Note: Descriptions are shown in the official language in which they were submitted.
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NANOSTRUCTURES COMPRISING COBALT PORPHYRIN-PHOSPHOLIPID
CONJUGATES AND POLYHISTIDINE-TAGS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. patent application no.
16/399,581,
filed on April 30, 2019, the disclosure of which is incorporated herein by
reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under grant
numbers
DP50D017898 and R21AI122964 awarded by the National Institutes of Health. The
government has certain rights in the invention.
FIELD OF THE DISCLOSURE
[0003] This disclosure relates general to the field of functionalized
nanostructures.
More particularly, the disclosure relates to nanostructures comprising cobalt-
porphyrin.
BACKGROUND OF THE DISCLOSURE
[0004] In the field of functionalized nanoparticles one of the challenges
is to easily
and reliably attach peptides and proteins to larger scaffolds. Targeted
nanoparticles require
effective ligands and unconjugated peptides themselves are weakly immunogenic.
Bioconjugate chemistry has provided a range of strategies, but most
nanoparticulate
conjugations suffer from limitations relating to one or more of the following:
1) low
conjugation yields and necessitated purification steps; 2) incompatibility
with biological
buffers, making labeling of intact nanoparticles impossible; 3) variable
labeling sites and
conjugated polypeptide conformations, creating an inhomogeneous particle
population of
varying efficacy; 4) necessity for complex and exogenous chemical approaches.
[0005] Standard approaches for ligand attachment to aqueous
nanoparticles make use
of maleimides, succinimidyl esters and carbodiimide-activated carboxylic
acids. These can
covalently react with amine and thiol groups of polypeptides. The use of
maleimide-lipids has
been explored extensively for antibody-conjugated immunoliposomes. Conjugation
yields
may reach as high as 90% from an overnight reaction, but subsequent quenching
of free
maleimide groups and additional purification is required. Proteins may require
a preparative
step of thiolation and purification prior to conjugation. Antibody orientation
is a major factor
influencing the conjugated antibody target binding efficacy, but these
approaches result in
numerous antibody labeling sites and indiscriminate orientations. Biorthogonal
synthetic
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strategies such as the click reaction have recently been applied to pre-formed
liposomes,
however these require the use exogenous catalysts and unconventional amino
acids.
[0006] Another approach that is suitable for smaller peptides which
are less prone to
permanent denaturation in organic solvents is to conjugate the peptides to a
lipid anchor. The
resulting lipopeptides can then be incorporated along with the other lipids
during the
liposome formation process. This approach has been used to generate synthetic
vaccines that
induce antibody production against otherwise non-immunogenic peptides.
However, due to
their amphipathic character, in that case the lipopeptides were difficult to
purify, with a yield
of 5-10%. It has also been shown that lipopeptides do not fully incorporate
into liposomes
during the formation process, resulting in aggregation.
SUMMARY OF THE DISCLOSURE
[0007] The present disclosure provides functionalized nanostructures.
The
nanostructures can be used for delivery of cargo, targeted delivery and/or
delivery of
presentation molecules. The nanostructures can be monolayers or bilayers which
enclose an
aqueous compartment therein. Bilayer structures enclosing an aqueous
compartment are
referred to herein as liposomes. The nanostructures can be monolayer or
bilayer coating on a
nanoparticle. The monolayer or bilayer comprises cobalt porphyrin-phospholipid
conjugate,
optionally phospholipids that are not conjugated to porphyrin, optionally
sterols, and
optionally polyethylene glycol (PEG). One or more targeting peptides or
polypeptides
(referred to herein as presentation molecules) having a polyhistidine tag are
incorporated into
the monolayer or bilayer such that a portion of the polyhistidine tag resides
in the monolayer
or bilayer and the presentation molecule is exposed to the exterior of the
monolayer or
bilayer. Instead of, or in addition to the cobalt porphyrin phospholipid
conjugate, cobalt
porphyrin can be used.
[0008] The nanostructures of the present disclosure can be loaded with
cargo for
delivery to sites that can be targeted by the polyhistidine tagged
presentation molecules. For
example, liposomes can be loaded with cargo for delivery to desired sites by
using
polyhistidine tagged presentation molecules.
[0009] Data presented here demonstrates that a bilayer containing a
cobalt-porphyrin,
such as a cobalt porphyrin-phospholipid (CoPoP) can stably bind polyhistidine-
tagged (also
referred to herein as "his-tagged") polypeptides (Fig. la). Other metallo-
porphyrins such as
zinc, nickel, and copper are not able to stably bind a his-tagged polypeptide.
This represents a
new binding paradigm, with at least some polyhistidines buried in the membrane
phase, as
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the porphyrins themselves form the hydrophobic portion of the bilayer and are
not accessible
to the external aqueous environment (Fig. lb). This leads to more stable
binding, allows for
significantly simpler non-covalent post-labeling paradigms following
nanoparticle formation,
and eliminates ambiguity regarding ligand orientation.
[0010] We show that lipid bilayers containing porphyin-phospholipid which
is
chelated with cobalt, but not other metals, can effectively capture his-tagged
proteins and
peptides. The binding follows a Co(II) to Co(III) transition and occurs within
the sheltered
hydrophobic bilayer, resulting in, for example, essentially irreversible
attachment in serum or
in million-fold excess of competing imidazole. Using this approach we inserted
homing
peptides into the bilayer of pre-formed empty and cargo-loaded liposomes to
enable site
targeting (such as tumor-targeting) without disrupting the bilayer integrity.
Peptides or
synthetic peptide can be bound to liposomes containing an adjuvant (such as
the lipid
monophosphoryl lipid A) for antibody generation for an otherwise non-antigenic
peptides.
[0011] The present disclosure provides monolayer or bilayer
structures, wherein the
monolayer or bilayer comprises porphyrins with cobalt chelated thereto such
that the cobalt
metal resides within monolayer or bilayer and the porphyrin macrocycle and
further has
molecules with a histidine tag non-covalently attached thereto, such that at
least a part of the
his-tag is within the monolayer or bilayer and coordinated to the cobalt metal
core. The
presentation molecules can be used for various applications including
targeting and
generation of immune responses. Liposomes or micelles formed by the present
layers may be
loaded with cargo for release at desired locations. The cobalt porphyrin maybe
be cobalt
porphyrin-phospholipid (CoPoP). The present layers may also be used as
coatings for other
nanostructures including metal nanoparticles, nanotubes and the like.
BRIEF DESCRIPTION OF THE FIGURES
[0012] Figure 1. His-tag binding to PoP-bilayers. (a) Schematic showing a
peptide
with a His-tag (green) binding to pre-formed CoPoP liposomes in aqueous
solution. (b)
Insertion of a His-tagged polypeptide into a bilayer containing CoPoP. Only a
single leaflet
of the bilayer is shown. (c) Chemical structure of metallo-PoPs used in this
study.
[0013] Figure 2. His-tagged protein binding to Co(III)-PoP liposomes.
(a) A heptahis-
tagged fluorescence protein comprising Cerulean (C) fused to Venus (V) reveals
binding to
PoP-bilayers. When C is excited, FRET occurs and V emits fluorescence (left),
but this is
inhibited when bound to the PoP-bilayer due to competing FRET with the
photonic bilayer
(middle). C fluorescence can be directly probed even when the protein is bound
to the bilayer
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(right). (b) Multispectral fluorescence images of fusion protein
electrophoretic mobility shift
following incubation with indicated metallo-PoP liposomes. (c) Binding
kinetics of the fusion
protein to the indicated metallo-PoP liposomes based on loss of C to V FRET.
(d) NMR peak
widths of the underlined proton of the vinyl group on CoPoP demonstrate
paramagnetic
broadening of Co (II) in deuterated chloroform (CDC13) but non-paramagnetic
peaks of
Co(III) following CoPoP-liposome formation in deuterated water. For each set
of bars, left to
right are bars for: CoPoP and 2H-PoP (e) Reversal of His-tagged peptide
binding to CoPoP
liposomes following addition of 2 M sodium sulfate. Liposomes were formed with
10 molar
% CoPoP or Ni-NTA phospholipid. For each set of bars, the bars from left to
right are: water,
and +2M sulfite.
[0014] Figure 3. Robust His-tagged protein binding to CoPoP liposomes
(a)
Multispectral electrophoretic mobility shift images of the fluorescent
reporter protein
incubated with liposomes containing the indicated lipid. (b) Binding stability
of reporter
protein bound to indicated liposomes in 1:1 serum. (c) Binding stability of
reporter protein
bound to indicated liposomes in excess free imidazole. Mean +/- std. dev. for
n=3.
[0015] Figure 4. Binding of a short His-tagged RGD peptide to CoPoP
liposomes. (a)
Binding of a short peptide labeled with FAM to metallo-PoP liposomes. (b)
Effect of His-tag
length on binding half-time to CoPoP liposomes. No binding "N.B." was observed
for the
peptide lacking a His-tag. Effect of liposome composition on binding half-time
to CoPoP
liposomes of indicated composition when incubated in PBS (c) or in 5 mg/mL BSA
(d).
Mean +/- std. dev. shown for triplicate measurements. In (c) and (d), for each
set of bars, the
bars from left to right are 50 and 0.
[0016] Figure 5. RGD-His targeting of cargo-loaded liposomes. (a)
Release of
entrapped sulforhodamine B in PoP liposomes during peptide binding. For each
set of bars,
the bars from left to right are: 8 hr, and 24 hr. (b) Targeted uptake of
sulforhodamine B-
loaded liposomes. Cells were incubated in the indicated conditions and uptake
was assessed
by examining sulforhodamine B fluorescence. For each set of bars, the bars
from left to right
are: MCF7 cells, and U87 cells (c) Confocal micrographs showing liposome
uptake. Cells
were incubated with the indicated liposome solutions for 2 hours, washed and
imaged. All
images were acquired with the same settings. (d) Biodistribution of
sulforhodamine B
entrapped in CoPoP liposomes with or without attachment of a His-tagged cyclic
RGD
targeting peptide 45 minutes following injection into nude mice bearing
subcutaneous U87
tumors. Mean +/- std. dev. for n=3. For each set of bars, the bars from left
to right are:
untargeted, and +cRGD-His.
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[0017] Figure 6. HIV peptide vaccination using immunogenic CoPoP
liposomes. (a)
BALB/c or athymic nude mice were immunized with CoPoP liposomes containing a
25 [tg of
MPL and 25 [tg of His-tagged MPER peptide derived from the HIV gp41 envelope
protein.
Sera titer was assessed with an ELISA using a biotinylated MPER peptide
lacking a His-tag
and probed with an anti-IgG secondary antibody. Arrows indicate time of
vaccinations. (b)
Anti-MPER titer in BALB/c mice vaccinated as indicated. Mice were vaccinated
on week 0
and week 2 and serum was collected on week 4. (c) Sustained anti-MPER titer in
mice
vaccinated with CoPoP liposomes containing MPL. Mean +/- std. dev. for n=4
mice per
group. The first two bars (joined) on the left are CoPoP and Ni-NTA (d)
Neutralization of
HIV infection in 293 cells in the presence of indicated antibodies. IgGs were
purified from
mouse sera using Protein A. Mean +/- std. dev. for n=3.
[0018] Figure 7. Stability of RGD-His peptide binding to liposomes.
(a) FAM-labeled
RGD-His peptide binding to liposomes containing 10 molar % Ni-NTA-lipid, Co-
NTA-lipid
or CoPoP. (b) Gel filtration following peptide binding. Only CoPoP liposomes
maintained
stable binding (c) Peptide stability following incubation with a 1:1 dilution
in fetal bovine
serum. Only CoPoP liposomes maintained stable binding.
[0019] Figure 8. Stable his-tagged protein binding to liposomes
containing CoPoP.
The reporter protein was incubated with liposomes containing CoPoP, free Co-
porphyrin or
2H-PoP, then incubated in serum and subjected to EMSA. The protein was then
imaged using
the FRET channel (ex: 430 nm, em: 525 nm). The lack of signal in the CoPoP
lane
demonstrates stable binding to the liposomes. The diminished signal in the Co-
porphyrin lane
demonstrates some binding of the his-tagged protein to the liposomes.
[0020] Figure 9. Time for 90 % peptide binding of RGD-His to CoPoP
liposomes of
different compositions. Effect of liposome composition on the time for 90 %
peptide binding
to CoPoP liposomes (10 molar % CoPoP), containing the indicated components
when
incubated in PBS with the RGD-His peptide. For each set of bars, the bars from
left to right
are: + cholesterol, and ¨ cholesterol.
[0021] Figure 10. RGD-His binding to CoPoP liposomes in the presence
of serum or
albumin. Liposomes of the indicated composition were incubated with the RGD-
his peptide
in the presence of 50% fetal bovine serum or 50 mg/mL bovine serum albumin.
The FAM-
labeled peptide emission was normalized by comparing the peptide emission when
bound to
CoPoP liposomes to 2H-PoP liposomes.
[0022] Figure 11. Membrane permeabilization by lipopeptides.
Sulforhodamine B
loaded liposomes were incubated with the indicated peptides (5 [tg/mL) at room
temperature
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and release was assessed using fluorescence. For each set of bars, the bars
from left to right
are: 8 hr, and 24 hr.
[0023] Figure 12. RGD-His peptide binding to liposomes containing 1
molar %
CoPoP and cell targeting. (a) Normalized peptide fluorescence upon incubation
with CoPoP
liposomes containing 1 molar % CoPoP. Emission was normalized by comparing
CoPoP
samples with 2H-PoP. (b) Cell uptake of sulforhodamine B liposomes containing
1 molar %
CoPoP, incubated with cells as indicated. For each set of bars, the bars from
left to right are:
MCF7 cells, and U87 cells.
[0024] Figure 13. Coating gold nanoparticles with a CoPoP his-tag
binding surface.
(a) Photograph of coating protocol to disperse gold nanosphere. Without the
PoP coating,
citrate stabilized gold aggregates following repeated centrifugation steps
(arrow). (b) Size of
nanospheres before and after lipid coating. Inset shows transmission electron
micrograph of
CoPoP gold with 50 nm scale bar. (c) Absorption spectra of citrate stabilized
gold and CoPoP
gold following RGD-His binding. (d) Confocal reflectance images showing uptake
of
targeted nanospheres. Cells were incubated with the indicated gold nanospheres
for 2 hours,
washed and then imaged. All images were acquired with the same settings.
[0025] Figure 14. (A) Psf25 (Pfs25 B) binding was measured by a
centrifugal
filtration assay. These data indicate 100% binding of the Psf25 protein to Co-
pop liposomes.
