JP7635149B2 - Nanostructures containing cobalt porphyrin-phospholipid conjugates and polyhistidine tags - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Patent Application No. 16/399,581, filed April 30, 2019, the disclosure of which is incorporated herein by reference.

This invention was made with Government support under Grant Nos. DP5OD017898 and R21AI122964 awarded by the National Institutes of Health. The Government has certain rights in this invention.

The present disclosure relates generally to the field of functional nanostructures. More specifically, the present disclosure relates to nanostructures comprising cobalt porphyrins.

In the field of functional 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 offered a wide range of strategies, but most nanoparticle conjugations suffer from limitations related to one or more of the following: 1) low conjugation rates and mandatory purification steps; 2) incompatibility with physiological buffers, making labeling of intact nanoparticles impossible; 3) variable labeling sites and conjugated polypeptide structures, generating heterogeneous particle populations with differing efficacies; 4) the need for complex, exogenous chemical approaches.

Standard approaches for attaching ligands to aqueous nanoparticles use maleimides, succinimidyl esters, and carbodiimide-activated carboxylic acids, which can covalently react with amine and thiol groups of polypeptides. For antibody-conjugated immunoliposomes, the use of maleimide-lipids has been widely explored. Conjugation rates can reach 90% in overnight reactions, but quenching of free maleimide groups and further purification are required afterwards. Proteins may require preparatory steps of thiolation and purification prior to conjugation. Although the orientation of the antibody is a major factor influencing the efficacy of conjugated antibody target binding, these approaches result in a large number of antibody labeling sites and indiscriminate orientation. Recently, bi-orthogonal synthetic strategies such as click reactions have been applied to preformed liposomes, but these require exogenous catalysts and the use of unconventional amino acids.

Another approach, suitable for smaller peptides that are less prone to permanent denaturation in organic solvents, is to conjugate the peptide to a lipid anchor. The resulting lipopeptide can then be incorporated with other lipids during the liposome formation process. This approach has been used to produce synthetic vaccines to elicit antibody production against non-immunogenic peptides. However, due to its amphiphilic nature, the yields were only 5-10% when purification of the lipopeptide was difficult. It has also been found that lipopeptides are not fully incorporated into liposomes during the formation process, leading to aggregation.

The present disclosure provides functional nanostructures. The nanostructures can be used for cargo delivery, targeted delivery and/or presentation molecule delivery. The nanostructures can be monolayers or bilayers that enclose an aqueous compartment therein. A bilayer structure that encloses an aqueous compartment is referred to herein as a liposome. The nanostructures can be monolayer or bilayer coatings on nanoparticles. The monolayer or bilayer comprises a cobalt porphyrin-phospholipid conjugate, optionally a phospholipid not conjugated to a porphyrin, optionally a sterol, and optionally polyethylene glycol (PEG). One or more targeting peptides or polypeptides with a polyhistidine tag (referred to herein as presentation molecules) are incorporated within the monolayer or bilayer, such that a portion of the polyhistidine tag is present in the monolayer or bilayer and the presentation molecule is exposed to the outside of the monolayer or bilayer. Cobalt porphyrin can be used instead of or in addition to the cobalt porphyrin-phospholipid conjugate.

The nanostructures of the present disclosure can be loaded with cargo for delivery to a site targeted by a polyhistidine-tagged presentation molecule. For example, liposomes can be loaded with cargo for delivery to a desired site by using a polyhistidine-tagged presentation molecule.

The data presented herein demonstrate that bilayers containing cobalt porphyrins, such as cobalt porphyrin-phospholipid (CoPoP), can stably bind polyhistidine-tagged (also referred to herein as "His-tagged") polypeptides (Figure 1a). Other metalloporphyrins, such as zinc, nickel, and copper, cannot stably bind His-tagged polypeptides. Since the porphyrin itself forms the hydrophobic portion of the bilayer and is inaccessible to the external aqueous environment, this represents a new binding paradigm with at least some polyhistidine embedded in the membrane phase (Figure 1b). This results in more stable binding and allows for a significantly simpler non-covalent post-labeling paradigm after nanoparticle formation, eliminating ambiguity regarding ligand orientation.

We show that lipid bilayers containing porphyrin-phospholipids chelated with cobalt, but not other metals, can efficiently capture His-tagged proteins and peptides. Binding follows a Co(II) to Co(III) transition within the protected hydrophobic bilayer, resulting in essentially irreversible attachment, e.g., in serum or in a million-fold excess of competing imidazole. Using this approach, we inserted homing peptides into the bilayers of preformed empty and cargo-containing liposomes, allowing site targeting (e.g., tumor targeting) without compromising bilayer integrity. Peptides or synthetic peptides can be conjugated to liposomes containing adjuvants (e.g., lipid monophosphoryl lipid A) to generate antibodies for otherwise non-antigenic peptides.

The present disclosure provides a monolayer or bilayer structure, the monolayer or bilayer comprising a porphyrin and cobalt chelated thereto, the cobalt metal being present within the monolayer or bilayer and the porphyrin macrocycle, and further having a molecule having a histidine tag non-covalently attached thereto, at least a portion of the His tag being present within the monolayer or bilayer and coordinated to the cobalt metal core. The presentation molecule can be used for a variety of applications, including targeting and generating an immune response. Liposomes or micelles formed with this layer can be loaded with cargo for release at a desired location. The cobalt porphyrin can be cobalt porphyrin-phospholipid (CoPoP). This layer can be used as a coating for other nanostructures, such as metal nanoparticles, nanotubes, etc.

FIG. 1. Binding of His-tag to PoP-bilayers. (a) Schematic showing that peptides bearing His-tags (green) bind to preformed CoPoP liposomes in aqueous solution.
(b) Insertion of a His-tagged polypeptide into a bilayer containing CoPoP. Only one piece of the bilayer is shown.
(c) Chemical structures of metal-PoPs used in this study.

[Figure 2] His-tagged proteins bound to Co(III)-PoP liposomes. (a) His-tagged (7) fluorescent proteins containing Cerulean (C) fused to Venus (V) demonstrate binding to the PoP bilayer. Upon excitation of C, FRET occurs, causing V to fluoresce (left), but this is inhibited upon binding to the PoP-bilayer, as FRET competes with the photonic bilayer (center). C fluorescence can be probed directly even when the protein is bound to the bilayer (right).
(b) Multispectral fluorescence images of the fusion protein electrophoretic mobility shift after incubation with the indicated metal-PoP liposomes.
(c) Binding kinetics of fusion proteins to the indicated metal-PoP liposomes based on loss of C to V FRET.
(d) NMR peak widths of the underlined protons of the vinyl groups of CoPoP demonstrate paramagnetic broadening of Co(II) in deuterated chloroform ( _CDCl3 ), but paramagnetic peaks of Co(III) after CoPoP-liposome formation in deuterated water. Each set of bar graphs, from left to right, are those of CoPoP and 2H-PoP.
(e) Reversal of His-tagged peptide binding to CoPoP liposomes after addition of 2M sodium sulfate. Liposomes were formed with 10 mol% CoPoP or Ni-NTA phospholipids. Each set of bars, from left to right, are water and +2M sulfite bars.

FIG. 3. Tight binding of His-tagged proteins to CoPoP liposomes. (a) Multispectral electrophoretic mobility shift images of fluorescent reporter proteins incubated with liposomes containing the indicated lipids.
(b) Binding stability of reporter proteins bound to the indicated liposomes (1:1 in serum).
(c) Binding stability of reporter proteins bound to the indicated liposomes (in excess free imidazole). Mean ± standard deviation (n = 3).

