th2 responses. These results indicated that PELC may have
applications in single-dose immunization and could play an
important role in influenza pandemic preparedness.
TABLE-US-00010 TABLE 8 GMT .+-. SE No adjuvant Formulated with PELC
Vaccine 0.5 .mu.g 5 .mu.g 0.5 .mu.g 5 .mu.g Week 2 <40 <40
<40 <40 Week 4 <40 <40 53 .+-. 21 211 .+-. 74*.sup.,#
Week 8 49 .+-. 26 56 .+-. 32 180 .+-. 59*.sup.,# 381 .+-.
54*.sup.,# Week 12 207 .+-. 56 144 .+-. 61 261 .+-. 134 1452 .+-.
154*.sup.,# Week 18 176 .+-. 55 125 .+-. 67 293 .+-. 195 695 .+-.
90*.sup.,# Week 26 64 .+-. 23 66 .+-. 34 92 .+-. 56 333 .+-.
39*.sup.,# The data are presented as geometric mean titers (GMTs)
with standard errors (SE) of eight mice per group. *P < 0.05:
comparison with the group of 0.5 .mu.g HA without adjuvant at the
same time point. .sup.#P < 0.05: comparison with the group of 5
.mu.g HA without adjuvant at the same time point. <40 means
undetectable in an initial dilution of 1:40.
Discussion
[0176] Alum-formulated H5N1 influenza vaccines have demonstrated
that a prime/boost vaccination schedule is required to generate
effective protection against either heterologous or homologous
viral strains. After priming, the virus-neutralizing antibody
levels were below the detection limit for groups that received
doses ranging from 0.001 to 3.75 .mu.g HA antigens with or without
alum. However, the antibody titers increased substantially and
virus-neutralizing antibodies were detectable after boosting with
the same amount of antigen. It was reported that after the
prime/boost immunization with inactivated H5N1, the induced levels
of protective antibodies were not statistically different between
the groups immunized with 0.2 .mu.g or 2 .mu.g HA antigens. In
addition, there were no differences between the groups immunized
with or without alum. It was also reported that the prime/boost
vaccination with inactivated H2N2 consisting of 1.5 .mu.g HA mixed
with alum induced a systemic response equivalent to that of a
non-adjuvanted 15 mg HA vaccine. Here, we demonstrated that a
single-dose administration of PELC-formulated virus significantly
induced virus-neutralizing antibodies and had a dose-dependent
effect on HI and VN responses. In contrast, no dose-dependent
responses were evident in the non-adjuvanted groups. In terms of
the longevity of the induced antibody responses, there was no
significant difference between the response to the virus alone and
to the versus formulated with PELC. In both cases, the total IgG
titers peaked at Week S and fluctuated, decreased slightly until
Week 26. Meanwhile, the HI and VN antibody levels reached a high
peak at Week 12 and then declined until Week 26. These results have
established a potential application of PELC-formulated vaccines in
pandemic influenza preparedness. PELC-adjuvanted immunization may
be particularly helpful in preventing a vaccine shortage since its
efficacy:permitted a decrease in antigen dosage and its single-dose
formulation eliminated the need for boosting.
[0177] Since O/W emulsions can quickly induce a strongly
immunocompetent environment at the site of injection, they are more
efficient than alum for human vaccination. Furthermore, recent
clinical data have demonstrated that pandemic H5N1 vaccines
formulated with O/W emulsions induce seroconversion and
cross-neutralization superior to that of non-adjuvanted and
alum-formulated vaccines. Despite the benefits of O/W emulsions,
one drawback to the use of Tween.RTM.80 as a hydrophilic emulsifier
is that it attacks cell walls and thus is potentially toxic. One
viable alternative is the hydrophilic emulsifier PEG-b-PLACL, which
has several advantages over Tween.RTM.80. Firstly, PEG-b-PLACL is
derived from the Food and Drug Administration (FDA)-approved PEG,
polylactides, and poly(3-caprolactone) and is thus expected to pass
all safety tests. Secondly, polymeric emulsifiers generally result
in more stable emulsions and more potent humoral and cellular
immune responses than small molecule-emulsified formulations.
Finally, degradable emulsifiers allow stabilization of emulsion
particles during storatte and allow disintegration of the system
after the injection. These characteristics of
PEG-b-PLACL-emulsified vaccines demonstrate the potential safety
and efficacy of PELC-formulated vaccines.
[0178] For over a decade, the advisory committees in the North
America have cautioned that influenza vaccine-induced antibodies
decline more rapidly in the elderly. It is believed that the aging
population is most susceptible to influenza infection. Recently, an
effective vaccination was correlated with the induction of Th1
cytokine IFN-.gamma., especially in the elderly, who undergo a
shift toward Th2 cytokine (such as IL-4) production and a relative
reduction in CTL activity as they age. Therefore, increasing
IFN-.gamma. induction via vaccination is thought to be an important
strategy for overcoming the age-related influenza susceptibility.
Toward this end, PELC may be a potent adjuvant due to its
stimulation of antigen-specific T-cell proliferation and
IFN-.gamma. secretion. PELC-formulated virus also upregulated
IFN-.gamma. and IL-4 in splenocytes and increased IgG1 and IgG2a
antibody levels more than virus alone. The vaccine formulation with
PELC however did not significantly skew the immune response toward
Th1 or Th2 (Table 6 and FIG. 15B). These results implied that the
antigens adjuvanted with PELC might dramatically enhance the
immunogenicity of vaccine candidates. Further investigations are
under way to examine the combinations of the PELC vaccine delivery
system and immunostimulatory adjuvants, such as CpG
oligodeoxynucleotide, in order to manipulate the immune response
and alter the Th1/Th2 balance. In conclusion, we found that 0.5
.mu.g HA of inactivated virus formulated with PELC induced treater
HI and VN activities than 5 .mu.g HA of virus alone. The use of an
adjuvanted, low dose, whole-virus influenza vaccine will open the
possibility of increasing the number of vaccine doses from the same
pool. T-cell proliferation as well as IFN-g and IL-4 secretions
were also significantly enhanced in the PELC-formulated 0.5 .mu.g
HA group, which indicated that this vaccination approach might
enhance protective immunity in the elderly. Taken together, these
results demonstrated that PELC may have applications in antigen
economization and preparation for an influenza pandemic.
[0179] The foregoing description of the exemplary embodiments of
the invention has been presented only for the purposes of
illustration and description and is not intended to be exhaustive
or to limit the invention to the precise forms disclosed. Many
modifications and variations are possible in light of the above
teaching. The embodiments and examples were chosen and described in
order to explain the principles of the invention and their
practical application so as to enable others skilled in the art to
utilize the invention and various embodiments and with various
modifications as are suited to the particular use contemplated.
Alternative embodiments will become apparent to those skilled in
the art to which the present invention pertains without departing
from its spirit and scope. Accordingly, the scope of the present
invention is defined by the appended claims rather than the
foregoing description and the exemplary embodiments described
therein.
[0180] Some references, which may include patents, patent
applications and various publications, are cited and discussed in
the description of this invention. The citation and/or discussion
of such references is provided merely to clarify the description of
the present invention and is not an admission that any such
reference is "prior art" to the invention described herein. All
references cited and discussed in this specification are
incorporated herein by reference in their entireties and to the
same extent as if each reference was individually incorporated by
reference.
* * * * *