(B) Particle size of CoPoP liposomes before and after Psf25 protein binding.
[0026] Figure 15. Anti-Psf25 IgG levels in CD-1 mice. Mice were vaccinated
with
Psf25 in CoPoP/MPL or ISA70 following intramuscular injections with (A) pre-
boost and
(B) after boost, three-week prime/three-week boost (5, 0.5 or 0.05 ug Pfs25
per injection).
IgG titers were measured by ELISA on a 96-well plates from mice vaccinated
with CoPoP
(Psf25) liposomes, or free Psf25 protein (with or without ISA70). Data show
mean +/- S.D.
with n=5 mice per group).
[0027] Figure 16. Anti-Psf25 IgG titers. Titers were defined as
reciprocal serum
dilution that produced an absorbance greater than 0.5 over background.
[0028] Figure 17. Illustration and characterization of different
length of NANP
peptide coating on CoPoP liposomes. (A) Different numbers of NANP repeated
peptide
containing 7x histidine (His) tag.(B) Mean diameter and polydispersity (PDI)
of CoPoP
liposomes conjugated with different length of NANP peptide were calculated by
dynamic
light scattering (n = 3). Error bars, SD. (C) peptide binding of NANP peptides
to CoPoP
liposomes and 2HCoPoP liposomes were measured by the microcentrifugal
filtration process
and BCA assay (n=3).
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[0029] Figure 18. Anti MPER IgG titers in mice pretreated with
CoPoP/phosphatidyl
serine liposomes bound to his-tagged MPER. Mice were pretreated MPER/CoPoP/PS
liposomes 4 weeks and 2 weeks prior to injection of MPER in CoPoP/MPLA
liposomes to
induce an antibody response against MPER.
[0030] Figure 19. Fluorescence of U87 cells following incubation with CoPoP
liposomes bound to various his-tagged CPPs.
[0031] Figure 20. After incubating at room temperature for 2 hours,
DS-Cavl bearing
a his-tag achieved near complete binding with CoPoP nanoparticles (Figure 20,
left). His-
tagged DS-Cavl did not bind significantly to PoP particles lacking cobalt
(2HPoP), and DS-
Cavl without the his-tag present did not achieve significant binding with
either particle. His-
tagged DS-Cavl did not induce liposome aggregation upon binding based on
liposome
diameter (Figure 20, center) or polydispersity (Figure 20, right).
[0032] Figure 21. CoPoP/DS-Cavl vaccination results in higher IgG
antibody titer
both before and after booster injections. At 35 days post-primary injection,
CoPoP vaccine
achieves the greatest RSV neutralization. CD-1 mice were intramuscularly
injected with 100
ng DS-Cavl on day 0 and day 21 and serum was collected on day 42 and assessed
for
neutralization.
[0033] Figure 22. Generation of his-tagged OspA.
[0034] Figure 23. Characterization of an OspA-based proteoliposome
formed using
.. CoPoP/PHAD liposomes. (A) Optimum binding mass ratio of OspA:CoPoP/PHAD
liposomes evaluated by native PAGE (histidine-MOPS buffer system pH 6.8). (B)
Kinetics of
OspA binding to CoPoP/PHAD liposomes incubated at 1:4 mass ratio at room
temperature.
(C) Specific binding of his-tagged OspA to CoPoP/PHAD liposomes measured by
microBCA
assay of supernatant obtained from high-speed centrifugation of liposomes. (D)
Hydrodynamic diameter and polydispersity index of liposomes measured using
dynamic light
scattering. (E) TEM images of CoPoP/PHAD liposomes with and without bound his-
tagged
protein. Acquisitions were made 3 hr after incubation of liposomes with
protein. Error bars
represent standard deviations for n=3 measurements.
[0035] Figure 24. Assessing epitope availability and stability of
antigen-
functionalized nanoliposomes. (A) Stability of nanoliposome-antigen particles
in 20% (v/v)
human serum based on fluorescence quenching assay. (B) Immunoprecipitation of
OspA-
bound liposomes by OspA-specific monoclonal antibody LA-2. (C) DY-490-OspA
uptake by
murine RAW 264.7 macrophage cells following 2 hr incubation with indicated
samples.
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Cytochalasin B was supplemented to medium 1 hr prior to incubation. Error bars
represent
standard deviations for n=3 experiments.
[0036] Figure 25. Evaluation of the immunogenicity of OspA-based
nanoliposomal
vaccine. (A) Anti-OspA IgG titers induced by CoPoP/PHAD liposomes compared to
other
commercial adjuvants. Detection of anti-OspA IgG antibodies by (B) indirect
immunofluorescence assay of permeabilized B. burgdorferi B31 using goat anti-
Mouse IgG
(H+L) secondary antibody DyLight 488 conjugate and by (C) immunoblot assay
using
whole cell lysates of B. burgdorferi B31 and B. azfelii. Horizontal lines
represent geometric
mean.
[0037] Figure 26. Thl-biased immune response of OspA-bound CoPoP/PHAD
liposomes. (A) IgG isotype profiling for post-immune sera (day 42) using
ELISA. (B)
Splenocyte stimulation study to detect interferon-gamma and interleukin-4
secretion after 72-
hr stimulation with OspA. Error bars represent standard deviations from n=3
triplicate
stimulation experiments. Horizontal lines indicate geometric mean.
[0038] Figure 27. Assessment of the borreliacidal activity of SNAP-induced
OspA
antibodies. (A) Serum bactericidal antibody assay performed using guinea pig
complement.
Survival percentage was derived from normalization of the number of
spirochetes after
overnight serum treatment to that immediately after incubation. Surviving B.
burgdorferi
B31-A3 were counted using dark-field microscopy. (B) Average 50% borreliacidal
activity
(serum dilution rate that effectively eliminated 50% of the bacteria) from
three different mice
sera. Error bars represent standard error of the mean. NI stands for no
inhibition. Statistical
significance (p < 0.05, indicated by asterisks) of differences between
bactericidal titers is
assessed by Kruskal-Wallis test with Dunn's post-hoc.
[0039] Figure 28. Longevity of anti-OspA IgG levels in SNAP-immunized
mice.
Prime and boost vaccinations of 100 ng OspA with CoPoP/PHAD liposomes were
administered on day 0 and 21, respectively. Endpoint titer is defined as the
reciprocal of
serum dilution at absorbance cut-off value of 0.5. Data points represent
geometric mean and
the error bars the 95% confidence interval.
[0040] Figure 29. Binding of his-tagged HA protein from H3N2 strain
Fr1478 to
CoPoP liposomes.
[0041] Figure 30. Shown in (A-F), three groups of mice (n=8) were
vaccinated with
100 ng of the H3 antigen Fr1478 (from flu strain A/canine/Illinois/11613/2015)
adjuvated
with CoPoP/MPLA, 2HPoP/MPLA, or Alum (aluminum hydroxide), and then inoculated
with A/Hong Kong/1/1968. (A) The IgG ELISA and (B) HAT assays performed with
serum
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samples taken throughout the vaccination period show that CoPoP/MPLA+Fr1478
particles
achieve the best antibody response prior to viral challenge. (C-D) Body weight
remained
stable across the 8-day challenge period, and both (E) viral load and (F)
white blood cell
count in lung tissue were minimal in CoPoP vaccinated mice, indicating
effective protection
.. against the virus. (G-I) A follow-up challenge study was performed to
investigate how
CoPoP/MPLA protection compared to CoPoP without MPLA and another adjuvant, the
montanide ISA720. Again, CoPoP/MPLA vaccinated mice exhibit the most
consistent body
weight retention and lowest viral load and white blood cell count in the
lungs.
[0042] Figure 31. While passive transfer of vaccinated mouse serum
yields lower
.. protection than direct vaccination, transfer of serum from mice vaccinated
with CoPoP yields
(A) significantly reduced weight loss, (B) a higher rate of survival, and (C)
lower clinical
scores. This data supports the hypothesis that the immune response resulting
from CoPoP
vaccination is significantly antibody-mediated.
[0043] Figure 32. Shown in (A), his-tagged influenza antigens from
various subtypes
.. were obtained and tested for binding affinity with CoPoP. The binding
potential of his-tagged
antigens appears to be independent of subtype, with most antigens achieving
approximately
100% binding with CoPoP after 3 hours of incubation at room temperature. (B)
Mice were
vaccinated with CoPoP incubated with one of the ten selected antigens, and the
serum was
collected and ELISA was used to quantify the binding reactions of the
resultant serum
antibodies. Antibody reactions against the antigen that was homologous with
the vaccine
antigen resulted in the highest IgG binding, with the nonspecific binding of
antibodies
elicited by other subtypes tended to average one to two orders of magnitude
lower binding.
[0044] Figure 33. Structures of some examples of synthetic adjuvants.
[0045] Figure 34. Liposomes were formed with 4:2:1:X
DPPC:Cholesterol:CoPoP:MPLA, where MPLA was each of these types of synthetic
versions, and X varied was either 5,4,3,2,1. Results are shown for anti-Pfs25
titer for CP
(PHAD), C3D6A (3D6A-PHAD), CP504 (PHAD-504), and CA (no MPLA).
DESCRIPTION OF THE DISCLOSURE
[0046] The present disclosure provides nanostructures comprising at
least a
monolayer. For example, the structures can comprise a monolayer or a bilayer
wherein the
monolayer or bilayer comprise porphyrin-phospholipid conjugates that have
cobalt chelated
thereto such that the cobalt resides within the bilayer. The bilayer
structures can form
liposomes. The structures can comprise two monolayers (bilayers), where the
hydrophobic
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groups of the two monolayers are opposed and the hydrophilic groups are
exposed to the
surface.
[0047] The disclosure herein regarding bilayers is also applicable to
monolayers. The
bilayers or monolayers are sometimes referred to herein as "membranes".
[0048] All ranges provided herein include all values that fall within the
ranges to the
tenth decimal place, unless indicated otherwise.
[0049] Some or all of the cobalt porphyrins in the monolayer or
bilayer can non-
covalently bind polyhistidine-tagged molecules, such that at least part of the
polyhistidine tag
resides within the bilayer and the tagged molecule is presented on the surface
of the bilayer.
In the present bilayers or monolayers, it is considered that one or more
histidine residues in
the polyhistidine tag are coordinated to the cobalt metal core within the
bilayer, thereby
providing stability to the structure. The histidine residues of a
polyhistidine tag may be
coordinated to the cobalt metal in the core of the porphyrin in the membrane.
The entire
histidine tag may reside within the bilayer. A porphyrin phospholipid
conjugate which has
cobalt metal conjugated thereto is referred to herein as CoPoP. Liposomes
wherein the
bilayer comprises CoPoP are referred to herein as CoPoP liposomes. The CoPoP
liposomes
can be functionalized with histidine tagged molecules. The term "his-tagged
molecules" as
used herein means molecules ¨ such as, for example, peptides, polypeptides, or
proteins ¨
which have a histidine tail. For example a peptide with a histidine tail is a
his-tagged
molecule. Such his-tag containing CoPoP liposomes are referred to herein as
his-tagged
CoPoP liposomes or his-tagged CoPoP.
[0050] The CoPoP monolayers or bilayers functionalized with his-
tagged presentation
molecules of the present disclosure provide a platform for presentation of
various molecules
of interest in the circulation or for delivery to desired locations or for
generation of specific
immune responses to those his-tagged molecules. These molecules are referred
to herein as
presentation molecules (PMs). Structures containing his-tagged CoPoP bilayers,
which have
PMs attached to the histidine tag exhibit desirable stability. The his-tagged
molecules are
non-covalently attached to (coordinated to) the CoPoP and can be prepared by
an incubation
process. Therefore, the process does not need removal of reactive moieties ¨
such as
maleimide and the like ¨ or exogenous catalysts or non-natural amino acids
that are used in
other types of conjugation chemistries.
[0051] The cobalt-porphyrin may be in a bilayer in self-assembling
liposomes
enclosing therewithin an aqueous compartment. Alternatively, it may be in a
single layer or
bilayer coating that coats other nanoparticles. Cobalt-porphyrin phospholipid
(CoPoP)
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behaves like a conventional lipid with respect to its amphipathic nature.
Therefore,
monolayers or bilayers comprising CoPoP can be used for coating of
nanoparticles by
methods that are known to those skilled in the art. In one embodiment, the
bilayer or
monolayer of the present disclosure may be present on other nanoparticles,
such as, for
example, in the form of a coating. In one embodiment, the bilayer or monolayer
containing
cobalt-porphyrin (e.g., cobalt porphyrin-phospholipid) is present as a coating
on gold or silica
nanoparticles, or other nanoparticles with a hydrophilic surface. In one
embodiment, the
coating may be in the form of monolayers. In one embodiment the monolayer or
bilayer
containing cobalt-porphyrin (e.g., cobalt porphyrin-phospholipid) is present
as a coating on
hydrophobic surfaces such as carbon nanotubes. In one embodiment, the
monolayers may
form micelles surrounding one or more hydrophobic molecules.
[0052] This disclosure provides a nanostructure comprising a
monolayer or a bilayer,
wherein the monolayer or bilayer comprises: i) optionally, phospholipids and
ii) porphyrin
which has cobalt coordinated thereto forming cobalt-porphyrin. Optionally, the
nanostructure
also has one or more polyhistidine-tagged presentation molecule. At least a
portion of the
polyhistidine tag resides in the hydrophobic portion of the monolayer or the
bilayer and one
or more histidines of the polyhistidine tag are coordinated to the cobalt in
the cobalt-
porphyrin. At least a portion of the polyhistidine-tagged presentation
molecule is exposed to
the outside of the nanostructure. The nanostructure can be in the form of a
liposome that
encloses an aqueous compartment. However, the nanostructure may also coat a
hydrophilic or
hydrophobic material such as a gold or silica nanoparticle. The cobalt
porphyrin may be
conjugated to a phospholipid to form a cobalt porphyrin-phospholipid
conjugate. The cobalt
porphyrin can make up from 1 to 100 mol % of the monolayer or the bilayer,
including 0.1
mol% values and ranges therebetween. For example, the cobalt porphyrin can
make up from
1 to 20 mole %, or from 5 to 10 mol% of the monolayer or the bilayer. If the
cobalt porphyrin
makes up 100% of the monolayer or the bilayer, then there are no phospholipids
present that
are not conjugated to cobalt porphyrin. The bilayer or the monolayer can also
comprise sterol
and/or polyethylene glycol. The sterol can be cholesterol.
[0053] The number of histidines in the polyhistidine-tag in the
monolayer or bilayer
can be from 2 to 20. For example, the number of histidines in the
polyhistidine-tag can be
from 6 to 10. For example, the number of histidines can be 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20.