FIG. 4 Binding of short His-tagged RGD peptides to CoPoP liposomes. (a) Binding of FAM-labeled short peptides to metal-PoP liposomes.
(b) Effect of His-tag length on binding half-life to CoPoP liposomes. No binding (N.B.) was observed with non-His-tagged peptides.
Effect of liposome composition on binding half-life to CoPoP liposomes of the indicated compositions upon incubation in PBS (c) or 5 mg/mL BSA (d). Mean values ± standard deviation for triplicate determinations. In (c) and (d), each set of bars is, from left to right, 50 and 0 bars.

FIG. 5: Release of sulforhodamine B encapsulated in POP liposomes during RGD-His targeting (a) peptide conjugation of cargo-containing liposomes. Each set of bars, from left to right, are 8 and 24 hour bars.
(b) Targeted uptake of liposomes containing sulforhodamine B. Cells were incubated under the indicated conditions and uptake was assessed by examining the fluorescence of sulforhodamine B. Each set of bar graphs, from left to right, are bar graphs of MCF7 and U87 cells.
(c) Confocal micrographs showing liposome uptake. Cells were incubated with the indicated liposome solutions for 2 h, washed, and imaged. All images were acquired with the same settings.
(d) Biodistribution of sulforhodamine B encapsulated in CoPoP liposomes with or without His-tagged cyclic RGD targeting peptide attached 45 min after injection into nude mice bearing subcutaneous U87 tumors. Mean ± SD (n=3). Each set of bars, from left to right, is a non-targeted and +cRGD-His bar.

FIG. 6: HIV peptide vaccination with immunogenic CoPoP liposomes. (a) BALB/c or athymic nude mice were immunized with CoPoP liposomes containing 25 μg MPL and 25 μg His-tagged MPER peptide (derived from HIV gp41 envelope protein). Serum titers were assessed by ELISA using biotinylated MPER peptide lacking the His tag and probed with anti-IgG secondary antibody. Arrows indicate the time of vaccination.
(b) Anti-MPER titers in BALB/c mice vaccinated as indicated. Mice were vaccinated at weeks 0 and 2 and sera were collected at week 4.
(c) Persistent anti-MPER titers in mice vaccinated with CoPoP liposomes containing MPL. Mean ± SD (n = 4 mice per group). The first two bars on the left (connected) are CoPoP and Ni-NTA.
(d) Neutralization of HIV infection of 293 cells in the presence of the indicated antibodies. IgG was purified from mouse serum using protein A. Mean values ± standard deviation (n = 3).

FIG. 7. Stability of RGD-His peptide conjugation to liposomes. (a) FAM-labeled RGD-His peptide conjugation to liposomes containing 10 mol % Ni-NTA-lipid, Co-NTA-lipid, or CoPoP.
(b) Gel filtration after peptide conjugation. Only CoPoP liposomes maintained stable conjugation.
(c) Peptide stability after incubation at 1:1 dilution with fetal bovine serum. Only CoPoP liposomes maintained stable association.

FIG. 8: Stable binding of His-tagged proteins to liposomes containing CoPoP. Reporter proteins were incubated with liposomes containing CoPoP, free Co-porphyrin or 2H-PoP, then incubated in serum and subjected to EMSA. The proteins were then imaged using the FRET channel (ex: 430 nm, em: 525 nm). The absence 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 proteins to the liposomes.

Figure 9: RGD-His 90% peptide binding time to CoPoP liposomes of different composition. Effect of liposome composition on the 90% peptide binding time to CoPoP liposomes (10 mol % CoPoP) containing the indicated components upon incubation with RGD-His peptide in PBS. Each set of bars, from left to right, is for (+) cholesterol and (-) cholesterol.

FIG. 10: RGD-His binding to CoPoP liposomes in the presence of serum or albumin. Liposomes of the indicated composition were incubated with RGD-His peptide in the presence of 50% fetal bovine serum or 50 mg/mL bovine serum albumin. Emission of FAM-labeled peptide was normalized by comparing peptide emission upon binding to CoPoP liposomes with 2H-PoP liposomes.

FIG. 11. Membrane permeabilization by lipopeptides. Liposomes loaded with sulforhodamine B were incubated with the indicated peptides (5 μg/mL) at room temperature and release was assessed using fluorescence. Each set of bars are from left to right at 8 and 24 h.

FIG. 12: Binding and cell targeting of RGD-His peptide to liposomes containing 1 mol% CoPoP. (a) Normalized peptide fluorescence upon incubation with CoPoP liposomes containing 1 mol% CoPoP. Emission was normalized by comparing CoPoP samples to 2H-PoP. (b) Cellular uptake of sulforhodamine B liposomes containing 1 mol% CoPoP incubated with cells as indicated. Each set of bar graphs, from left to right, are for MCF7 and U87 cells.

FIG. 13: Coated gold nanoparticles with CoPoP His tag-bound surfaces. (a) Photograph of the coating protocol to disperse gold nanoparticles. In the absence of POP coating, citrate-stabilized gold aggregates after repeated centrifugation steps (arrows). (b) Size of nanoparticles before and after lipid coating. Inset shows transmission electron micrograph of CoPoP Gold with a scale bar of 50 nm. (c) Absorption spectra of citrate-stabilized gold and CoPoP Gold after RGD-His conjugation. (d) Confocal reflectance images showing uptake of targeted nanoparticles. Cells were incubated with the indicated gold nanoparticles for 2 h, washed and then imaged. All images were acquired with the same settings.

[Figure 14] (A) Psf25 (Pfs25_B) binding was measured by centrifugal filtration analysis. These data show 100% binding of Psf25 protein to Co-PoP liposomes. (B) Particle size of CoPoP liposomes before and after Psf25 protein binding.

FIG. 15. Anti-Psf25 IgG levels in CD-1 mice. Mice were vaccinated with CoPoP/MPL or Psf25 in ISA70 after intramuscular injection. (A) Pre-boost and (B) post-boost (3-week prime/3-week boost [5, 0.5, or 0.05 ug Pfs25 per injection) IgG titers were measured by ELISA in 96-well plates from mice vaccinated with CoPoP(Psf25) liposomes, or free Psf25 protein (with or without ISA70). Data represent mean ± SD (n=5 mice per group).

FIG. 16: Anti-Psf25 IgG titers. Titers are defined as the reciprocal of the serum dilution that produces an absorbance greater than 0.5 above background.

FIG. 17: Description and characteristics of coating of NANP peptides of various lengths on CoPoP liposomes. (A) NANP repeat peptides of various numbers containing a 7× histidine (His) tag.
(B) The mean diameter and polydispersity (PDI) of CoPoP liposomes conjugated with NANP peptides of various lengths were calculated by dynamic light scattering (n=3). Error bars, standard deviation.
(C) Peptide binding of NANP peptide to CoPoP liposomes and 2HCoPoP liposomes was measured by a microcentrifuge filtration process and BCA analysis (n=3).

[Figure 18] Anti-MPER IgG titers in mice pretreated with CoPoP/phosphatidylserine liposomes conjugated with His-tagged MPER. To induce antibody responses against MPER, mice were pretreated with MPER/CoPoP/PS liposomes 4 and 2 weeks before injection of MPER in CoPoP/MPLA liposomes.

[Figure 19] Fluorescence of U87 cells after incubation with CoPoP liposomes bound to various His-tagged CPPs

[Figure 20]
After 2 hours of incubation at room temperature, DS-Cav1 with a His tag achieved nearly complete binding to CoPoP nanoparticles (Figure 20, left). His-tagged DS-Cav1 did not significantly bind to PoP particles lacking cobalt (2HPoP), and DS-Cav1 without a His tag achieved no significant binding to either particle. His-tagged DS-Cav1 did not induce liposome aggregation upon binding, based on liposome diameter (Figure 20, center) or polydispersity (Figure 20, right).

[Figure 21]
CoPoP/DS-Cav1 vaccination results in higher IgG antibody titers both before and after booster injections. At 35 days after the first injection, the CoPoP vaccine achieves maximum RSV neutralization. CD-1 mice were injected intramuscularly with 100 ng of DS-Cav1 on days 0 and 21, and serum was collected on day 42 and assessed for neutralization.

[Figure 22]
Generation of His-tagged OspA.

FIG. 23. Characterization of OspA-based proteoliposomes formed using CoPoP/PHAD liposomes.
(A) Optimal binding mass ratio of OspA:CoPoP/PHAD liposomes assessed by native PAGE (histidine-MOPS buffer system, pH 6.8).
(B) Kinetics of OspA binding to CoPoP/PHAD liposomes incubated at room temperature in a 1:4 mass ratio.
(C) Specific binding of His-tagged OspA to CoPoP/PHAD liposomes as measured by microBCA assay of the 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 His-tagged protein binding, acquired 3 h after incubation of liposomes with protein. Error bars represent standard deviation of n=3 measurements.

FIG. 24. Evaluation of 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 with the OspA-specific monoclonal antibody LA-2.
(C) DY-490-OspA uptake by mouse RAW 264.7 macrophage cells after 2 hours of incubation with the indicated samples. Cytochalasin B was supplemented to the medium 1 hour prior to incubation. Error bars represent standard deviation of n=3 experiments.

FIG. 25. Evaluation of the immunogenicity of OspA-based nanoliposomal vaccines.
(A) Anti-OspA IgG titers induced by CoPoP/PHAD liposomes compared with other commercially available adjuvants. (B) Detection of anti-OspA IgG antibodies by indirect immunofluorescence assay of ^{permeabilized} B. burgdorferi B31 using goat anti-mouse IgG (H+L) secondary antibody DyLight® 488 conjugate. (C) Detection of anti-OspA IgG antibodies by immunoblot assay using whole cell lysates of B. burgdorferi B31 and B. azfelii.
The horizontal line represents the geometric mean.

Figure 26: Th1-biased immune response of OspA-conjugated CoPoP/PHAD liposomes. (A) IgG isotype profiling of post-immunization sera (day 42) using ELISA. (B) Splenocyte stimulation assay to detect interferon-gamma and interleukin-4 secretion after 72 hours of stimulation with OspA. Error bars represent standard deviation from n=3 triple stimulation experiments.
The horizontal line indicates the geometric mean.

FIG. 27. Evaluation of borreliacidal activity of SNAP-induced OspA antibodies.
(A) Serum bactericidal antibody assay performed with guinea pig complement. Viability was obtained by normalizing the number of spirochetes after overnight serum treatment with the number of spirochetes immediately after incubation. Surviving B. burgdorferi B31-A3 were counted using dark-field microscopy.
(B) Mean 50% borreliacidal activity (serum dilution effectively eliminating 50% of the bacteria) from three different mouse sera. Error bars indicate standard error of the mean. NI represents no inhibition. Statistical significance of differences between bactericidal titers (p<0.05, indicated by asterisks) is assessed by Kruskal-Wallis test with Dunn's post-hoc.

[Figure 28]
Persistence of anti-OspA IgG levels in SNAP-immunized mice. Prime and boost vaccinations of 100 ng OspA in CoPoP/PHAD liposomes were administered on days 0 and 21, respectively. Endpoint titers are defined as the reciprocal of the serum dilution at an absorbance cutoff of 0.5. Data points represent geometric means and error bars represent 95% confidence intervals.

[Figure 29]
Binding of His-tagged HA protein from H3N2 strain Fr1478 to CoPoP liposomes.

[Figure 30]
As shown in (A)-(F), three groups of mice (n=8) were vaccinated with 100 ng of H3 antigen Fr1478 (derived from flu strain A/canine/Illinois/11613/2015) adjuvanted with CoPoP/MPLA, 2HPoP/MPLA, or Alum (aluminum hydroxide), followed by A/Hong Kong/1/1968.
(A) IgG ELISA and (B) HAI assays performed with serum samples collected throughout the vaccination period show that CoPoP/MPLA+Fr1478 particles achieve the best antibody responses prior to viral challenge.
(C-D) Body weight remained stable throughout the 8-day challenge period, and (E) viral load and (F) white blood cell counts in lung tissue were both minimal in CoPoP-vaccinated mice, indicating effective protection against the virus.
(GI) Follow-up challenge studies were performed comparing CoPoP/MPLA protection to CoPoP without MPLA and another adjuvant, Montanide ISA 720. Again, CoPoP/MPLA vaccinated mice show the most consistent weight retention and the lowest viral load and white blood cell counts in the lungs.

[Figure 31]
Although passive transfer of vaccinated mouse serum provides less protection than direct vaccination, transfer of serum from mice vaccinated with CoPoP results in (A) significantly reduced weight loss, (B) higher survival rates, and (C) lower clinical scores. This data supports the hypothesis that the immune response resulting from CoPoP vaccination is significantly antibody-mediated.

[Figure 32]
As shown in (A), His-tagged influenza antigens from various subtypes were obtained and tested for binding affinity with CoPoP. The binding capacity of His-tagged antigens was 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 10 selected antigens, serum was collected, and the binding response of the resulting serum antibodies was quantified using ELISA. Antibody responses against antigens that were homologous to the vaccine antigens yielded the highest IgG binding, while nonspecific binding of antibodies elicited by other subtypes tended to show, on average, 1-2 orders of magnitude lower binding.

[Figure 33]
Structures of some examples of synthetic adjuvants.

[Figure 34]
Liposomes were formed with 4:2:1:X DPPC:cholesterol:CoP:MPLA, where MPLA was each of the synthetic versions of these types and X was varied and was either 5, 4, 3, 2, or 1. Results are shown for anti-Pfs25 titers for CP (PHAD), C3D6A (3D6A-PHAD), CP504 (PHAD-504), and CA (no MPLA).

The present disclosure provides a nanostructure comprising at least a monolayer. For example, the structure can comprise a monolayer or a bilayer, the monolayer or bilayer comprising a porphyrin-phospholipid conjugate to which cobalt is chelated such that the cobalt is present within the bilayer. The bilayer structure can form a liposome. The structure can comprise two monolayers (bilayers), the hydrophobic groups of the two monolayers facing each other and the hydrophilic groups exposed to the surface.

The disclosure herein regarding bilayers may also apply to monolayers. The bilayer or monolayer may also be referred to herein as a "membrane."

All ranges provided herein include all numbers within the range to ten decimal places unless otherwise stated.

Some or all of the cobalt porphyrin in the monolayer or bilayer can be non-covalently bound to a polyhistidine-tagged molecule, with at least a portion of the polyhistidine tag being present in the bilayer, and the tagged molecule being presented on the surface of the bilayer. In the bilayers or monolayers of the invention, it is believed that one or more histidine residues in the polyhistidine tag coordinate to a cobalt metal core in the bilayer, thereby providing stability to the structure. The histidine residues of the polyhistidine tag may coordinate to the cobalt metal in the core of the porphyrin in the membrane. The entire histidine tag may be present in the bilayer. A porphyrin-phospholipid conjugate having a cobalt metal conjugated thereto is referred to herein as CoPoP. A liposome whose bilayer comprises CoPoP is referred to herein as a CoPoP liposome. CoPoP liposomes can be functionalized with histidine-tagged molecules. As used herein, the term "His-tagged molecule" refers to a molecule, such as a peptide, polypeptide, or protein, that has a histidine tail. For example, a peptide that has a histidine tail is a His-tagged molecule. CoPoP liposomes that contain such a His tag are referred to herein as His-tagged CoPoP liposomes or His-tagged CoPoP.