[0054] The liposomes may be spherical or non-spherical. The size of
the liposomes
can be from 50 to 1000 nm or more. In one embodiment, the liposomes have a
size (e.g., a
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longest dimension such as, for example, a diameter) of 50 to 1000 nm,
including all integer
nm values and ranges therebetween. For example, the size may be from 50 to 200
nm or from
20 to 1000 nm. If the liposomes are not spherical, the longest dimension can
be from 50 to
1000 nm. These dimensions can be achieved while preserving the nanostructure
width of the
monolayer of the bilayer. The liposomes can carry cargo in the aqueous
compartment. The
cargo, or part thereof, can also, or alternatively, be incorporated in the
monolayer or the
bilayer.
[0055] In one embodiment, this disclosure provides a liposome
comprising: a
monolayer or a bilayer, wherein the monolayer or bilayer comprises cobalt-
porphyrin
phospholipid conjugate, optionally phospholipids that are not conjugated to
cobalt porphyrin,
and a polyhistidine-tagged presentation molecule, wherein at least a portion
of the
polyhistidine tag resides in the hydrophobic portion of the monolayer or the
bilayer and one
or more histidines of the polyhistidine tag are coordinated to the cobalt in
the cobalt-
porphyrin phospholipid conjugates. At least a portion of the polyhistidine-
tagged presentation
molecule is exposed to the outside of the nanostructure. The nanostructure,
such as a
liposome, can enclose an aqueous compartment. The monolayer or the bilayer
need not
contain any phospholipids that are not conjugated to cobalt porphyrin and in
this case only
has cobalt porphyrin phospholipid conjugates. Cargo can be present in the
aqueous
compartment. The cargo need not reside exclusive in the aqueous compartment
and a part
thereof can reside in the monolayer or the bilayer.
[0056] The disclosure also provides a monolayer or a bilayer, wherein
the monolayer
or bilayer comprises phospholipid monomers and porphyrin having cobalt
coordinated
thereto (forming cobalt-porphyrin). The monolayer or the bilayer has
associated therewith
one or more polyhistidine-tagged presentation molecules, wherein at least a
portion of the
polyhistidine tag resides in the hydrophobic portion of the monolayer or the
bilayer. One or
more histidines of the polyhistidine tag are coordinated to the cobalt in the
cobalt-porphyrin
and at least a portion of the polyhistidine-tagged presentation molecule is
outside of the
bilayer or the monolayer. In various examples, the monolayer or the bilayer
encloses an
aqueous compartment or forms a coating on a nanoparticle ¨ such as a gold or
silica
nanoparticle.
[0057] The disclosure provides a nanostructure comprising a core, and
a monolayer or
a bilayer coating on the core, wherein the monolayer or bilayer comprises
phospholipids, and
porphyrin having cobalt coordinated thereto forming cobalt-porphyrin. The
nanostructure can
have one or more polyhistidine-tagged presentation molecules, wherein at least
a portion of
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the polyhistidine tag resides in the hydrophobic portion of the monolayer or
the bilayer and
one or more histidines of the polyhistidine tag are coordinated to the cobalt
in the cobalt-
porphyrin. At least a portion of the polyhistidine-tagged presentation
molecule is exposed to
the outside of the nanoparticle. The core of the nanostructure can be a
nanoparticle such as a
gold or silica nanoparticle.
[0058] The liposomes, or nanoparticles having a coating or monolayer
or bilayer, as
described herein can have presentation molecules thereon, which can be
antigenic molecules
and/or targeting molecules. The presentation molecules can also provide
targeting ability
and/or imaging or other functionalities.
[0059] Liposomes or other nanostructures comprising his-tagged polypeptides
and
CoPoP compositions exhibit high serum-stability with respect to binding of the
his-tagged
polypeptide to the liposome. In one embodiment, when incubated with serum
(such as diluted
serum) at room temperature, more than 60% of the his-tagged peptide remains
bound to the
CoPoP-containing bilayer after 24 hours incubation. In one embodiment, more
than 85% of
the his-tagged peptide remains bound to the CoPoP layer after incubation with
serum for 24
hours.
[0060] The CoPoP liposomes or the his-tagged CoPoP liposomes can be
loaded with
cargo ¨ which typically resides in the aqueous compartment, but may reside
entirely or
partially embedded in the bilayer ¨ if it is hydrophobic or has a hydrophobic
component. In
addition to having presentation molecules on the surface, these structures can
be used to load
cargo in the aqueous compartment within the structures, or in the bilayer. The
release of
cargo from the CoPoP-liposomes can be triggered by near infrared (NIR) light.
The cargo can
be released at desired locations ¨ such as by being internalized in targeted
cells or by light
triggered release.
[0061] The cobalt-porphyrin of the monolayers or bilayers is a porphyrin
having a
cobalt (Co) cation conjugated to the porphyrin. The porphyrin can be
conjugated to a
phospholipid (referred to herein as a cobalt porphyrin-phospholipid or cobalt
porphyrin-
phospholipid conjugate).
[0062] The porphyrin portion of the cobalt-porphyrin or cobalt-
porphyrin conjugate
making at least part of some of the bilayer of the liposomes or other
structures comprise
porphyrins, porphyrin derivatives, porphyrin analogs, or combinations thereof.
Exemplary
porphyrins include hematoporphyrin, protoporphyrin, and tetraphenylporphyrin.
Exemplary
porphyrin derivatives include pyropheophorbides, bacteriochlorophylls,
Chlorophyll A,
benzoporphyrin derivatives, tetrahydroxyphenyl chlorins, purpurins,
benzochlorins,
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naphthochlorins, verdins, rhodins, keto chlorins, azachlorins,
bacteriochlorins,
tolyporphyrins, and benzobacteriochlorins. Exemplary porphyrin analogs include
expanded
porphyrin family members (such as texaphyrins, sapphyrins and hexaphyrins) and
porphyrin
isomers (such as porphycenes, inverted porphyrins, phthalocyanines, and
naphthalocyanines).
For example, the cobalt-porphyrin can be a vitamin B12 (cobalamin) or
derivative.
[0063] In one embodiment, the PoP is pyropheophorbide-phospholipid.
The structure
of pyropheophorbide-phospholipid is shown below:
0
II
FI)01\1+
0-
0
1NH N-
[0064] In one embodiment, the layer (monolayer or bilayer) has only CoPoP
which
has his-tagged presentation molecules embedded therein. In this embodiment,
the only
phospholipid in the layer is CoPoP (i.e., CoPoP is 100 mol %). In one
embodiment, the layer
(monolayer or bilayer) has only CoPoP and porphyrin conjugated phospholipids
(PoP),
wherein CoPoP has histidines embedded therein, with the histidines having a
peptide or other
presentation molecules attached thereto. In certain embodiments, there are no
other
phospholipids, but the layer (monolayer or bilayer) may optionally contain
sterols and/or
PEG-lipid.
[0065] In one embodiment, in addition to the CoPoP, the bilayer or
monolayer also
has phospholipids which are not conjugated to porphyrin and therefore, not
coordinated with
Co. Such phospholipids may be referred to herein as "additional
phospholipids". The bilayer
or monolayer may also comprise sterol and PEG-lipid. In one embodiment, the
bilayer or
monolayer consists essentially of, or consists of CoPoP, phospholipids that
are not conjugated
to porphyrins, and optionally sterol and/or PEG, wherein the PEG may be
conjugated to lipid.
In one embodiment, the only metal-PoP in the bilayer is CoPoP, which has his-
tagged
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presentation molecules embedded therein. In one embodiment, the only metal in
the bilayer is
Co.
[0066] In one embodiment, the bilayer of the liposomes comprises
CoPoP and PoP. In
addition to the CoPoP and the PoP, the bilayer can have additional
phospholipids. The bilayer
or monolayer may further comprise sterol and/or PEG. The PEG may be conjugated
to lipid.
In one embodiment, the bilayer consists essentially of, or consists of CoPoP,
PoP, additional
phospholipids, and optionally sterol and/or PEG, wherein the PEG may be
conjugated to
lipid. In one embodiment, the only metal-PoP in the bilayer is CoPoP. In one
embodiment,
the only metal in the bilayer is Co.
[0067] In one embodiment, the CoPoP is present in the nanoparticles from
0.1 to 10
mol % with the remainder 99.9 to 90 mol % being made up by additional lipids,
with the
percent being of the entire bilayer lipids. For example, the combination of
CoPoP can be
present from 0.1 to 10 mol %, sterol can be present from 0.1 to 50 mol %,
optionally,
attenuated lipid A derivatives such as monophosphoryl lipid A or 3-deacylated
monophosphoryl lipid A or a related analog can be present from 0 to 20 mol %
or 0.1 to 20
mol %, and the remainder can be made up by additional phospholipids. The
phospholipids are
DOPC, DSPC, DMPC or combinations thereof, and sterol, if present, can be
cholesterol.
[0068] In one embodiment, the combination of CoPoP and PoP may be
present in the
nanoparticles from 0.1 to 10 mol % with the remaining 99.9 to 90 mol% being
made up by
additional phospholipids. For example, the combination of CoPoP and PoP can be
present
from 0.1 to 10 mol %, sterol can be present from 0 to 50 mol % or 0.1 to 50
mol%, optionally
PEG can be present from 0 to 20 mol % or 0.1 to 20 mol%, and the remainder can
be made
up by phospholipids. The phospholipids can be DOPC, DSPC, DMPC or combinations
thereof and sterol, if present, can be cholesterol.
[0069] As used herein, "phospholipid" is a lipid having a hydrophilic head
group
having a phosphate group connected via a glycerol backbone to a hydrophobic
lipid tail. The
phospholipid comprises an acyl side chain of 6 to 22 carbons, including all
integer number of
carbons and ranges therebetween. In certain embodiments, the phospholipid in
the porphyrin
conjugate is 1-palmitoy1-2-hydroxy-sn-glycero-3-phosphocholine. The
phospholipid of the
porphyrin conjugate may comprise, or consist essentially of
phosphatidylcholine,
phosphatidylethanoloamine, phosphatidylserine and/or phosphatidylinositol.
[0070] In certain embodiments, the porphyrin is conjugated to the
glycerol group on
the phospholipid by a carbon chain linker of 1 to 20 carbons, including all
integer number of
carbons therebetween.
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[0071] In various embodiments, in addition to the porphyrin
conjugates disclosed
herein, the bilayer of the liposomes also comprises other phospholipids. The
fatty acid chains
of these phospholipids may contain a suitable number of carbon atoms to form a
bilayer. For
example, the fatty acid chain may contain 12, 14, 16, 18 or 20 carbon atoms.
In different
embodiments the bilayer comprises phosphatidylcholine,
phosphatidylethanoloamine,
phosphatidylserine and/or phosphatidylinositol.
[0072] The present bilayers and monolayers may also comprise sterols.
The sterols
may be animal sterols or plant sterols. Examples of sterols include
cholesterol, sitosterol,
stigmasterol, and cholesterol. In embodiments, cholesterol may be from 0 mol %
to 50 mol
%, or 0.1 to 50 mol %. In other embodiments, cholesterol may be present from 1
to 50 mol%,
5 to 45 mol%, 10 to 30 mol%.
[0073] In certain embodiments, the bilayer or monolayer further
comprises PEG-
lipid. The PEG-lipid can be DSPE-PEG such as DSPE-PEG-2000, DSPE-PEG-5000 or
other
sizes of DSPE-PEG. The PEG-lipid is present in an amount of 0 to 20 mol %
including all
percentage amounts therebetween to the tenth decimal point. The average
molecular weight
of the PEG moiety can be between 500 and 5000 Daltons and all integer values
and ranges
therebetween.
[0074] In certain embodiments, the bilayer or monolayer further
comprises an
adjuvant such as attenuated lipid A derivatives such as monophosphoryl lipid A
or 3-
deacylated monophosphoryl lipid A.
[0075] The histidine tag (his-tag) may carry a variety of
presentation molecules of
interest for various applications. At least one or both ends of the his-tag
can reside close to
the outer surface of the liposome. In one embodiment, at least one end of the
polyhistidine tag
is covalently attached to a presentation molecule. In one embodiment, the his-
tag is a string
of at least 2 histidines. In one embodiment, the his-tag is a string of 2-20
histidines. In one
embodiment, the his-tag is a string of from 4-12 histidines and all integer
numbers
therebetween. In one embodiment, it is from 6-10 histidines. In one
embodiment, it is 6, 7, 8,
9 or 10 histidines. In one embodiment, one end of the his-tag is free and a
peptide or other
molecule is attached to the other end. It is considered that at least a part
of the his-tag is
located within the bilayer such that it is coordinated to the cobalt metal
core.
[0076] The liposomes of the present disclosure (without the his-
tagged molecules)
can be substantially spherical and have a size (e.g., a longest dimension such
as, for example,
a diameter) of 30 nm to 250 nm, including all integers to the nm and ranges
therebetween. In
one embodiment, the size of the liposomes is from 100-175 nm. In one
embodiment, at least
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50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or 100% of the liposomes in the
composition
have a size of from 30 to 250 nm or from 100 to 175 nm. The liposomes or
nanostructures
can be more than 200 nm. In one embodiment, the nanostructures are more than
1000 nm. In
one embodiment, the nanostructures are from 200 to 1000 nm. The liposomes or
nanostructures may be spherical or non-spherical. In one embodiment, the
largest dimensions
of the nanostructure are less than 200 nm, while preserving the nanostructure
width of the
monolayer or bilayer. In one embodiment, the size of the nanostructure exceed
200 nm in
some dimensions, while preserving the nanostructure width of the monolayer or
bilayer. In
one embodiment, the size of the nanostructure exceed 1000 nm in some
dimensions, while
preserving the nanostructure width of the monolayer or bilayer.
[0077] In one aspect, the disclosure provides a composition
comprising liposomes or
other structures of the present disclosure or a mixture of different liposomes
or other
structures. The compositions can also comprise a sterile, suitable carrier for
administration to
individuals including humans, such as, for example, a physiological buffer
such as sucrose,
dextrose, saline, pH buffering (such as from pH 5 to 9, from pH 7 to 8, from
pH 7.2 to 7.6,
(e.g., 7.4)) element such as histidine, citrate, or phosphate. In one
embodiment, the
composition comprises at least 0.1% (w/v) CoPoP liposomes or his-tagged-CoPoP
liposomes
or other structures. In various embodiments, the composition comprises from
0.1 to 100
mol% CoPoP liposomes or his-tagged CoPoP liposomes or other structures such as
bilayer
coated nanoparticles. In one embodiment, the composition comprises from 0.1 to
99 mol%
CoPoP liposomes having his-tagged presentation molecules associated therewith.
[0078] In one embodiment, the compositions of the present disclosure
are free of
maleimide or succinimidyl ester reactive groups. In one embodiment, the tagged
molecule to
be attached to the membrane does not have a non-natural amino acid.
[0079] The presentation molecule bearing the his-tag may be a small
molecule or a
macromolecule. In one embodiment, the molecule is a peptide or a peptide
derivative. In one
embodiment, the molecule is a polypeptide, polynucleotide, carbohydrate or
polymer. The
his-tag may be chemically conjugated to the molecule of interest. The his-tag
may be
incorporated into the primary amino acid sequence of a polypeptide. In one
embodiment, the
molecule is an antigen, such as a peptide (2-50 amino acids and all peptides
of amino acid
lengths between 2 and 50) or a polypeptide (50 -1,000 amino acids and all
polypeptides of
amino acid lengths between 50 -1,000) or a protein (larger than 1,000 amino
acids). The
peptide, polypeptide or protein can have only naturally occurring amino acids,
or can be a
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mixture of naturally occurring and non-naturally occurring amino acids, or can
have only
non-naturally occurring amino acids.
[0080] The presentation molecules attached to the his-tag may be
antigenic
molecules, targeting molecules, therapeutic molecules, diagnostic molecules or
molecules
providing any other type of functionality. The tagged molecules may be used
for targeting
i.e., to guide the structures bearing the monolayers or bilayers to its
targeted locations. For
example, a peptide ligand can be attached to the his-tag such that the ligand
guides liposomes
(or other structures) to cells that have receptors or recognition molecules
for the ligands. In
one embodiment, the attached peptide could provide alternative or additional
functionality ¨
such as, for example, the attached peptide could provide therapeutic,
diagnostic, or
immunogenic functionality.
[0081] In specific embodiments, the presentation molecule may be a
targeting
molecule such as an antibody, peptide, aptamer or other molecules such as
folic acid. The
term "targeting molecule" is used to refer to any molecule that can direct the
bilayer bearing
structure such as liposome, to a particular target, for example, by binding to
a receptor or
other molecule on the surface of a targeted cell. Targeting molecules may be
proteins,
peptides, nucleic acid molecules, saccharides or polysaccharides, receptor
ligands or other
small molecules. The degree of specificity can be modulated through the
selection of the
targeting molecule. For example, antibodies typically exhibit high
specificity. These can be
polyclonal, monoclonal, fragments, recombinant, single chain, or nanobodies,
many of which
are commercially available or readily obtained using standard techniques.
[0082] The presentation molecule can be an antigenic molecule ¨ i.e.,
a molecule
bearing antigenic epitopes. In one embodiment, the molecule is a peptide. In
one
embodiment, the peptide is a RGD bearing peptide sequence. Such sequences may
be 7-20
amino acids or longer bearing an epitope. The peptide may be a fragment of, or
may comprise
an epitope of a polypeptide or protein that is part of a microorganism, such
as a pathogenic
microorganism (e.g., virus, bacteria, parasites, or fungi). Examples include
respiratory
syncytial virus, Borrelia burgdorferi (referred to herein as Lyme borreliae),
influenza
viruses, and the like. The peptide may be a fragment of a popypeptide or
protein that is
generally not immunogenic, such as, for example, a viral protein that is not
known to be
practically immunogenic. The peptide may be fragment of, or may comprise an
epitope of, a
HIV antigen, such as an HIV outer envelope protein. In one embodiment, the HIV
antigen is
gp41. For example, the peptide can be membrane proximal external-region (MPER)
of the
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gp41 envelope. Additional examples of antigens include, but are not limited
to, DS-Cavl for
RSV, OspA for Lyme disease, and hemagglutinin and neuraminidase for influenza.
[0083] In one embodiment, the present disclosure provides antigenic
compositions.
The compositions comprise bilayer bearing structures in which an antigen
having a histidine
tail is non-covalently conjugated to the cobalt porphyrin (or cobalt porphyrin
phospholipid)
such that the his-tag is embedded in the bilayer and one or more epitopes of
the antigen are
exposed on the surface. The compositions may comprise adjuvants and other
carriers known
in the art. Examples of adjuvants include complete Freund's adjuvant,
incomplete Freund's
adjuvant, monophosphoryl lipid A (MPL), aluminum phosphate, aluminum
hydroxide, alum,
phosphorylated hexaacyl disaccharide (PHAD), Sigma adjuvant sytem (SAS),
AddaVax
(Invitrogen), or saponin. Other carriers like wetting agents, emulsifiers,
fillers etc. may also
be used.
[0084] A wide variety of cargo may be loaded into the liposomes or
other structures
of the present disclosure. The cargo can be delivered to desired locations
using near infrared
light. For example, bioactive or therapeutic agents, pharmaceutical
substances, or drugs can
be encapsulated within the interior of the CoPoP liposome. This includes water-
soluble drugs
and also drugs that are weak acids or bases that can be loaded via chemical
gradients and
concentrated in the aqueous core of the liposome. Thus, in various
embodiments, the
liposome comprises an active agent encapsulated therein, such as a therapeutic
agent and/or a
diagnostic agent, which can be a chemotherapy agent such as doxorubicin. The
chemotherapeutic agent doxorubicin could be actively loaded and released with
NIR
irradiation providing for robust and direct light-triggered release using
CoPoP liposomes.
[0085] In one embodiment, the ratio of lipid to drug (or any other
cargo agent) is from
10:1 to 5:1. In various embodiments, the ratio of lipid to drug/cargo ratio is
10:1, 9:1, 8:1,
.. 7:1, 6:1, or 5:1. The lipid used for calculating the ratios includes all
the lipid including
phospholipid that is part of the porphyrin phospholipid conjugate, additional
phospholipids,
or sterol, and lipid conjugated to PEG, if present. Although at times, cargo
is described as a
drug in the disclosure, the description is equally applicable to any agent
contained for
treatment and/or delivery to a desired location, and the term "drug" is
intended to refer to any
agent. The agent may be contained, in whole or in part, within on in the PoP-
liposomes-
whether present in the aqueous compartment, the bilayer or both.
[0086] In one embodiment, the cargo loaded within the liposome or
other carriers is a
therapeutic agent. The term "therapeutic agent" is art-recognized and refers
to any chemical
moiety that is a biologically, physiologically, or pharmacologically active
substance.
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Examples of therapeutic agents, also referred to as "drugs", are described in
well-known
literature references such as the Merck Index, the Physicians Desk Reference,
and The
Pharmacological Basis of Therapeutics, and they include, without limitation,
medicaments;
vitamins; mineral supplements; substances used for the treatment, prevention,
diagnosis, cure
or mitigation of a disease or illness; substances which affect the structure
or function of the
body; or pro-drugs, which become biologically active or more active after they
have been
placed in a physiological environment. Various forms of a therapeutic agent
may be used
which are capable of being released from the subject composition into adjacent
tissues or
fluids upon administration to a subject. Drugs that are known be loaded via
active gradients
include doxorubicin, daunorubicin, gemcitabine, epirubicin, topotecan,
vincristine,
mitoxantrone, ciprofloxacin and cisplatin. Therapeutic cargo also includes
various antibiotics
(such as gentamicin) or other agents effective against infections caused by
bacteria, fungi,
parasites, or other organisms. These drugs can be loaded and released in CoPoP
liposomes.
[0087] In one embodiment, the cargo loaded in the liposome is a
diagnostic agent. A
"diagnostic" or "diagnostic agent" is any chemical moiety that may be used for
diagnosis. For
example, diagnostic agents include imaging agents, such as, for example, those
containing
radioisotopes such as indium or technetium; contrasting agents containing
iodine or
gadolinium; enzymes such as, for example, horse radish peroxidase, GFP,
alkaline
phosphatase, or beta.-galactosidase; fluorescent substances such as, for
example, europium
derivatives; luminescent substances such as, for example, N-methylacrydium
derivatives or
the like.
[0088] The cargo may comprise more than one agent. For example, cargo
may
comprise a combinations of diagnostic, therapeutic, immunogenic, and/or
imaging agents,
and/or any other type of agents. The same agent can have multiple
functionalities. For
example, an agent can be diagnostic and therapeutic, or an agent can be
imaging and
immunogenic and the like.
[0089] The structures formed by the layers of the present disclosure
are serum stable.
For example, in vitro, the his-tag binding stability to the CoPoP bilayers is
stable when
incubated in 50% bovine serum at room temperature for 24 hours. Thus, these
structures can
be stable under serum or concentrated or diluted serum conditions.
[0090] The present disclosure also provides methods for using
structures bearing the
bilayers as described herein. In one embodiment, this disclosure provides a
method of
eliciting an immune response in a host. The immune response may generate
antibodies. The
method comprises administering to an individual a composition comprising a
structure
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bearing Co PoP bilayers to which is conjugated a histidine tagged antigen. The
compositions
may be administered by any standard route of immunication including
subcutaneous,
intradermal, intramuscular, intratumoral, or any other route. The compositions
may be
administered in a single administration or may be administered in multiple
administrations
including booster shots. Antibody titres can be measured to monitor the immune
response.
[0091] The present nanostructures can be used for reducing antibody
titer against
desired antigens. For example, if immunogenicity is desired to be reduced,
nanostructures in
which PS (or other) containing phospholipids are present can be used.
Compositions
comprising these nanostructures can be administered for reducing
immunogenicity.
[0092] In one aspect, the disclosure provides a method of delivery of
agents contained
as cargo in the liposomes or other nanostructures to desired locations. The
agent may be
contained, in whole or in part, within or in the CoPoP liposomes ¨ whether
present in the
aqueous compartment, the bilayer or both. The method comprises 1) providing a
composition comprising liposomes or other structures bearing the bilayers of
the present
disclosure optionally comprising cargo (such as an active agent); 2) allowing
the liposomes to
reach a selected or desired destination; 3) irradiating the liposome with
radiation having a
wavelength of near-infrared under conditions such that at least a portion of
the cargo is
released from the liposome. The cargo can alternatively, or additionally reach
the interior of
the cell by the liposomes being internalized and then releasing the cargo upon
action of
intracellular processes.
[0093] The liposomes may be irradiated with near-infrared light from
a laser of power
50 to 1000 mW/cm2, including all integer values to the mW/cm2 and ranges
therebetween, at
a wavelength of from 650 to 1000 nm, including all integer values to the nm
and ranges
therebetween. In another embodiment, the wavelength is from 650 to 800 nm,
including all
integer values to the nm and all ranges therebetween. The liposomes may be
irradiated for up
to 30 minutes or less. In various embodiments, the liposomes in vitro or in
vivo may be
irradiated from 0.5 to 30 minutes and all values to the tenth decimal place
therebetween. In
one embodiment, the liposomes are irradiated with a 658 nm laser diode for up
to 10 minutes.
In other embodiments, the liposomes are irradiated with wavelengths of 665 or
671 nm. The
infrared radiation can be delivered to the desired area directly by shining
laser light on the
area or fiber optic probes may be used. In the case of a tumor, the fiber
optic probe can be
inserted into the tumor (i.e., via a catheter or endoscopic device) to provide
irradiation to a
localized area.
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[0094] In one aspect, the disclosure provides a method of preparing
bilayers
comprising CoPoPs. Freebase PoP can be produced by esterifying a monocarboxlic
acid
porphyrin such as pyropheophorbide-a with 2-palmitoy1-2-hydroxy-sn-glycero-3-
phosphocholine (lyso-C16-PC), Avanti #855675P) using 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine in chloroform at
a 1:1:2:2
lyso-C16-PC:Pyro:EDC:DMAP molar ratio by stirring overnight at room
temperature. The
PoP is then purified by silica gel chromatography. CoPoP can be generated by
contacting
porphyrin-phospholipid conjugate with a molar excess (e.g., 10-fold molar
excess) of a cobalt
salt (e.g., cobalt (II) acetate tetrahydrate) in a solvent (e.g., methanol) in
the dark.
[0095] In one embodiment, this disclosure provides a method for coating a
nanoparticle with a cobalt-porphyrin (e.g., CoPoP) bilayer or monolayer. The
method
generally comprises hydrating nanoparticles with a lipid solution in order to
disperse the
particles in water.
[0096] For delivery of cargo to desired locations or for general
administration, the
composition comprising the liposomes in a suitable carrier can be administered
to individuals
by any suitable route. In one embodiment, it is administered by intravenous
infusion such
that it will enter the vasculature (circulatory system). The composition may
be administered
systemically or may be administered directly into the blood supply for a
particular organ or
tissue or tumor. When irradiated by NIR, the contents of the PoP liposomes may
be released
within the circulatory system and may then enter the surrounding tissue.
[0097] In the following Statements, various examples of
nanostructures,
compositions, and methods of the present disclosure are described:
1. A nanostructure (e.g., a liposome) comprising: a) a monolayer or bilayer,
wherein the
monolayer or bilayer comprises: i) optionally, phospholipid, and ii) porphyrin
having cobalt
coordinated thereto forming cobalt-porphyrin; and b) optionally, a
polyhistidine-tagged
presentation molecule, where at least a portion of the polyhistidine tag
resides in the
hydrophobic portion of the monolayer or the bilayer or monolayer and one or
more histidines
of the polyhistidine tag are coordinated to the cobalt in the cobalt-
porphyrin, where at least a
portion of the polyhistidine-tagged presentation molecule is exposed to the
outside of the
nanostructure (e.g., liposome), and where, in the case of liposomes, the
liposome encloses an
aqueous compartment.
2. A nanostructure (e.g., liposome) of Statement 1, where the cobalt porphyrin
is conjugated
to a phospholipid to form a cobalt porphyrin-phospholipid conjugate.
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3. A nanostructure (e.g., liposome) of Statement 2, where the cobalt porphyrin-
phospholipid
conjugate makes up from 1 to 25 mol % of the monolayer or the bilayer.
4. A nanostructure (e.g., liposome) of Statement 3, where the cobalt porphyrin-
phospholipid
conjugate makes up from 5 to 10 mol % of the monolayer or bilayer.
5. A nanostructure (e.g., liposome) of any one of Statements 1 to 4, where the
bilayer further
comprises a sterol (e.g., cholesterol).
6. A nanostructure (e.g., liposome) of any one of Statements 1 to 4, where the
bilayer further
comprises phosphatidylserine and, optionally, cholesterol.
7. A nanostructure (e.g., liposome) of any one of Statements 1 to 4, where the
polyhistidine-
tag comprises 6 to 10 histidine residues.