CoPoP monolayers or bilayers functionalized with His-tagged presentation molecules of the present disclosure provide a platform for presenting various molecules of interest in the circulation, or for delivery to a desired location, or for generating a specific immune response to those His-tagged molecules. These molecules are referred to herein as presentation molecules (PMs). Structures comprising His-tagged CoPoP bilayers with PMs attached to histidine tags exhibit desirable stability. The His-tagged molecules are non-covalently attached (coordinated) to the CoPoP and can be prepared by an incubation process. Therefore, this process does not require the removal of reactive components used in other types of conjugation chemistries, such as maleimides, or exogenous catalysts or unnatural amino acids.

The cobalt porphyrin may be present in the bilayer of a self-assembled liposome with an internal aqueous compartment. Alternatively, it may be present in a monolayer or bilayer coating that coats other nanoparticles. Cobalt porphyrin-phospholipid (CoPoP) behaves like a conventional lipid with respect to its amphiphilic nature. Therefore, a monolayer or bilayer containing CoPoP can be used to coat nanoparticles by methods known to those skilled in the art. In one embodiment, the bilayer or monolayer of the present disclosure may be present on other nanoparticles (e.g., in the form of a coating). In one embodiment, a bilayer or monolayer containing cobalt porphyrin (e.g., cobalt porphyrin-phospholipid) is present as a coating on gold nanoparticles or silica nanoparticles, or other nanoparticles with hydrophilic surfaces. In one embodiment, the coating may be in the form of a monolayer. In one embodiment, a monolayer or bilayer containing cobalt porphyrin (e.g., cobalt porphyrin-phospholipid) is present as a coating on a hydrophobic surface, such as a carbon nanotube. In one embodiment, the monolayer may form micelles surrounding one or more hydrophobic molecules.

This disclosure provides a nanostructure comprising a monolayer or bilayer, the monolayer or bilayer comprising i) optionally a phospholipid, and ii) a porphyrin coordinated to cobalt to form a cobalt porphyrin. Optionally, the nanostructure also comprises one or more polyhistidine-tagged presentation molecules. At least a portion of the polyhistidine-tag is present in a hydrophobic portion of the monolayer or bilayer, and one or more histidines of the polyhistidine-tag are coordinated to cobalt in the cobalt porphyrin. At least a portion of the polyhistidine-tagged presentation molecule is exposed on the exterior of the nanostructure. The nanostructure may take the form of a liposome surrounding an aqueous compartment. However, the nanostructure may be coated with a hydrophilic or hydrophobic material, such as gold or silica nanoparticles. The cobalt porphyrin may be conjugated to a phospholipid to form a cobalt porphyrin-phospholipid conjugate. The cobalt porphyrin may comprise 1-100 mol % of the monolayer or bilayer, including all 0.1 mol % values and ranges therebetween. For example, the cobalt porphyrin may comprise 1-20 mol % or 5-10 mol % of the monolayer or bilayer. When the cobalt porphyrin comprises 100% of the monolayer or bilayer, there are no phospholipids that are not conjugated to the cobalt porphyrin. The bilayer or monolayer may include a sterol and/or polyethylene glycol. The sterol may be cholesterol.

The number of histidines in the polyhistidine tag in the monolayer or bilayer may be between 2 and 20. For example, the number of histidines in the polyhistidine tag may be between 6 and 10. For example, the number of histidines may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

The liposomes may be spherical or non-spherical. The size of the liposomes may be 50-1000 nm or more. In one embodiment, the liposomes have a size (e.g., a maximum dimension, e.g., a diameter) of 50-1000 nm (including all integer nm values and ranges therebetween). For example, the size may be 50-200 nm, or 20-1000 nm. If the liposome is not spherical, its maximum dimension may be 50-1000 nm. These dimensions can be achieved while maintaining the nanostructure width of the monolayer of the bilayer. The liposomes can carry cargo in their aqueous compartments. The cargo, or a portion thereof, may also or instead be entrapped within the monolayer or bilayer.

In one embodiment, the present disclosure provides a liposome comprising a monolayer or bilayer, wherein the monolayer or bilayer comprises a cobalt porphyrin-phospholipid conjugate, optionally a phospholipid not conjugated to the cobalt porphyrin, and a polyhistidine-tagged presentation molecule, wherein at least a portion of the polyhistidine-tag is present in a hydrophobic portion of the monolayer or bilayer, and one or more histidines of the polyhistidine-tag are coordinated to the cobalt of the cobalt porphyrin-phospholipid conjugate. At least a portion of the polyhistidine-tagged presentation molecule is exposed to the exterior of the nanostructure. The nanostructure (e.g., liposome) can enclose an aqueous compartment. The monolayer or bilayer need not include a phospholipid not conjugated to the cobalt porphyrin, in which case it will only have a cobalt porphyrin-phospholipid conjugate. Cargo can be present in the aqueous compartment. The cargo need not be present exclusively in the aqueous compartment, but part of it may be present in the monolayer or bilayer.

The present disclosure also provides a monolayer or bilayer comprising a phospholipid monomer and a porphyrin coordinated to cobalt (forming a cobalt porphyrin). The monolayer or bilayer has associated therewith one or more polyhistidine-tagged presentation molecules, at least a portion of the polyhistidine tag being present in a hydrophobic portion of the monolayer or bilayer. One or more histidines of the polyhistidine tag are coordinated to the cobalt of the cobalt porphyrin, and at least a portion of the polyhistidine-tagged presentation molecule is present on the exterior of the bilayer or monolayer. In various examples, the monolayer or bilayer encapsulates an aqueous compartment or forms a coating on a nanoparticle (e.g., a gold nanoparticle or a silica nanoparticle).

The present disclosure provides a nanostructure comprising a core and a monolayer or bilayer coating on the core, wherein the monolayer or bilayer comprises a phospholipid and a porphyrin coordinated to cobalt to form a cobalt porphyrin. The nanostructure can have one or more polyhistidine-tagged presentation molecules, wherein at least a portion of the polyhistidine-tag is present in a hydrophobic portion of the monolayer or bilayer and one or more histidines of the polyhistidine-tag are coordinated to the cobalt of the cobalt porphyrin. At least a portion of the polyhistidine-tagged presentation molecule is exposed on the exterior of the nanoparticle. The core of the nanostructure can be a nanoparticle, such as a gold nanoparticle or a silica nanoparticle.

The liposomes, or nanoparticles having a coating or monolayer or bilayer, can have a presentation molecule thereon, which can be an antigenic molecule and/or a targeting molecule, as described herein. The presentation molecule can also provide targeting capability and/or imaging or other functionality.

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 (e.g., diluted serum) at room temperature, greater than 60% of the His-tagged peptide remains bound to the CoPoP-containing bilayer after 24 hours of incubation. In one embodiment, greater than 85% of the His-tagged peptide remains bound to the CoPoP layer after 24 hours of incubation with serum.

CoPoP liposomes or His-tagged CoPoP liposomes can be loaded with cargo. The cargo typically resides in the aqueous compartment, but if it is hydrophobic or has a hydrophobic component, it may reside fully or partially embedded in the bilayer. In addition to having display molecules on the surface, these structures can be used to load cargo in the aqueous compartments within the structure or in the bilayer. Release of cargo from CoPoP liposomes can be triggered by near-infrared (NIR) light. The cargo can be released at the desired location (e.g., by internalization into the target cell or by light-triggered release).

A monolayer or bilayer cobalt porphyrin 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).