8. A nanostructure (e.g., liposome) of any one of Statements 1 to 4, where
size of the
liposome is 50 nm to 200 nm.
9. A nanostructure (e.g., liposome) of any one of Statements 1 to 4, where the
nanostructure
(e.g., liposome) comprises a cargo and, in the case of liposomes, at least a
portion of the
cargo resides in the aqueous compartment of the liposome.
10. A nanostructure (e.g., liposome) of any one of the preceding Statements,
where the
presentation molecule is a peptide of from 4 to 50 amino acids, said number of
amino acids
not including the histidines of the his-tag.
11. A nanostructure (e.g., liposome) of any one of the preceding Statements,
wherein the
presentation molecule is a protein from 4 to 500 kDa.
12. A nanostructure (e.g., liposome) of any one of the preceding Statements,
where the
presentation molecule is an antigenic molecule and the monolayer or the
bilayer further
comprises an adjuvant incorporated therein.
13. A nanostructure (e.g., liposome) of Statement 12, where the adjuvant is
attenuated lipid
A derivative.
14. A nanostructure (e.g., liposome) of Statement 13, where the attenuated
lipid A derivative
is monophosphoryl lipid A or 3-deacylated monophosphoryl lipid A.
is. A nanostructure comprising: a) a core; and b) a monolayer or a bilayer on
said core,
wherein the monolayer or bilayer comprises: i) optionally, phospholipid
monomers, and ii)
.. porphyrin having cobalt coordinated thereto forming cobalt-porphyrin (e.g.,
CoPoP); and c)
optionally, a polyhistidine-tagged presentation molecule, where at least a
portion of the
polyhistidine tag resides in the hydrophobic portion of the monolayer or the
bilayer, one or
more histidines of the polyhistidine tag are coordinated to the cobalt in the
cobalt-porphyrin,
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and at least a portion of the polyhistidine-tagged presentation molecule is
exposed on the
outside of the nanostructure.
16. A nanostructure of Statement 15, where the core is a gold nanoparticle.
17. A method of targeted delivery of a cargo comprising: a) administering to
an individual a
composition comprising nanostructures (e.g., liposomes) of any one of
Statements 9 to 16 or
a combination of nanostructures (e.g., liposomes) of any one of Statements 9
to 16 in a
pharmaceutical carrier; and b) after a suitable period of time to allow the
nanostructures (e.g.,
liposomes) to reach a desired location in the individual, exposing the
liposomes to near
infrared radiation of a wavelength from 650 to 1000 nm to effect release of
the cargo from
the liposomes.
18. A method of Statement 17, where the individual is a human or non-human
animal.
19. A method for generating an immune response in a host individual comprising
administering to the individual a composition comprising nanostructures (e.g.,
liposomes) of
any one of Statements 1 to 16 or a combination nanostructures (e.g.,
liposomes) of any one of
Statements 1 to 16 of in a pharmaceutical carrier, where the presentation
molecule comprises
an immunogenic epitope.
20. A method of Statement 19, where the presentation molecule is a peptide,
polypeptide or
protein derived from a pathogenic microorganism.
21. A method of any one of Statements 19 or 20, where the individual is a
human or non-
human animal.
[0098] The following examples are presented to illustrate the present
disclosure. They
are not intended to limiting in any manner.
EXAMPLE 1
[0099] This example describes the synthesis and functionalization of
cobalt
porphyrin-phospholipid (CoPoP) bilayers with histidine-tagged ligands and
antigens.
[0100] Materials and Methods. Materials were obtained from Sigma
unless otherwise
noted. Peptides were obtained from commercial vendors that determined purity
by HPLC and
confirmed identity by mass spectrometry:
[0101] Table 1. Properties of peptides
Name Sequence Expecte Observed Purity
Source
d mass mass
RGD-His 5-FAM-GRGDSPKGAGAKG-HHHHHHH 2475.52 2475.60 99.1% GenScript
(SEQ ID NO:1)
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Free RGD GRGDSPK (SEQ ID NO:2) 715.76 715.8 99.3%
GenScript
RGD-palm 5-FAM- 1882.15 N.D.
96.7% GenScript
GRGDSPKGAGAKG(lys(palmiticacid) (SEQ
ID NO:3)
cRGD-His Cyclo(RGDY(D-)K(-Suc-PRG12- 2280.5 2280.1 94.7% Anaspec
HHHHHHH)) (SEQ ID NO:4)
0-His 5-FAM-KKGGGG (SEQ ID NO:5) 860.9 861.68 95.3%
Biomatik
2-His 5-FAM-KKGGGGHH(SEQ ID NO:6) 1135.18 1135.63 96.8%
Biomatik
4-His 5-FAM-KKGGGGHHHH (SEQ ID NO:7) 1409.46 1409.20 93.6%
Biomatik
6-His 5-FAM-KKGGGGHHHHHH (SEQ ID NO:8) 1683.75 1683.40 92.8%
Biomatik
8-His 5-FAM-KKGGGGHHHHHHHH (SEQ ID 1958.03 1957.30 92.9%
Biomatik
NO:9)
10-His 5-FAM-KKGGGGHHHHHHHHHH (SEQ 2232.32 2231.64 90.9%
Biomatik
ID NO:10)
MPER-His NEQELLELDKWASLWNGGKGG-
3304.52 3304.75 93.6% GenScript
HHHHHHH (SEQ ID NO:11)
MPER- NEQELLELDKWASLWNGGK-Biotin (SEQ 2584.91 2285.55
90.1% GenScript
Biotin ID NO:12)
[0102] For protein binding, the recombinant heptahistidine-tagged
cerulean-venus
fusion reporter protein was produced in Escherichia coil and was purified and
characterized
as previously described. Stoichiometry approximations were based on the
assumption that
each -100 nm liposome contains 80,000 lipids.
[0103] Generation of PoP-lipid, PoP-liposomes and PoP-gold. Freebase
(2H) PoP
sn-l-palmitoyl sn-2-pyropheophorbide phosphtatidylcholine was synthesized as
previously
described. CoPoP was generated by stirring 100 mg 2H-PoP with 10 fold molar
excess of
cobalt (II) acetate tetrahydrate in 4 mL methanol for 17 hours in the dark.
Reaction
.. completion and product purity was monitored by TLC (>90% purity). The
solvent was then
removed by rotary evaporation and PoP was extracted with
chloroform:methanol:water
(1:1.8:1) 3 times. The chloroform layer was collected, the solvent was removed
by rotary
evaporation and the product was freeze-dried in 20% water in tert-butanol to
give 81.5 mg
(77 % yield) (Identity was confirmed with mass spectrometry). Other metallo-
PoPs were
synthesized using the same method. For Ni-PoP, Ni (II) acetate tetrandrate was
used and
incubated for 17 hours. For Zn-PoP, Zn (II) acetate dehydrate was used and
incubated for 17
hours. For Mn-PoP, Mn (II) acetate was used and incubated for 30 hours. For Cu-
PoP, Cu (II)
acetate was used and incubated in tetrahydrofuran for 3 hours.
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[0104] PoP-liposomes were formulated at a 1 mg scale. After
dissolving lipids in
chloroform in a test tube, the solvent was evaporated and the film was further
dried under
vacuum overnight. Lipids were rehydrated with 1 mL of phosphate buffered
saline (PBS),
sonicated, subjected to 10 freeze-thaw cycles and then extruded through 100 nm
polycarbonate membranes (VWR # 28157-790) with a handheld extruder (Avanti #
610000).
For protein and peptide binding analysis, liposomes were formed with 10 mol %
PoP along
with 85 mol % DOPC (Avanti # 850375P), and 5 mol % PEG-lipid (Avanti #
880120P). Ni-
NTA liposomes included 10 molar % Ni-NTA lipid dioleoyl-glycero-Ni-NTA (Avanti
#
790404P) as well as 10 molar % 2H-PoP. Liposomes incorporating free Co-
porphyrin
included 10 molar % Co-pyropheophorbide with 85 mol % DOPC and 5 mol % PEG-
lipid.
Co-NTA-liposome was prepared using liposomes containing 10 mol % dioleoyl-
gycero-NTA
(Avanti # 790528P). Liposomes were incubated with 20 mg/mL cobalt (II)
chloride for 2
hours and then dialized in PBS. Sulforhodamine B loading liposomes contained
10 mol %
PoP, 35 mol % cholesterol (Avanti # 700000P), 55 mol % DOPC and PEG-lipid as
indicated.
A solution of sulforhodamine B (VWR # 89139-502) was used to hydrate the lipid
film,
which was then freeze-thawed then sonicated. Unentrapped dye was removed with
a 10 mL
Sephadex G-75 (VWR # 95016-784) column followed by dialysis in PBS. For
bilayer
integrity and quantitative cell binding studies, 50 mM dye was used, whereas
microscopy
studies used 10 mM dye.
[0105] For gold coating, 60 nm citrate-stabilized gold nanospheres (Ted
Pella #
15709-20) were used to hydrate a 1 mg lipid film composed of 45 mol %
distearoyl
phosphocholine (Avanti # 850365P), 45 mol % distearoyl phosphoglycerol (Avanti
#
840465X) and 10 mol % PoP. Following brief vortexing and sonication, the
samples were
repeatedly centrifuged at 1500 relative centrifugal force (rcf) for 15 min.
The supernatant was
discarded and the pellet was resuspended and re-centrifuged 2 more times. PoP
gold was
resuspended in water for further analysis.
[0106] Polypeptide binding. 11.tg of fluorescent reporter protein was
incubated with
201.tg of liposomes in 200 [IL PBS in a 96 well plate. Fluorescence in the
FRET channel (ex:
430 nm, em: 525 nm) was measured periodically with a fluorescence microplate
reader
(Tecan Infinite II). Data were normalized to the FRET signal in the protein
without addition
of liposomes. EMSA experiments were performed with 2.51.tg protein incubated
with 501.tg
liposomes followed by electrophoresis in a 0.75% agarose gel with 50 V applied
for 90
minutes and imaging with an IVIS Lumina II system with the indicated
excitation and
emission filters. For serum stability test, 3mg protein was pre-incubated with
60mg liposome
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in 40u1 PBS. After 24 incubation, 40u1 FBS was added and incubated for another
8h (hours).
For imidazole displacement experiments, 11.ig of reporter protein was bound to
201.ig
liposomes in PBS. Imidazole was then titrated and binding was assessed with
fluorescence.
For serum stability, 11.ig of reporter protein was bound to 201.ig liposomes
in 100 [IL PBS
and then an equal volume of fetal bovine serum (VWR # 82013-602) was added and
binding
was monitored with fluorescence. Peptide binding was assessed with RGD-His FAM
fluorophore quenching following incubation of 500 ng peptide with 201.ig
liposomes.
[0107] Targeting experiments. U-87 and MCF-7 cell lines were obtained
from ATCC
and cultured according to vendor protocol. 2x104 cells were seeded overnight
in 96-well-plate
wells. 500 ng RGD-His peptide was bound with 201.ig of sulforhodamine B loaded
liposomes
and liposomes were incubated with cells for 2 h. Media was removed, cells were
washed with
PBS 3 times and then cells and liposomes were lysed with a 1 % Triton X-100
solution.
Liposomal uptake was assessed by measuring the fluorescence of sulforhodamine
B.
[0108] For confocal imaging, 104 cells were seeded overnight in a
Nunc chamber
slide (Nunc # 155411) in DMEM with 10% fetal bovine serum (FBS). 201.ig of
liposomes
were added to the serum containing media and incubated for 2h. Media was
removed and the
cells were washed with PBS 3 times. Fresh media was added and cells were
imaged with
microscopy using a Zeiss LSM 710 confocal fluorescence microscope. Gold
imaging was
carried out in the same way but 633 nm light was used for both excitation and
emission for
back scatter imaging. After peptide binding, gold was centrifuged to remove
any unbound
RGD peptide.
[0109] For in vivo experiments, animal procedures were conducted in
accordance
with the policies and approval of the University at Buffalo Institutional
Animal Care and Use
Committee (IACUC). 5-week old female athymic nude mice (Jackson Labs) were
inoculated
on the flank with U87 cells and mice were treated when tumor growth reached 4-
5 mm
diameter. Mice were intravenously injected with 200 !IL of sulforhodamine B-
loaded
liposomes (1 mg/mL llipid) targeted with or without cRGD-his. 45 minutes after
injection
mice were sacrificed, organs were extracted, weighed, mechanically homogenized
in a 0.2 %
Triton X-100 solution and fluorescence was assessed to determine
biodistribution.
[0110] Vaccinations. Unless otherwise indicated, 8-week-old female BALB/c
mice
(Harlan Laboratories) received hind ventral footpad injections on days 0 and
14 containing 25
1.ig of MPER peptide in 50 [EL of sterile PBS. Where indicated, injections
also included 251.ig
MPL (Avanti # 699800P) or TDB (Avanti # 890808P) in liposomes comprising
DOPC:Cholesterol:MPL:PoP at a molar ratio of 50:30:5:5. For Freund's adjuvant,
the peptide
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was mixed directly in Fruend's complete adjuvant (Fisher # PI-77140) and
injected. 4 weeks
following the first injection, or as indicated, blood was collected from the
submandibular vein
and serum was obtained following blood clotting and centrifugation at 2000 rcf
for 15 min
and stored at -80 C.
[0111] Anti-MPER titer was assessed by ELISA in 96-well streptavidin-coated
plates
(GBiosciences #130804). 11.ig of His-tag-free MPER-biotin in 100 [IL of PBS
containing
0.1% Tween 20 (PBS-T) was incubated in the wells for 2 h at 37 C. Wells were
then washed
5 times with PBS-T and mouse sera was serially diluted in PBS containing 0.1%
casein
(PBS-C) and incubated for 30 min at 37 C. Wells were washed 5 times with PBS-
T then 100
[IL of goat anti-mouse IgG-HRP (GenScript # A00160) diluted in PBS-C was added
to the
wells to provide a final concentration of containing 1 pg/mL secondary
antibody and
incubated for 30 min at 37 C. The wells were washed 5 times with PBS-T then
100pL
tetramethylbenzidine substrate solution (Amresco # J644) was added to each
well and
incubated for 20 min at 37 C. The reaction was stopped by 100 [IL 1M HC1 and
absorption
was measured at 450 nm. Titers were defined as the reciprocal dilution at
which the
absorbance at 450 nm exceeded the identical dilution of non-serum background
by greater
than 0.05 absorbance units. Every sample was averaged from duplicate
measurements.