The porphyrin portion of the cobalt porphyrin or cobalt porphyrin conjugate that constitutes at least a portion of the liposomal bilayer or other structure includes porphyrin, porphyrin derivative, porphyrin analog, or combinations thereof. Representative porphyrins include hematoporphyrin, protoporphyrin, and tetraphenylporphyrin. Representative porphyrin derivatives include pyropheophorbides, bacteriochlorophylls, chlorophyll A, benzoporphyrin derivatives, tetrahydroxyphenylchlorins, purpurins, benzochlorins, naphthochlorins, verdins, rhodins, ketochlorins, azachlorins, bacteriochlorins, triporphyrins, and benzobacteriochlorins. Representative porphyrin analogs include extended porphyrin family members (e.g., texaphyrins, sapphyrins, and hexaphyrins) and porphyrin isomers (e.g., porphycenes, inverted porphyrins, phthalocyanines, and naphthalocyanines). For example, the cobalt porphyrin may be vitamin B ₁₂ (cobalamin) or a derivative thereof.

In one embodiment, the POP is a pyropheophorbide-phospholipid. The structure of a pyropheophorbide-phospholipid is shown below.

In one embodiment, the layer (monolayer or bilayer) comprises only CoPoP having a His-tagged presentation molecule incorporated therein. In this embodiment, the only phospholipid in the layer is CoPoP (i.e., 100 mol % CoPoP). In one embodiment, the layer (monolayer or bilayer) comprises only CoPoP and porphyrin-conjugated phospholipids (POPs), where the CoPoP has a histidine embedded therein that has a peptide or other presentation molecule attached thereto. In some embodiments, no other phospholipids are present, but the layer (monolayer or bilayer) may optionally comprise a sterol and/or a PEG-lipid.

In one embodiment, in addition to the CoPoP, the bilayer or monolayer also includes a phospholipid that is not conjugated to a porphyrin and therefore not coordinated to Co. Such phospholipids are also referred to herein as "additional phospholipids". The bilayer or monolayer may include a sterol and a PEG-lipid. In one embodiment, the bilayer or monolayer consists essentially of CoPoP, phospholipids not conjugated to a porphyrin, and optionally a sterol and/or PEG, or consists of CoPoP, phospholipids not conjugated to a porphyrin, and optionally a sterol and/or PEG, where PEG may be conjugated to a lipid. In one embodiment, the only metal-PoP in the bilayer is CoPoP, with a His-tagged presentation molecule embedded therein. In one embodiment, the only metal in the bilayer is Co.

In one embodiment, the liposomal bilayer comprises CoPoP and PoP. In addition to CoPoP and PoP, the bilayer may comprise additional phospholipids. The bilayer or monolayer may further comprise a sterol and/or PEG. The PEG may be conjugated to a lipid. In one embodiment, the bilayer consists essentially of CoPoP, PoP, additional phospholipids, and optionally a sterol and/or PEG, or consists of CoPoP, PoP, additional phospholipids, and optionally a sterol and/or PEG, where PEG may be conjugated to a lipid. In one embodiment, the only metal-PoP in the bilayer is CoPoP. In one embodiment, the only metal in the bilayer is Co.

In one embodiment, the CoPoP is present in the nanoparticle at 0.1-10 mol %, with the remaining 99.9-90 mol % being made up of additional lipids (the percentages being relative to the total lipids in the bilayer). For example, the CoPoP combination can be present at 0.1-10 mol %, the sterol can be present at 0.1-50 mol %, and optionally an attenuated lipid A derivative (e.g., monophosphoryl lipid A, or 3-deacylated monophosphoryl lipid A) or related analogue can be present at 0-20 mol % or 0.1-20 mol %, with the remainder being made up of additional phospholipids. The phospholipids can be DOPC, DSPC, DMPC or combinations thereof, and a sterol (if present; the sterol can be cholesterol).

In one embodiment, the combination of CoPoP and PoP may be present in the nanoparticle at 0.1-10 mol %, with the remaining 99.9-90 mol % being constituted by additional phospholipids. For example, the combination of CoPoP and PoP may be present at 0.1-10 mol %, a sterol may be present at 0-50 mol % or 0.1-50 mol %, and optionally PEG may be present at 0-20 mol % or 0.1-20 mol %, with the remainder being constituted by phospholipids. The phospholipids may be DOPC, DSPC, DMPC or combinations thereof, and a sterol (if a sterol is present; the sterol may be cholesterol).

As used herein, a "phospholipid" is a lipid with a hydrophilic head having a phosphate group linked to a hydrophobic lipid tail via a glycerol backbone. The phospholipid comprises an acyl side chain having 6 to 22 carbon atoms, including all integer values of carbon and ranges therebetween. In one embodiment, the phospholipid in the porphyrin conjugate is 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine. The phospholipid of the porphyrin conjugate may comprise, consist essentially of, or consist of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and/or phosphatidylinositol.

In one embodiment, the porphyrin is conjugated to the glycerol group of the phospholipid by a carbon chain linker having 1 to 20 carbon atoms (including all integer values of carbon therebetween).

In various embodiments, the liposome bilayer comprises other phospholipids in addition to the porphyrin conjugates disclosed herein. The fatty acid chains of these phospholipids may contain the appropriate number of carbon atoms to form a bilayer. For example, the fatty acid chains may contain 12, 14, 16, 18, or 20 carbon atoms. In various embodiments, the bilayer comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and/or phosphatidylinositol.

The bilayers and monolayers of the present invention may include a sterol. The sterol may be an animal sterol or a plant sterol. Examples of sterols include cholesterol, sitosterol, stigmasterol, and cholesterol. In one embodiment, cholesterol may be 0 mol% to 50 mol%, or 0.1 to 50 mol%. In other embodiments, cholesterol may be present at 1 to 50 mol%, 5 to 45 mol%, or 10 to 30 mol%.

In some embodiments, the bilayer or monolayer further comprises a PEG-lipid. The PEG-lipid may be DSPE-PEG (e.g., DSPE-PEG-2000, DSPE-PEG-5000, or other sizes of DSPE-PEG). The PEG-lipid is present in an amount of 0-20 mol %, including all % amounts therebetween to the tenth decimal place. The average molecular weight of the PEG component may be between 500-5000 Daltons and all integer values and ranges therebetween.

In one embodiment, the bilayer or monolayer further comprises an adjuvant, such as monophosphoryl lipid A or an attenuated lipid A derivative, such as 3-deacylated monophosphoryl lipid A.

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 may be near the outer surface of the liposome. In one embodiment, at least one end of the polyhistidine-tag is covalently attached to the presentation molecule. In one embodiment, the His tag is a chain of at least two histidines. In one embodiment, the His tag is a chain of 2-20 histidines. In one embodiment, the His tag is a chain of 4-12 histidines and all integer values of histidines therebetween. In one embodiment, it is 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 the other end has a peptide or other molecule attached. It is believed that at least a portion of the His tag is located within the bilayer so as to coordinate to the cobalt metal core.

Liposomes of the present disclosure (without His-tagged molecules) may be substantially spherical and may have a size (e.g., a maximum dimension, such as a diameter) of 30 nm to 250 nm (including all integer values and ranges to nm therebetween). In one embodiment, the liposomes have a size of 100-175 nm. In one embodiment, at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% of the liposomes in the composition have a size of 30-250 nm or 100-175 nm. The liposomes or nanostructures may be larger than 200 nm. In one embodiment, the nanostructures are larger than 1000 nm. In one embodiment, the nanostructures are 200-1000 nm. The liposomes or nanostructures may be spherical or non-spherical. In one embodiment, the maximum dimension of the nanostructures is less than 200 nm while maintaining a monolayer or bilayer nanostructure width. In one embodiment, the nanostructure size exceeds 200 nm in one dimension while maintaining a monolayer or bilayer nanostructure width. In one embodiment, the nanostructure size exceeds 1000 nm in one dimension while maintaining a monolayer or bilayer nanostructure width.

In one aspect, the disclosure provides compositions comprising the liposomes or other structures of the disclosure, or a mixture of different liposomes or other structures. The compositions can also include a sterile, suitable carrier for administration to an individual, including a human, such as a physiological buffer, such as sucrose, dextrose, saline, a pH buffering component (e.g., to achieve a pH of 5-9, a pH of 7-8, a pH of 7.2-7.6 (e.g., 7.4) (e.g., histidine, citrate, or phosphate), and the like. 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 0.1-100 mol % CoPoP liposomes or His-tagged-CoPoP liposomes or other structures (e.g., bilayer-coated nanoparticles). In one embodiment, the composition comprises 0.1-99 mol% CoPoP liposomes having His-tagged presentation molecules associated therewith.

In one embodiment, the composition of the present disclosure does not have a maleimide or succinimidyl ester reactive group. In one embodiment, the tagging molecule attached to the membrane does not have an unnatural amino acid.

A presentation molecule with a His tag may be a small molecule or a large molecule. In one embodiment, the molecule is a peptide or peptide derivative. In one embodiment, the molecule is a polypeptide, a polynucleotide, a carbohydrate or a 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 the polypeptide. In one embodiment, the molecule is an antigen, such as a peptide (2-50 amino acids and all peptides between 2 and 50 amino acids in length) or a polypeptide (50-1000 amino acids and all polypeptides between 50 and 1000 amino acids in length) or a protein (greater than 1000 amino acids). The peptide, polypeptide or protein may have only naturally occurring amino acids, or may consist of a mixture of naturally occurring and non-naturally occurring amino acids, or may have only non-naturally occurring amino acids.

The presentation molecule attached to the His tag may be an antigenic molecule, a targeting molecule, a therapeutic molecule, a diagnostic molecule, or a molecule that provides another type of function. The tagged molecule may be used for targeting, i.e., to guide a structure having a monolayer or bilayer to its target site. For example, a peptide ligand may be attached to the His tag to guide a liposome (or other structure) to a cell that has a receptor or recognition molecule for the ligand. In one embodiment, the attached peptide may provide an alternative or additional function. For example, the attached peptide may provide a therapeutic, diagnostic, or immunogenic function.

In certain embodiments, the presentation molecule may be a targeting molecule (e.g., an antibody, a peptide, an aptamer, or other molecule such as folate). The term "targeting molecule" is used to refer to a molecule that can direct a bilayer structure, such as a liposome, to a specific target (e.g., by binding to a receptor or other molecule on the surface of a target cell). The targeting molecule may be a protein, peptide, nucleic acid molecule, sugar or polysaccharide, receptor ligand, or other small molecule. The degree of specificity can be modulated through the choice of the targeting molecule. For example, antibodies usually exhibit high specificity. They may be polyclonal, monoclonal, fragments, recombinant, single chain, or nanobodies, many of which are commercially available or can be easily obtained using standard techniques.

The presentation molecule may be an antigenic molecule (i.e., a molecule having an antigenic epitope). In one embodiment, the molecule is a peptide. In one embodiment, the peptide is an RGD-containing peptide sequence. Such a sequence may be 7-20 amino acids or longer that have an epitope. The peptide may be a fragment of a polypeptide or protein that is part of a microorganism, such as a pathogenic microorganism (e.g., a virus, a bacterium, a parasite, or a fungus), or may include an epitope of such a polypeptide or protein. Examples include respiratory syncytial virus (RSV), Borrelia burgdorferi (referred to herein as Lyme disease Borrelia), influenza virus, and the like. The peptide may be a fragment of a polypeptide or protein that is not generally immunogenic (e.g., a viral protein that is not known to be immunogenic in nature). The peptide may be a fragment of an HIV antigen, such as the HIV outer envelope protein, or may include an epitope of an HIV antigen. In one embodiment, the HIV antigen is gp41. For example, the peptide may be the membrane proximal external region (MPER) of the gp41 envelope. Further examples of antigens include, but are not limited to, DS-Cav1 for RSV, OspA for Lyme disease, and hemagglutinin and neuraminidase for influenza.

In one embodiment, the present disclosure provides an antigenic composition. The composition includes a structure having a bilayer, in which an antigen having a histidine tail is non-covalently conjugated to a cobalt porphyrin (or a cobalt porphyrin phospholipid) such that the His tag is embedded in the bilayer and one or more epitopes of the antigen are surface exposed. The composition may include adjuvants and other carriers known in the art. Examples of adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, monophosphoryl lipid A (MPL), aluminum phosphate, aluminum hydroxide, alum, phosphorylated hexaacyl disaccharide (PHAD), Sigma adjuvant system (SAS), or saponin. Other carriers, such as wetting agents, emulsifiers, and fillers, can also be used. AddaVax (Invitrogen), or saponin. Other carriers such as wetting agents, emulsifiers, fillers, etc. may also be used.

A wide variety of cargoes can be loaded into the liposomes or other structures of the present disclosure. The cargoes can be delivered to a desired location using near infrared light. For example, bioactive or therapeutic agents, pharmaceutical agents, or drugs can be encapsulated inside CoPoP liposomes. This includes water-soluble drugs and drugs that are weak acids or bases that can be loaded via a chemical gradient and concentrated in the aqueous core of the liposome. Thus, in various embodiments, the liposomes contain an active agent encapsulated therein (e.g., a therapeutic and/or diagnostic agent, which may be a chemotherapeutic agent such as doxorubicin). The chemotherapeutic agent doxorubicin can be actively loaded and released with NIR irradiation, providing a strong and direct light-triggered release with CoPoP liposomes.

In one embodiment, the lipid to drug (or any other cargo substance) ratio is 10:1 to 5:1. In various embodiments, the lipid to drug/cargo ratio is 10:1, 9:1, 8:1, 7:1.6:1, or 5:1. The lipids used to calculate the ratio include all lipids, including phospholipids that are part of the porphyrin-phospholipid conjugate, additional phospholipids, or sterols, and PEG-conjugated lipids (if present). Sometimes in this disclosure the cargo is described as a drug, but the description can equally apply to any substance included for treatment and/or delivery to a desired site, and the term "drug" refers to any substance. The substance may be included in whole or in part inside or within the PoP-liposome, which may be present in the aqueous compartment, the bilayer, or both.

In one embodiment, the cargo loaded in the liposome or other carrier is a therapeutic agent. The term "therapeutic agent" is an art-recognized term and refers to any chemical moiety that is biologically, physiologically, or pharmacologically active. Examples of therapeutic agents, also referred to as "drugs", are described in well-known references such as the Merck Index, Physicians Desk Reference, and the Pharmacological Basis of Therapeutics, and include, but are not limited to, medicines; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure, or mitigate disease or illness; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a physiological environment. Various forms of therapeutic agents can be used that are capable of being released from the composition into adjacent tissues or fluids upon administration to a subject. Known drugs that are loaded by activity gradient include doxorubicin, daunorubicin, gemcitabine, epirubicin, topotecan, vincristine, mitoxantrone, ciprofloxacin, and cisplatin. Therapeutic cargo also includes various antibiotics (e.g., gentamicin) or other substances that are effective against infections caused by bacteria, fungi, parasites, or other organisms. These drugs can be loaded into CoPoP liposomes and released.

In one embodiment, the cargo loaded into the liposome is a diagnostic agent. A "diagnostic agent" or "diagnostic agent" is any chemical moiety that can be used for diagnosis. For example, diagnostic agents include imaging agents, such as those containing radioisotopes such as indium or technetium; contrast agents containing iodine or gadolinium; enzymes, such as horseradish peroxidase, GFP, alkaline phosphatase, or β-galactosidase; fluorescent substances, such as europium derivatives; and luminescent substances, such as N-methylacridium derivatives.

The cargo may be two or more substances. For example, the cargo may include a combination of diagnostic agents, therapeutic agents, immunogenic agents, and/or imaging agents, and/or other types of substances. It is also possible for the same substance to have multiple functions. For example, a substance may be diagnostic and therapeutic, or a substance may be imaging and immunogenic, etc.