[0112] Viral entry experiments. Viral entry experiments were carried
out as
previously described. In short, HIV-1 was produced by co-transfection of pHXB2-
env and
pNL4-3.HSA.R-E- in 293T cells. 2 days post-transfection, the cell media was
passed through
a 0.451.tm filter and centrifuged. The viral pellet was dried, re-suspended in
600 [IL of PBS
and stored at ¨80 C. The infectious titer of HIV-1 stock was determined by X-
Gal staining
as previously described.
[0113] Sera from 3 mice immunized with MPER and CoPoP liposomes was
pooled
and IgG was isolated using immobilized Protein G beads (VWR # P1203 98)
according to
vendor protocol. Concentration was determined with absorption with the
Bradford assay. 2F5
was obtained from the free NIH AIDS reagent program. lx104 TZM-bl receptor
cells per well
were plated to a 96-well plate the day before infection. HIV (multiplicity of
infection of 0.1)
was incubated with antibodies for 30 min at 37 C, added to the cells and
spinoculated at
1000 rcf for 1 h at 25 C followed by further incubation for 2 days at 37'C in
a 5% CO2
incubator. Cell viability was then measured using a CellTiter-Fluor Assay
(Promega)
according to manufacturer protocol. Viral entry level was then measured by a
luciferase assay
system (ONE-Glo, Promega) according to manufacturer protocol and was
normalized to the
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virus only sample. Data were further normalized to cellular viability (all
groups exhibited
viability within 10% of the control untreated cells).
[0114] Results
[0115] His-tagged protein binding to CoPoP liposomes. A series of sn-
l-palmitoyl sn-
.. 2-pyropheophorbide phosphtatidylcholine chelates was generated with the
transition metals
Co, Cu, Zn, Ni and Mn (Fig. lc). PoP bilayers were then formed with 10 molar %
metallo-
PoP along with 85 molar % dioleoylphosphocholine (DOPC) and 5 molar %
polyethylene
glycol-conjugated distearoylphosphoethanolamine (PEG-lipid) via extrusion into
100 nm
liposomes. His-tagged protein binding to PoP bilayers was assessed with a
fluorescent protein
.. reporter. As shown in Fig. 2a, the system comprised a fusion protein made
up of two linked
fluorescent proteins; Cerulean (blue emission) and Venus (green emission). Due
to their
linked proximity and spectral overlap, Cerulean serves as a Forster resonance
energy transfer
(FRET) donor for Venus, so that Cerulean excitation results in FRET emission
from Venus.
Cerulean was tagged at its C-terminus with a heptahistidine tag. However, if
bound to a PoP
.. bilayer, energy transfer from Cerulean is diverted to the bilayer itself,
which is absorbing in
the Cerulean emission range and thus competes with FRET to Venus. On the other
hand,
because Venus is not directly attached to the photonic bilayer, it is not
completely quenched
upon direct excitation, which enables tracking of the bound fusion protein.
[0116] A 3-color electrophoretic mobility shift assay (EMSA) was
developed to
.. assess reporter fusion protein binding to various PoP liposomes. 2.5 [tg
protein was incubated
with the 50 [tg of various PoP liposomes for 24 hours and then subjected to
agarose gel
electrophoresis. As shown in the top image in Fig. 2b, when the PoP-liposomes
were imaged
only the free base (2H) liposomes were readily visualized, along with the Zn-
PoP liposomes
to a lesser degree. This demonstrates that the metals have a quenching effect
on the PoP and
.. confirms they were stably chelated in the bilayer. As expected, the
liposomes exhibited
minimal electrophoretic mobility due to their relatively large size. Next, the
same gel was
imaged using Cerulean excitation and Venus emission to probe for inhibition of
FRET, which
would be indicative of the fusion protein binding to PoP liposomes. All the
samples exhibited
the same amount of FRET and migrated the same distance as the free protein
with the
.. exception of the protein incubated with CoPoP liposomes, in which case FRET
disappeared
completely (middle image). To verify the presence of the protein, Venus was
directly excited
and imaged. Only with the CoPoP liposomes was the reported protein co-
localized with the
liposomes. Together, these images demonstrate that the protein bound
quantitatively to
CoPoP liposomes. Solution-based studies confirmed this finding (Fig. 2c). Of
all the types of
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PoP liposomes examined, only the CoPoP ones induced a dramatic decrease in the
FRET
efficiency between Cerulean and Venus, due to liposomal binding. The binding
required
approximately a day to fully complete, although the time to achieve 50%
binding (the tv2)
was just 3 hours. It was shown by molecular dynamics simulations of a 2H-PoP
bilayer that
the center of the porphyrins (where metal chelation would occur) are
inaccessible to the
aqueous phase surrounding the bilayer. Thus, this slow binding can be
attributed to a His-tag
that is partially obscured by the rest of the protein as well as having to
making its way into
the sheltered hydrophobic bilayer.
[0117] Polyhistidine Coordination with CoPoP. The mechanism
underlying His-tag
binding to immobilized metals involves metal coordination with the nitrogenous
imidazole
groups of histidine residues. The absence of His-tag binding to liposomes
formed with Ni(II),
Cu(II), Zn(II) and Mn(II) PoP likely relates to axial ligand binding affinity
or the
coordination number within the porphyrin. For instance, it has been proposed
that Ni(II) and
Cu(II) porphyrin chelates can coordinate completely with the 4 surrounding
macrocyclic
nitrogens atoms without axial ligands. For the Zn (II) and Mn (III)
porphyrins, the ligand
binding strength is likely insufficient to confer stable polyhistidine
binding.
[0118] To determine the electronic state of the CoPoP, paramagnetism
was assessed.
Because Co(II) is paramagnetic, but Co (III) porphyrins are low-spin and
diamagnetic, NMR
was used to probe for peak broadening induced by paramagnetic species. As
shown in Fig.
2d, based on the hydrogens of each carbon of the vinyl group within the PoP,
wide peak
broadening was observed only for the CoPoP, and only in organic solvent. When
CoPoP was
formed into aqueous liposomes, the peaks narrowed, indicative of oxidation to
diamagnetic
Co (III) within the bilayer. To further verify this mechanism, the reducing
agent sodium
sulfite was added to CoPoP liposomes after they quantitatively bound a
fluorescently-labeled
His-tagged peptide. As shown in Fig. 2e, 2 M sulfite induced peptide release
from CoPoP
liposomes. Liposomes were also formed with commercially available Ni-NTA
lipid. The His-
tagged peptide did not bind as avidly to the Ni-NTA liposomes. Upon addition
of sulfite to
the system, no release of the peptide was observed, as would be expected with
Ni (II) which
cannot readily be reduced. Together, these data suggest that CoPoP transitions
from Co (II) to
Co (III) upon forming CoPoP liposomes and the polyhistidine imidazole groups
coordinate in
the bilayer with chelated Co (III) in the PoP.
[0119] Stable His-tag binding to CoPoP liposomes. The fluorescence
reporter protein
was then used to compare the binding of His-tagged proteins to liposomes
incorporating
either CoPoP or Ni-NTA-lipid (Fig. 3a). Ni-NTA liposomes included 10 molar %
2H-PoP to
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enable protein binding determination based on FRET. By EMSA, the protein
migrated
unimpeded when incubated without liposomes or when incubated with 2H-PoP
liposomes in
both the FRET channel and protein channel. When incubated with Ni-NTA
liposomes,
migration of the protein was only slightly inhibited, indicating that the
protein binding did not
withstand the conditions of electrophoresis. The FRET channel was unquenched,
confirming
a lack of binding to the Ni-NTA liposomes. In contrast, when incubated with
the CoPoP
liposomes, the protein stably bound with a complete disappearance of the FRET
channel and
decreased electrophoretic mobility that was consistent with the protein
remaining bound to
liposomes.
[0120] For biomedical applications, an intractable obstacle of using Ni-NTA-
lipid is
that it does not maintain stable His-tag binding in biological media such as
serum. To
examine whether liposomes could maintain binding in the presence of serum,
fetal bovine
serum was added at a 1:1 volume ratio to a solution of liposomes that had
bound the His-
tagged protein. As shown in Fig. 3b, Ni-NTA liposomes did not fully sequester
all the
protein, which is consistent with the weak binding exhibited in the EMSA
result.
Furthermore, following serum addition, all binding was abrogated over a 24
hour period. In
the same conditions CoPoP liposomes stably sequestered the His-tagged reporter
protein
without substantial protein release.
[0121] Since the histidine side chain comprises an imidazole group,
an imidazole
competition assay was used to compare the Ni-NTA and CoPoP liposomes binding
stability
with His-tagged polypeptides. As shown in Fig. 3c, CoPoP liposomes maintained
over 75%
binding to the reporter protein even at concentration approaching 1 M
imidazole. This
represents an approximate 10 million fold imidazole excess over the 100 nM
protein
concentration used in the binding study. In contrast, the Ni-NTA liposomes
released over
90% the His-tag in the presence of just 30 mM imidazole. The drastically
stronger binding of
the CoPoP liposome to the His-tag may be attributed to at least 2 factors; the
superior stable
chelation of Co(III) to imidazole groups and the protected hydrophobic
environment of the
CoPoP bilayer which limits access to competing external molecules.
[0122] Liposomes formed with Ni-NTA-lipid, the cobalt-chelated Co-NTA-
lipid, and
CoPoP could bind a fluorescent peptide in solution (Figure 7a). However, the
binding of Co-
NTA and Ni-NTA was not maintained during gel filtration chromatography (Figure
7b).
Liposomes formed with Co-NTA and Ni-NTA, but not CoPoP, released the peptide
when
incubated in serum (Figure 7c). This demonstrates the significance of bilayer-
confined
polyhistidine binding. We next examined whether or not CoPoP was required for
stable
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binding in serum, or whether a simple liposome-inserted cobalt porphyrin (Co-
pyro) could be
sufficient. After initial binding, incubation with serum caused the
polypeptide to become
displaced from the liposomes (Figure 8). This result is consistent with recent
demonstrations
that membrane-inserted porphyrins, but not PoP, rapidly exchange with serum
components
and exit the liposome. Together these results point to the essential role of
CoPoP in order to
stably bind His-tagged polypeptides.
[0123] Peptide binding to CoPoP liposomes
[0124] Peptide targeting has attracted interest for use as disease
and tissue-specific
"zip codes". The short RGD tripeptide, which is found in fibronectin and
vibronectin, is a
promising targeting ligand for its effective binding to the integrin av133
expressed on tumor
endothelial cells. CoPoP liposomes were examined to verify whether they can be
delivered to
molecular receptors on target cells via a His-tagged ligand approach with the
short linear
amino acid sequence GRGDSPKGAGAKG-HEIREIHHH (SEQ ID NO:1). Carboxy
fluorescein (FAM) was labeled on the N-terminus to enable detection of binding
to PoP-
liposome via FRET. It has been shown that linear RGD peptides can be labeled
with
fluorophores without disrupting integrin binding. As shown in Figure 4a, when
this peptide
was incubated with various metallo-PoP liposomes, only the CoPoP ones bound
the peptide.
Compared to protein-binding, peptide-binding was about five times faster.
Presumably, the
smaller size, faster molecular motion and decreased steric hindrance of the
peptide enabled
more rapid interdigitation into the bilayer to interact with and irreversibly
bind the CoPoP.
Based on previous estimates that each ¨100 nm liposome contains approximately
80,000
lipids, this equates to 8000 CoPoPs and 750 peptides per liposome. Since each
peptide
contained 7 histidine residues, the ratio of CoPoP to histidine in the bilayer
was 1:0.66. For
conventional His-tag binding to Ni-NTA, of all the residues in the His-tag,
just the ith and
i+2 or i+5 histidine residues are believed to be involved in coordinating with
the metal. The
porphyrin and polyhistidine density within the CoPoP bilayer is likely higher
and therefore
the coordination mechanism may be different.
[0125] The effect of His-tag length on peptide binding to CoPoP
liposome was
examined. A series of N-terminus FAM-labeled peptides was synthesized with
varying
lengths of His-tag attached to the C-terminus. As demonstrated in Figure 4b,
when the His-
tag was omitted from the peptide, no peptide binding was observed. With 2
histidine residues,
the binding was slow, with a binding tv2 of nearly 10 hours. As the His-tag
length increased,
binding speed rapidly increased. With 6 residues, corresponding to the common
hexahistidine
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tag, binding tuzwas less than one hour. By increasing the His-tag length to 10
residues,
binding ti/2 decreased to 20 minutes.
[0126] Next, lipid composition was varied to determine the effect of
membrane
fluidity on His-tag binding. Liposomes were formed with 90 mol % of either
DSPC, DMPC
or DOPC along 10 mol % CoPoP, Alternatively, 50 mol % cholesterol was
incorporated in
the bilayer with a corresponding reduction in the amount of standard lipid
used. DSPC forms
rigid, gel-phase bilayers at room temperature, whereas DMPC and DOPC have
lower
transition temperatures and are in the liquid crystal phase. Cholesterol
occupies space in the
bilayer and can have a moderating effect on membrane fluidity. Interestingly,
no major
differences were observed in the peptide binding rate to membranes of
different
compositions, with or without cholesterol (Fig. 4c). The peptide binding
process might occur
in a multi-step process and that once the peptide begins insertion into the
bilayer, cooperative
effects of the polyhistidine are not impacted by lipid composition. However,
in 5 mg/mL
bovine serum albumin (BSA), dramatic differences between the membranes with
and without
cholesterol were observed (Fig. 4d). The slower binding in cholesterol-free
liposomes was
likely due to greater interaction of BSA with the membrane interfering with
peptide binding.
Binding half-times were not reached with BSA at 50 mg/mL and serum completely
inhibited
binding (Fig. 9 Fig. 10).
[0127] Biotargeting of cargo-loaded liposomes. Given the binding
efficacy of the
His-tagged peptide to liposomes, bilayer integrity was assessed to determine
whether peptide
binding induces membrane destabilization. The aqueous core of liposomes was
loaded with
the fluorophore sulforhodamine B, a water soluble dye, at self-quenching
concentrations to
probe for membrane permeabilization. As shown in Fig. 5a, dye-loaded liposomes
did not
release a substantial amount of dye over the 8 hour period in which the
peptide had fully
bound to the liposomes. At 24 hours, the CoPoP liposomes with the peptide
bound released
less than 10% of the dye. Thus, His-tag insertion and binding process is
sufficiently gentle
and non-disruptive so that the bilayer integrity and entrapped cargo remains
intact. The
analogous palmitoylated lipopeptide resulted in permeabilization of cargo-
loaded liposomes
upon incubation (Fig. 11), further demonstrating the robustness of the His-tag
approach.