The structures formed by the layers of the present disclosure are serum stable. For example, in vitro, the binding stability of the His tag to the CoPoP bilayer is stable even when incubated in 50% bovine serum at room temperature for 24 hours. Thus, these structures are stable under serum or concentrated serum or diluted serum conditions.

The present disclosure also provides methods of using the structures having a bilayer described herein. In one embodiment, the disclosure provides a method of eliciting an immune response in a host. The immune response may generate antibodies. The method includes administering to an individual a composition comprising a structure having a CoPoP bilayer conjugated with a histidine-tagged antigen. The composition can be administered by any standard route for immunization, including subcutaneous, intradermal, intramuscular, intratumoral, or any other route. The composition can be administered in a single dose or in multiple doses, including booster shots. Antibody titers can be measured to monitor the immune response.

The nanostructures of the invention can be used to reduce antibody titers against a desired antigen. For example, where it is desired to reduce immunogenicity, nanostructures having phospholipids present therein, including PS (or otherwise), can be used. Compositions containing these nanostructures can be administered to reduce immunogenicity.

In one aspect, the present disclosure provides a method for delivering a substance contained as cargo in a liposome or other nanostructure to a desired location. The substance may be contained in whole or in part in or within a CoPoP liposome, which may be present in the aqueous compartment, the bilayer, or both. The method includes: 1) preparing a composition comprising a liposome or other structure having a bilayer of the present disclosure, optionally containing a cargo (such as an active agent); 2) allowing the liposome to reach a selected or desired location; and 3) irradiating the liposome with radiation having a near infrared wavelength under conditions such that at least a portion of the cargo is released from the liposome. Alternatively or additionally, the cargo may reach the interior of a cell by internalization of the liposome, followed by intracellular processes that release the cargo.

The liposomes may be irradiated with near infrared light from a laser with a wavelength of 650-1000 nm (including all integer values and ranges up to mW/cm2 in between) and a power output of 50-1000 mW/ ^cm2 (including all integer values and ranges up to mW/ ^cm2 in between). In another embodiment, the wavelength is 650-800 nm, including all integer values and ranges up to mW/cm2 in between. The liposomes may be irradiated for up to 30 minutes or less. In various embodiments, the liposomes may be irradiated in vitro or in vivo for a period of 0.5-30 minutes and all decimal points 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 a wavelength of 665 nm or 671 nm. Infrared light may be delivered directly to the desired area by directing a laser beam at the area or a fiber optic probe may be used. In the case of tumors, a fiber optic probe can be inserted (ie, by catheter or endoscopic device) into the tumor for localized irradiation.

In one aspect, the disclosure provides a method for preparing a bilayer comprising CoPoP. Free base PoP can be prepared by esterification of a monocarboxylate porphyrin such as pyropheophorbide-a with 2-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-C16-PC, Avanti #855675P) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine in a molar ratio of lyso-C16-PC:Pyro:EDC:DMAP=1:1:2:2 in chloroform by stirring overnight at room temperature. The PoP is then purified by silica gel chromatography. CoPoP can be made by contacting a 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.

In one embodiment, the present disclosure provides a method for coating nanoparticles with a cobalt porphyrin (e.g., CoPoP) bilayer or monolayer. The method generally involves hydrating the nanoparticles with a solution of lipids to disperse the particles in water.

To deliver the cargo to a desired location or for general administration, a composition comprising the liposomes in a suitable carrier can be administered to an individual by any suitable route. In one embodiment, it is administered by intravenous infusion so that it enters the vasculature (circulatory system). The composition may be administered systemically or directly into the blood supply to a specific organ or tissue or tumor. When irradiated with NIR, the contents of the PoP liposomes may be released in the circulatory system and then enter the surrounding tissues.

In the following description, various examples of the nanostructures, compositions and methods of the present disclosure are disclosed.
1. Nanostructures (e.g., liposomes),
a) a monolayer or bilayer having i) optionally a phospholipid, and ii) a porphyrin coordinated to cobalt to form a cobalt porphyrin; and b) optionally a polyhistidine-tagged presentation molecule, where at least a portion of the polyhistidine-tag is present in a hydrophobic portion of the monolayer or bilayer and one or more histidines of the polyhistidine-tag are coordinated to the cobalt of the cobalt porphyrin, where at least a portion of the polyhistidine-tagged presentation molecule is exposed to the exterior of the nanostructure (e.g., a liposome), and where the liposome is a liposome, the liposome surrounds an aqueous compartment;
A nanostructure comprising:
2. The nanostructure (eg, liposome) according to claim 1, wherein the cobalt porphyrin is conjugated to a phospholipid to form a cobalt porphyrin-phospholipid conjugate.
3. The nanostructure (eg, liposome) according to claim 2, wherein the cobalt porphyrin-phospholipid conjugate constitutes 1 to 25 mol % of the monolayer or bilayer.
4. The nanostructure (eg, liposome) according to claim 3, wherein the cobalt porphyrin-phospholipid conjugate constitutes 5-10 mol % of the monolayer or bilayer.
5. The nanostructure (eg, liposome) of any one of claims 1 to 4, wherein the bilayer further comprises a sterol (eg, cholesterol).
6. The nanostructure (eg, liposome) of any one of claims 1 to 4, wherein the bilayer further comprises phosphatidylserine and, optionally, cholesterol.
7. The nanostructure (eg, liposome) according to any one of claims 1 to 4, wherein the polyhistidine tag comprises 6 to 10 histidine residues.
8. The nanostructure (eg, liposome) according to any one of 1 to 4 above, wherein the size of the liposome is 50 nm to 200 nm.
9. The nanostructure (e.g., liposome) of any one of claims 1 to 4, wherein the nanostructure (e.g., liposome) comprises a cargo and, if a liposome, at least a portion of the cargo is present within an aqueous compartment of the liposome.
10. The nanostructure (e.g., liposome) according to any one of 1 to 9 above, wherein the presentation molecule is a peptide consisting of 4 to 50 amino acids (the number of amino acids does not include histidines of a His tag).
11. The nanostructure (eg, liposome) according to any one of 1 to 10 above, wherein the presentation molecule is a protein of 4 to 500 kDa.
12. The nanostructure (e.g., liposome) according to any one of claims 1 to 11, wherein the presentation molecule is an antigen molecule and the monolayer or bilayer further comprises an adjuvant incorporated therein.
13. The nanostructure (e.g., liposome) according to claim 12, wherein the adjuvant is an attenuated lipid A derivative.
14. The nanostructure (eg, liposome) according to claim 13, wherein the attenuated lipid A derivative is monophosphoryl lipid A or 3-deacylated monophosphoryl lipid A.
15. A nanostructure comprising:
a) a core; and b) a monolayer or bilayer on the core, the monolayer or bilayer having i) optionally phospholipid monomers, and ii) a porphyrin coordinated to cobalt to form a cobalt porphyrin (e.g., CoPoP); and c) optionally a polyhistidine-tagged presentation molecule, where at least a portion of the polyhistidine-tag is present in a hydrophobic portion of the monolayer or bilayer, one or more histidines of the polyhistidine-tag are coordinated to the cobalt of the cobalt porphyrin, and where at least a portion of the polyhistidine-tagged presentation molecule is exposed to the exterior of the nanostructure;
A nanostructure comprising:
16. The nanostructure according to claim 15, wherein the core is a gold nanoparticle.
17. A method for targeted delivery of a cargo, comprising:
a) administering to an individual a composition comprising a nanostructure (e.g., liposome) as described in any one of claims 9 to 16, or a combination of nanostructures (e.g., liposomes) as described in any one of claims 9 to 16, in a pharmaceutical carrier; and b) after a suitable amount of time has passed to allow the nanostructures (e.g., liposomes) to reach a desired location in the individual, exposing the liposomes to near infrared radiation of wavelengths between 650 and 1000 nm to release cargo from the liposomes.
18. The method according to claim 17, wherein the individual is a human or a non-human animal.
19. A method of generating an immune response in a host individual, comprising administering to the individual a composition comprising a nanostructure (e.g., liposome) according to any one of 1 to 16 above, or a combination nanostructure (e.g., liposome) according to any one of 1 to 16 above, in a pharmaceutical carrier, wherein the presentation molecule comprises an immunogenic epitope.
20. The method according to claim 19, wherein the presentation molecule is a peptide, polypeptide or protein derived from a pathogenic microorganism.
21. The method according to claim 19 or 20, wherein the individual is a human or a non-human animal.