[0128] Next, RGD-decorated liposomes were assessed whether they could bind
to
their molecular targets with the established cell-line pair of U87
glioblastoma cells (RGD-
binding) and MCF7 breast cancer cells (RGD non-binding). Following
sulforhodamine B
entrapment, liposomes were first incubated with the His-tagged RGD peptide and
then with
both cell lines. Approximately 550 peptides were attached to each liposome.
Liposomal
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uptake was assessed by examining the fluorescence in the cells following
washing and lysis
(to remove any effects of cargo self-quenching). As shown in Fig. 5b, high
liposome uptake
was observed in U87 cells incubated with targeted CoPoP liposomes, whereas
negligible
binding occurred with MCF7 cells. As expected, without the RGD targeting
ligand, no uptake
occurred in either cell line. Inclusion of PEG-lipid in the liposome
formulation resulted in
liposomes that did not target to either cell line. It is likely that the
presence of the PEG had an
effect of obstructing the peptide, which is directly tethered to the bilayer
surface. Binding
could be inhibited with the presence of excess free RGD peptide, confirming
the targeting
specificity of the approach. Confocal microscopy substantiated these binding
results (Fig.
5c). Although the FAM-labeled peptide was quenched by the PoP liposomes,
sufficient signal
remained to verify the binding of both the targeting peptide and the liposomal
cargo. Both
cargo and the peptide were internalized and remained co-localized in U87
cells. When the
CoPoP liposomes and targeting peptide were ad-mixed immediately prior to
incubation with
U87 cells, the targeting peptide itself bound to U87 cells but did not have
time to attach to the
liposomes, which remained untargeted. The same result was observed for 2H-PoP
liposomes
which did not bind the peptide. Peptide binding was maintained when liposomes
were formed
with only 1 molar % CoPoP (Fig. 12a) and maintained selective binding to U87
cells (Fig.
12b). Cargo-loaded liposomes incubated a His-tagged cRGD moiety were
intravenously
injected into nude mice bearing U87 tumors. As shown in Fig 5d, 45 minutes
after
intravenous injection, the targeted liposomes accumulated in tumors with 2.5
fold activity
compared to the untargeted liposomes. These data show that CoPoP liposomes can
be loaded
with cargo in the core of the liposome, be labeled with a His-tagged targeting
peptide without
inducing cargo leakage, and be directed to molecular receptors expressed on
cells expressing
specific surface proteins in vitro and in vivo.
[0129] Liposomes represent only a subset of all the types of nanomaterials
used in
biomedical applications. CoPoP was assessed as a generalized surface coating
with selective
adhesion for His-tags. Gold nanoparticles were used as a model nanoparticle
since these have
are used in numerous biological applications. Using an established protocol to
lipid-coat gold
nanospheres, a citrate-stabilized 60 nm gold dispersion was used to hydrate a
thin film of
PoP-lipid. Upon repeated centrifugation and re-suspension, the citrate was
displaced, causing
the nanospheres to aggregate (Fig. 13a). However, in the presence of PoP-
lipid, the
nanospheres became coated and remained dispersible. Compared to citrate-
stabilized gold,
PoP-coated nanospheres had a slightly larger hydrodynamic size, corresponding
to a bilayer
coating on the gold (Fig. 13b). The presence of the coating following His-tag
binding did not
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influence the plasmonic peak of the gold at 540 nm, demonstrating the mild
nature of the
ligand binding (Fig. 13c). As shown in Figure 13d, only RGD-His CoPoP-coated
gold
nanoparticles targeted U87 cells and free RGD inhibited the binding as
determined by
backscatter microscopy. CoPoP gold alone, as well as 2H-PoP-coated gold with
the RGD-His
peptide were ineffective at targeting U87 cells.
[0130] Development of antigenic liposomes. Many of the monoclonal
antibodies that
broadly neutralize HIV viral entry, such as 2F5, Z13 and 4E10, target a
conserved linear
epitope in the membrane proximal external region (MPER) of the gp41 envelope
protein,
making the MPER a prime target for HIV peptide vaccines. However, it is
exposed only
.. during viral entry and attempts to use MPER peptides to generate
neutralizing antibodies
have faced challenges. This has given rise to the paradigm that vaccination
strategies should
consider antibody interaction with the lipid bilayer in which the MPER is
presented. We have
made use of liposomes containing the Toll-like receptor 4 (TLR-4) agonist
monophosphoryl
lipid A (MPL) combined with liposome-bound MPER peptide sequences. However,
the use
of a simple anchoring techniques based on biding of MPER His-tagged
polypeptides to Ni-
NTA liposomes generated low antibody titers. We set out to examine if the same
approach
could be enhanced with CoPoP liposomes.
[0131] A liposomal-peptide vaccination system was used with the MPER-
His
sequence NEQELLELDKWASLWNGGKGG-HHHEIHHH (SEQ ID NO:11). The MPER-
His peptide was bound to CoPoP liposomes containing MPL. A single injection
containing
[tg MPER-His and 25 [tg of MPL was administered to BALB/c mice and to athymic
nude
mice. This elicited a titer on the order of 104 in both BALB/c mice and nude
mice,
demonstrating a strong humoral immune response (Fig. 6a). This may be
significant since
HIV infects helper T cell populations, making B cell mediated responses
important.
25 Following a booster injection, the anti-MPER titer in athymic nude mice
was unaffected, but
in healthy mice there was a titer increase by an order of magnitude,
demonstrating a T cell-
mediated memory effect. Thus, the vaccination protocol resulted in both B cell
and T cell-
mediated immunity.
[0132] Next, various vaccine components were examined to better
determine the
specificity of the immune response (Fig. 6b). The MPER-His peptide did not
elicit any
antibodies when injected on its own, in Freund's complete adjuvant or along
with 2H-PoP
liposomes containing MPL. Interestingly, when the peptide was administered
with CoPoP
liposomes lacking MPL, no antibodies were generated whatsoever. Another lipid
adjuvant,
trehalose dibehenate (TDB) also failed to elicit any antibody production. TDB
does not act on
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TLR-4, which underscores the importance of MPL in immune activation of the
liposomal
vaccine system. When MPER-His bound to Ni-NTA liposomes were used, a weak
antibody
titer of less than 103 was achieved, consistent with previous reports.
However, when CoPoP
lipsoomes were used, a stronger response by 2 orders of magnitude was
observed.
Presumably, the stable binding of the peptide to the liposomes in vivo is
directly responsible
for this effect. The CoPoP immunization strategy was effective, with antibody
titers
persisting for at least 3 months, whereas no antibodies were detected with Ni-
NTA liposomes
after one month (Fig. 6c).
[0133] Post vaccination sera from mice was pooled and purified using
Protein G
agarose to yield purified IgG. This was then used to assess inhibition of
viral entry by HIV
(Fig. 6d). When the purified IgG from vaccinated mice was used at a final
concentration of
0.2 mg/mL, viral entry was inhibited by more than 75 %. This efficacy of
inhibition is greater
than that of the broadly neutralizing monoclonal antibody 2F5 when incubated
at a
concentration of 21.1g/mL but less than and 20 1.1g/mL. These data show the
potential for a
vaccination approach making use of CoPoP liposomes with HIV-derived peptides
in order to
induce antibody generation that can prevent viral entry.
EXAMPLE 2
[0134] In this example, a vaccine was developed that made use of his-
tagged Pfs25, a
recombinant protein derived from Plasmodium falciparum and liposomes
containing CoPoP
and MPLA.
[0135] Liposome Preparation. For generation of CoPoP and 2H-PoP
liposomes, 1,2-
dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol (CHOL),
monophosphoryl
lipid A (MPLA) and CoPoP (or 2H-PoP) were dissolved in chloroform at the
indicated molar
ratio (Table 2). A dried lipid film was formed after N2 stream and vacuum
overnight and was
rehydrated in PBS to a final lipid concentration of 3 mg/mL. The liposomal
suspension was
subjected to 11 times freeze/thaw cycles using ice cold CO2/acetone and a
water bath
followed by extrusion 10 times at 60 C through 200 nm polycarbonate
membranes.
[0136] The Psf25 insect protein (Recombinant subunit Pfs25 purified
from super sf9
cells, concentration determined by BCA assay as 1 mg/mL) was combined with
liposomes
overnight (20 h) at 4 C. Psf25 incubated in H20 served as a control. BSA was
combined
with liposomes as a negative control. The binding of Psf25 protein and
liposomes was
determined by a micro-centrifugal filtration method.
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[0137] Table 2. Formulation of Psf25 CoPoP/MPLA liposomes for each
mouse
injection
Each mice injection DMPC CHO CoPoP MPLA Psf25
dose
Molar ratio 55 35 5 5
g/50 1 84.6 30.7 12.1 20.0 5/0.5/0.05
nmole 127.75 79.39 11.34 11.34 0.25/0.025/0.0025
[0138] Microcentrifugal filtration for binding. Psf25 protein with
CoPoP, Psf25
protein with standard PoP (no cobalt) and Psf25 protein with water were placed
into Nanosep
Centrifugal Devices (100K, OMEGA). The device was rinsed with QH20 and
centrifuged at
1100 rpm for 3 min before use. Each of the samples was added and spun for 3
min at 1400
rpm. The device was washed twice with QH20 and centrifuged at 1400 rpm for 3
min
(minutes). The sample from the bottom filtrate was collected and the protein
concentration
was analyzed by BCA assay measuring absorbance at 562 nm (Thermo cat. 23235).
[0139] Vaccinations. On days 0 and 21, CD-1 mice (8 week females,
Envigo)
received intramuscular injections (i.m.) of 5, 0.5 and 0.05 1.ig Psf25
protein. Where indicated,
the injections also included 201.ig MPLA incorporated into the liposomes
(Avanti No.
699800P). ISA720 was also used as an adjuvant incubated with 51.ig Psf25
protein. The
treatment groups and flow chart are shown in Table 3.
[0140] Table 3. Treatment groups for serum IgG titer (n=10 mice per
group).
CoPoP/MPLA Psf25 (ug) ISA720
Group 1 (G1) 5
Group 2(G2) 0.5
Group 3 (G3) 0.05
Group 4 (G4) 0
Group 5 (G5) 5
Group 6 (G6) 5
[0141] Serum anti-Psf25 IgG level by ELISA. Anti-Psf25 titer was
assessed by
enzyme-linked immunosorbent assay (ELISA) in 96-well plates (Thermo,
Maxisorp). His-
tagged Psf25 (0.1m) in 100 pi coating buffer (3.03 g Na2CO3 and 6.0 g
NaHCO3/1L distilled
water, pH 9.6) was incubated in the wells for overnight at 4 C. Wells were
washed with PBS
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containing 0.1 % Tween (PBS-T) for 3 times and block with PBS containing 0.1 %
casein
(PBS-C) and then incubated for 2 h. Wells were wash with then washed with PBS-
T for 3
times and goat anti-mouse IgG-HRP (GenScript No. A00160) was diluted in PBS-C
to
become 1iAg/m1 and added to each well. The wells were washed again with PBS-T
for 6 times
before the addition of tetramethylbenzidine (Amresco No. J644).
[0142] Results.
[0143] Characterization of liposomes and protein binding binding
ability after Psf25
protein coated. The average size of the CoPoP liposomes before and after Psf25
protein
binding was measured by dynamic light scattering was 122.9 and 139 nm,
respectively (Fig.
14A), with similar polydispersity indexes around 0.05, showing favorable
liposome size after
binding with high monodispersity. Furthermore, to investigate if the Psf25
protein bound
efficiency and specifically to CoPoP liposomes, we incubated Pfs25 with CoPoP
liposomes
or 2H-PoP liposomes at 4 C overnight, followed by microcentrifugal filtration
to determine
protein binding to liposomes. Based on the amount of Pfs25 in the filtrate as
detected by
BCA assay, most of the Psf25 bound CoPoP liposomes (99.27%) but not the 2H-Pop
liposomes (12.33%). The binding of Pfs25 to CoPoP liposomes was ¨99% (Figure
14A).
[0144] Anti-Pfs25 IgG levels.
[0145] CD-1 mice were vaccinated with the CoPoP/MPLA/Pfs25 liposomes
conjugated with or without Psf25(with concentration of 5, 0.5 and 0.05 ps);
via i.m. or with
free Psf25 (with or without ISA70) on day 0 (prime) and day 21 (boost). On day
20 and 42,
serum was collected and anti-Psf25 IgG titers were determined by ELISA (Fig.
15A and Fig.
15B). Mice preboost with the CoPoP/MPLA/Psf25 liposomes showed similar IgG
level in
serum in both 5 j_tg and 0.5m Psf25 conjugated groups. Different dilutions
(starting at
1/1000) of anti-Psf25 IgG- serum sample were tested by the Psf25 (plant) ELISA
for each
treatment groups (Table 3). Here, the cutoff point was set at O.D. equal to
0.5. The data
shows that the IgG titer changed from 1/5000 to 1/45000 after boosting in Gl,
as well as in
G2, which represent CoPoP/MPLA/Pfs25 liposomes conjugated with 5 or 0.5 j_tg
Psf25
protein (Table 3). On the other hand, the IgG titer of CoPoP/MPLA/Pfs25
liposomes
conjugated with 0.05 i_tg Psf25 protein switch from 1/1000 to 1/15000 after
boosting, similar
results were shown in G6, which represent the 5 g Psf25 protein incubated with
ISA70. This
data is also reflected in Fig. 16, which shows titers of 0.05 ug Pfs25 in
CoPoP/MPLA
liposomes producing a higher titer than 5 pg Pfs25 in ISA720.
EXAMPLE 3
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[0146] In this example, a liposomal vaccine was developed that made
use of synthetic
his-tagged peptides containing repeating NANP sequences, which are derived
from the
circumsporozoite protein of Plasmodium falciparum. The liposomes had the same
composition as Example 1, but peptides instead of protein were used. The
peptides examined
are shown in Fig. 17A. The average size of the CoPoP liposomes before and
after different
NANP binding was measured by dynamic light scattering was 122.9 and 139 nm,
respectively (Figure 17B), with similar polydispersity indexes around 0.05,
showing
favorable liposome size after binding with high monodispersity. Furthermore,
to investigate if
the NANP peptide bound efficiency and specifically to CoPoP liposomes, we
incubated
NANP peptide with CoPoP liposomes or 2H-PoP liposomes at 4 C overnight,
followed by
microcentrifugal filtration to determine peptide binding to liposomes. Based
on the amount of
peptide in the filtrate as detected by BCA assay, a large amount of the NANP
bound CoPoP
liposomes (-80% in different length of peptides) but not the 2H-Pop liposomes
(less than
20%). The binding of NANP to CoPoP liposomes was ¨80% in all different length
of
peptides. (Fig. 17C)
EXAMPLE 4
[0147] In this example, CoPoP liposomes containing phosphatidylserine
(PS) was
used to reduce antibody response to his-tagged presentation molecules.
Liposomes were
formed with 10:30:30:30 CoPoP:phosphatidylserine:DOPC:Cholesterol via thin
film
hydration and extrusion. His-tagged NIPER was then bound to the liposomes.
These PS
liposomes were injected via footpad to mice 4 weeks and 2 weeks prior to
vaccination with
CoPoP/MPLA liposomes decorated with NIPER. As shown in Fig. 18, pretreatment
with PS
liposomes resulted in decreased IgG titers against NIPER.
EXAMPLE 5
[0148] In this example, CoPoP liposomes were targeted to cells via his-
tagged cell
penetrating peptides. The following his-tagged cell penetrating peptides were
obtained:
HEIREIHHHGRKKRRQRRRPPQ (SEQ ID NO:13) (TAT peptide);
HEIREIHREIRRRRRRRR (SEQ ID NO:14) (R8 peptide);
EIREIREIRHRQIKIWFQNRRMKWKK (SEQ ID NO:15) (PEN peptide). As shown in Fig.
19, following straightforward aqueous incubation with CoPoP liposomes
containing
[2:5:30:63] [PoP:CoP:CHOL:DNIPC], these liposomes could bind and get uptaken
following
1 hour incubation with U87 cells.
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EXAMPLE 6
[0149] In this example, CoPoP liposomes were used with a pre-fusion
RSV antigen (a
his-tagged antigen, DS-Cavl for RSV). His-tagged antigens can be used with the
CoPoP/PHAD system for generating functional antibodies.
[0150] The antigen DS-Cavl is a pre-fusion form of the F protein on the
surface of
the RSV virus. His-tagged DS-Cavl has been show to induce neutralizing
antibodies to RSV
A2.
[0151] CoPoP liposomes bind his-tagged proteins with simple mixing of
antigen and
liposomes, so that the resulting liposomes become decorated with
conformationally-intact
antigens, presented in a uniform orientation. The antigen binding is non-
covalent stable in
biological environments. It was shown (with another his-tagged antigen, Pfs25,
a malaria
transmission blocking antigen) that CoPoP liposomes result in improved antigen
delivery to
antigen presenting cells in vitro and in vivo.
[0152] It was showed that his-tagged DS-Cavl effectively binds to
CoPoP liposomes.
A 50 [iL volume of antigen-liposome solution is prepared using CoPoP and PoP
(also
referred to as 2H) and diluted to 200 [iL. Reference samples of liposomes
without antigen,
and antigen without liposomes, were also prepared. Samples were centrifuged at
20,000 rcf
for 90 minutes, and the supernatant was extracted from the resulting liposome
pellet. An
equal volume of BCA was added, to a total volume of 400 [iL. Samples were then
incubated
in a water bath at 60 C for 10 minutes. A volume of 100 [iL of each sample
are transferred to
each of 3 wells of a 96-well plate and the absorbance was measured at 562 nm.
The
absorbance difference between the antigen-liposome supernatant and liposome-
only reference
samples were used to calculate the percentage binding, with higher absorbance
corresponding
with a higher protein concentration in the supernatant and thus a lower
binding.
[0153] Mice were then injected with RSV antigen in conjunction with one of
six
adjuvants: CoPoP with PHAD, CoPoP without PHAD, 2HPoP with PHAD, aluminum
hydroxide (Alum), Sigma Adjuvant System (SAS), and AddaVax. When mice were
vaccinated with RSV, all mice injected with the RSV vaccine containing
CoPoP/PHAD
particles exhibited detectable IgG antibody titers prior to the booster
vaccination (Fig 21A).
In contrast, only a total of two mice from the remaining five groups exhibited
detectable IgG
titers. Two weeks after the booster vaccination the IgG titers of mice
vaccinated with CoPoP,
2HPoP, and SAS all showed significant increase; however, CoPoP with PHAD
remained the
most effective formulation for inducing antibody production (Fig 21B). The
mean antibody
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titer for the CoPoP/PHAD group exceeded the other five groups, and the
CoPoP/PHAD was
the only group without mice that were unresponsive to the vaccine after the
booster injection.
[0154] An RSV neutralization assay was performed using serum taken
from mice 35
days after the primary injection (Fig 21C,D). The results of the assay
indicate that the
antibodies produced in response to the CoPoP/PHAD vaccine are significantly
more effective
at neutralizing RSV type A and B than any other vaccine group. From the data,
it was
concluded that CoPoP/PHAD performs favorably compared to alternative adjuvants
such as
Alum, SAS, and AddaVax. Furthermore, the neither the CoPoP liposomes nor PHAD
adjuvant component of CoPoP alone are sufficient to increase effectiveness;
these results can
only be attributed to the combined effects of the novel CoPoP/PHAD
formulation.
EXAMPLE 7
[0155] In this example, CoPoP liposomes were used with OspA, a Lyme
disease
antigen. His-tagged antigens can be used with the CoPoP/PHAD system for
generating
functional antibodies.
[0156] OspA is a lyme disease antigen. A his-tagged OspA based on the B31
strain
protein sequence without the lipid tail was generated, and purified it as
shown in Figure 22.
[0157] Spontaneous formation of the proteoliposomes occurs via
insertion of the
histidine tag into the hydrophobic bilayer and subsequent coordination of the
imidazole
moiety to the metal center. Binding conditions for were evaluated using native
electrophoresis, which allows physical separation of liposome-bound and free
proteins due to
the limiting pore size of the acrylamide gel. Observed optimum binding mass
ratio of 4:1 of
PHAD:OspA (Figure 23A) is consistent with previous studies using the malaria
antigen,
Pfs25. Incubating 80 [tg/mL OspA with an equal volume of 320 [tg/mL CoPoP
liposomes
requires three hours of incubation at room temperature (Figure 23B). Visual
inspection of the
acrylamide gel showed that kinetics of binding started to plateau after 15 min
likely due to
steric hindrance for subsequent binding to nearby unconjugated sites. It can
be deduced that
the initial phase of binding occurred at a fast rate based from the relative
band intensities for
time points, 0 and 15 min. At the particleization conditions, specific binding
is estimated to
be about 80% based from microBCA assay of the supernatant obtained from high
speed
centrifugation (Figure 23C). Using the same method, low non-specific binding
was observed
for PoP/PHAD liposome, which lacks the chelating metal. A non-hi stidine
tagged protein,
lysozyme, failed to bind to neither CoPoP/PHAD nor PoP/PHAD liposomes. Based
from
DLS measurements, post-incubation liposomal size remains essentially the same
for both
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CoPoP/PHAD and PoP/PHAD liposomes (Figure 23D). Moreover, transmission
electron
micrographs revealed that CoPoP/PHAD liposomes retained its shape after
antigen binding
(Figure 23E).
[0158] Serum stability study (Figure 24A) showed that incubation of
the
proteoliposomes with human at 37 C did not significantly increase the
fluorescence signal
after 12 days. This basically indicates that OspA remains mainly associated to
the
metallochelating liposomes within the duration of the study. CoPoP liposome
exhibited more
than a hundred-fold increase in stability compared to nickel-chelating
liposomes due to the
strategic spatial location of the metallochelating bond within the hydrophobic
bilayer. Serum-
stable antigen binding ensures integrity of the proteoliposomes during transit
to draining
lymph nodes.
[0159] Previous studies using analogous nanoparticle systems
demonstrated variation
of the immunogenicity and protective efficacy with the point of attachment to
the
nanoparticle scaffold. This highlights the importance of proper antigenic
epitope presentation
on the particle surface. In this study, polyhistidine tag was appended at the
N-terminus
opposite to the locality of protective epitopes to ensure epitope
accessibility on the liposomal
surface and avoid possible occlusion of the important C-terminal epitopes.
This configuration
basically mimics the lipoprotein integration to the outer membrane of Lyme
borreliae .
Assessment of the epitope availability using immunoprecipitation method
indicated exposure
of LA-2 epitope on the surface-bound OspA (Figure 24B). The negative control,
which is a
monoclonal antibody for a non-homologous protein, exhibited low PoP
fluorescence signal.
These results indirectly corroborated findings in CD spectroscopy
measurements, which
elucidated the secondary structure of recombinant OspA. Furthermore, LA-2
antibody-OspA
recognition suggested that structural integrity of the binding antigen is
maintained after
attaching to liposomes.
[0160] Practical utility of liposomes as antigen delivery vesicles is
attributed from its
enhanced uptake by antigen presenting cells. Nanoparticle internalization
studies using
murine macrophage-like cells (Figure 24C) showed high antigen uptake only with
the
surface-functionalized liposome (CoPoP/PoP liposome). Minimal uptake was
observed with
the free non-adjuvanted or mixed non-associated (i.e., PoP/PHAD) forms of the
antigen.
These results corroborate studies demonstrating enhanced antigen
internalization in the
nanoparticulate form. The macrophage selective uptake of PoP/PHAD liposomes in
the
mixed form further implicates that physical association of antigen to
liposomes may be
necessary for liposomal adjuvanticity. Co-delivery of surface-exposed antigen
and
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immunostimulant to immune cells is an important facet of the CoPoP/PHAD
liposome.
Cellular uptake mechanism via phagocytosis is supported by the diminished
uptake for both
antigen and liposome in the presence of cytochalasin B.
[0161] Assessment of the immunogenicity using ELISA indicates that
this novel
.. vaccine platform can stimulate strong immune response at low antigen dose.
Homologous
prime-boost vaccination with CoPoP/PHAD liposomes elicited OspA-specific IgG
antibody
titer higher than other commercial adjuvants (Figure 25A). Furthermore,
surface labeling of
B. burgdorferi B31 cells (Figure 25B) suggested that the generated OspA-
specific antibodies
can recognize epitopes on the cell surface, supporting results obtained from
immunoprecipitation assay. Fluorescence micrographs further reveals low
functionality of the
anti-OspA antibodies induced from immunization with alum. Immunoblot using
antisera
from CoPoP/PHAD immunization showed that OspA-specific antibodies recognize
different
strains of Lyme borreliae but to different extents (Figure 22C). Different
band intensity
depicts antigenic heterogeneity of OspA across different genospecies. The
negative control,
B. hermsii, which lacks the ospa gene, has no apparent band. Minimal non-
specific bands
were observed for E. coil lysate with histidine-tagged OspA. Lower molecular
weight
observed for the OspA band is due to the substitution of the lipidation signal
sequence with a
shorter polyhistidine segment.
[0162] Inclusion of the synthetic version of monophosphoryl lipid A,
PHAD, which is
a TLR-4 agonist, in the liposomal formulation skewed the immune response
towards Thl,
switching the isotype to IgG2a. CoPoP/PHAD liposomes produced higher levels of
OspA-
specific IgG2a antibodies than IgG1 (Figure 23A). Alum, on the other hand,
induced higher
levels of IgG1 isotype. Predominance of IgG2a isotype is vital as this isotype
exhibits higher
bactericidal activity and greater capacity to activate complement than IgG1
isotype. The Thl-
biased immune response observed for CoPoP/PHAD liposomes correlates with the
higher
stimulation of interferon-gamma than interleukin-4 in the splenocyte study
(Figure 23B).
[0163] The ability of OspA-specific antibodies to eradicate
spirochetes was
demonstrated using bactericidal assay. Generated antibodies from CoPoP/PHAD-
OspA
immunization exhibited higher bactericidal activity compared to alum or
PoP/PHAD
liposomes (Figure 27). Calculated 50% borreliacidal titer for CoPoP/PHAD
liposomes is
significantly higher than alum.
[0164] An OspA-based transmission-blocking vaccine heavily relies on
the high
levels of circulating antibodies. ELISA titers calculated at different time
points remained
fairly the same after one year, indicating high durability of the antibody
response in Fig 28.
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EXAMPLE 8
[0165] In this example, CoPoP liposomes were used with hemagglutinin
antigens and
neuraminidase for influenza. His-tagged antigens can be used with the
CoPoP/PHAD system
for generating functional antibodies.
[0166] Influenza hemagglutinin (HA) is the major surface antigen on the flu
virus and
a major vaccine target. We obtained his-tagged H3 strain HA. As shown in
Figure 29, his-
tagged HA bound effectively to CoPoP liposomes.
[0167] A 50 pL volume of antigen-liposome solution is prepared using
CoPoP and
PoP (also referred to as 2H) and diluted to 200 [EL. Reference samples of
liposomes without
antigen, and antigen without liposomes, were also prepared. Samples were
centrifuged at
20,000 rcf for 90 minutes, and the supernatant was extracted from the
resulting liposome
pellet. An equal volume of BCA was added, to a total volume of 400 [EL.
Samples were then
incubated in a water bath at 60 C for 10 minutes. A volume of 100 pL of each
sample were
transferred to each of 3 wells of a 96-well plate and the absorbance was
measured at 562 nm.
The absorbance difference between the antigen-liposome supernatant and
liposome-only
reference samples were used to calculate the percentage binding, with higher
absorbance
corresponding with a higher protein concentration in the supernatant and thus
a lower
binding.
[0168] As shown in Figure 30, immunization with CoPoP liposomes and
his-tagged
HA was highly potent.
[0169] A passive serum transfer experiment established that the
vaccine-mediated
protection was at least in part due to antibody generation (Figure 31).
[0170] The capacity for multiplexing was then assessed using multiple
his-tagged
HAs. As shown in Figure 32A, various HA and NA proteins bound to CoPoP
liposomes.
When immunized, all the HAs could generate specific antibodies with minimal
cross-
reactivity (Figure 32B).
EXAMPLE 9
[0171] This example demonstrates that different types of synthetic
MPLA can be
used in the present compositions and methods, including PHAD, PHAD-504, and
3D6A-
PHAD. The structures for these adjuvants are shown in Figure 33. Liposomes
were formed
with 4:2:1:X DPPC:Cholesterol:CoPoP:MPLA, where MPLA was each of these types
of
synthetic versions, and X varied was either 5,4,3,2,1.
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[0172] As shown in Figure 34, inclusion of all types of MPLA could
generate
liposomes and result in antibody generation that was generally higher than
omission of
MPLA ("CA") bleow. His-tagged Pfs25 was mixed in a 1:4 mass ratio of
antigen:CoPoP and
mice were immunized on day 0 and day 21. Serum was collected on day 42 and
serum was
assessed for Pfs25 binding IgG ELISA.
[0173] While the invention has been described through specific
embodiments, routine
modifications will be apparent to those skilled in the art and such
modifications are intended
to be within the scope of the present disclosure.
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