The following examples are presented to illustrate the present disclosure and are not intended to be limiting in any way.

This example describes the synthesis and functionalization of cobalt porphyrin-phospholipid (CoPoP) bilayers with histidine-tagged ligands and antigens.

Materials and Methods Materials were obtained from Sigma unless otherwise noted. Peptides were obtained from commercial vendors and purity was determined by HPLC and identity was confirmed by mass spectrometry.

For protein binding, recombinant heptahistidine-tagged Cerulean-Venus fusion reporter proteins were produced in E. coli, purified, and characterized as previously described. Stoichiometry estimates are based on the assumption that each ∼100 nm liposome contains 80,000 lipid molecules.

Preparation of PoP-lipids, PoP-liposomes, and PoP-gold. Free base (2H)PoP sn-1-palmitoyl sn-2-pyropheophorbide phosphatidylcholine was synthesized as described above. CoPoP was prepared by stirring 100 mg of 2H-PoP with a 10-fold molar excess of cobalt(II) acetate tetrahydrate in 4 mL of methanol for 17 h in the dark. Completion of the reaction and purity of the product were monitored by TLC (>90% purity). The solvent was then removed by rotary evaporation and PoP was extracted three times with chloroform:methanol:water (1:1.8:1). The chloroform layer was collected, the solvent removed by rotary evaporation, and the product was lyophilized in a solution containing 20% water in tert-butanol to give 81.5 mg of product (77% yield) (identity was confirmed by mass spectrometry). Other metal-PoPs were synthesized using the same method. For Ni-PoP, Ni(II) acetate tetrahydrate was used and incubated for 17 hours. For Zn-PoP, Zn(II) acetate anhydride 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.

PoP-liposomes were formulated at 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 a 100 nm polycarbonate membrane (VWR #28157-790) using a mini-extruder (Avanti #610000). For protein and peptide binding analysis, liposomes were formed using 10 mol% PoP, 85 mol% DOPC (Avanti #850375P), and 5 mol% PEG-lipid (Avanti #880120P). Ni-NTA liposomes contained 10 mol% Ni-NTA lipid dioleoyl-glycero-Ni-NTA (Avanti #790404P) and 10 mol% 2H-PoP. Liposomes incorporating free Co-porphyrin contained 10 mol% Co-pyropheophorbide with 85 mol% DOPC and 5 mol% PEG-lipid. Co-NTA-liposomes were prepared using liposomes containing 10 mol% dioleoyl-glycero-NTA (Avanti #790528P). Liposomes were incubated with 20 mg/mL cobalt(II) chloride for 2 hours and then dialyzed against PBS. Sulforhodamine B loaded 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, followed by freeze-thawing and sonication. Unencapsulated dye was removed by a 10 mL Sephadex G-75 (VWR #95016-784) column followed by dialysis in PBS. 50 mM dye was used for bilayer integrity and quantitative cell binding studies, and 10 mM dye was used for microscopy studies.

For gold coating, 60 nm citrate-stabilized gold nanoparticles (Ted Pella #15709-20) were used to hydrate a 1 mg lipid film consisting of 45 mol% distearoylphosphocholine (Avanti #850365P), 45 mol% distearoylphosphoglycerol (Avanti #840465X) and 10 mol% PoP. After brief vortexing and sonication, the sample was repeatedly centrifuged for 15 min at 1500 relative centrifugal force (rcf). The supernatant was discarded and the pellet was resuspended and recentrifuged two more times. The PoP gold was resuspended in water for further analysis.

Polypeptide binding
1 μg of fluorescent reporter protein was incubated with 20 μg of liposomes in 200 μL of PBS in a 96-well plate. Fluorescence of the FRET channel (ex: 430 nm, em: 525 nm) was measured periodically using a fluorescence microplate reader (Tecan Infinite II). Data were normalized to the FRET signal in protein without added liposomes. EMSA experiments were performed by incubating 2.5 μg of protein with 50 μg of liposomes, followed by electrophoresis in a 0.75% agarose gel (applied 50V for 90 min) and imaging with the indicated excitation and emission filters by an IVIS Lumina II system. For serum stability studies, 3 mg of protein was pre-incubated with 60 mg of liposomes in 40 ul PBS. After 24 h of incubation, 40 uL of FBS was added and incubated for another 8 h. For imidazole displacement experiments, 1 μg of reporter protein was coupled to 20 μg of liposomes in PBS. Imidazole was then titrated and binding was assessed by fluorescence. For serum stability, 1 μg of reporter protein was coupled to 20 μg of liposomes in 100 μL of PBS, after which an equal volume of fetal bovine serum (VWR #82013-602) was added and binding was monitored by fluorescence. After incubation of 500 ng of peptide with 20 μg of liposomes, peptide binding was assessed by RGD-His FAM fluorophore quenching.

Targeting Experiments
U-87 and MCF-7 cell lines were obtained from ATCC and cultured according to the supplier's protocol. 2 × ¹⁰⁴ cells were seeded overnight in a well of a 96-well plate. 500 ng of RGD-His peptide was conjugated to 20 μg of sulforhodamine B-loaded liposomes, and the liposomes were incubated with the cells for 2 h. The medium was removed, the cells were washed three times with PBS, and then the cells and liposomes were lysed with 1% Triton X-100 solution. Liposome uptake was assessed by measuring sulforhodamine B fluorescence.

For confocal imaging, ¹⁰⁴ cells were seeded overnight on Nunc chamber slides (Nunc #155411) in DMEM containing 10% fetal bovine serum (FBS). 20 μg of liposomes were added to the serum-containing medium and incubated for 2 h. The medium was removed and cells were washed three times with PBS. Fresh medium was added and cells were imaged by microscopy using a Zeiss LSM 710 confocal fluorescence microscope. Gold imaging was performed in the same manner, except that 633 nm light was used for both excitation and emission for backscatter imaging. After peptide conjugation, the gold was centrifuged to remove unbound RGD peptide.

For in vivo experiments, animals were treated in accordance with the policies and approval of the Institutional Animal Care and Use Committee (IACUC) of the University at Buffalo. Five-week-old female athymic nude mice (Jackson Labs) were inoculated with U87 cells in the flank and treated when tumor growth reached 4-5 mm in diameter. Mice were injected intravenously with 200 μL of sulforhodamine B-loaded liposomes (1 mg/mL lipid) targeted with or without cRGD-His. Mice were sacrificed 45 min after injection and organs were extracted, weighed, mechanically homogenized in 0.2% Triton X-100 solution, and fluorescence was assessed to determine biodistribution.

Vaccination. Unless otherwise stated, 8-week-old female BALB/c mice (Harlan Laboratories) were injected with 25 μg of MPER peptide in 50 μL of sterile PBS in the posterior ventral footpad on days 0 and 14. Where indicated, injections also contained 25 μg of MPL (Avanti #699800P) or TDB (Avanti #890808P) in liposomes, which contained a molar ratio of DOPC:cholesterol:MPL:PoP of 50:30:5:5. For Freund's adjuvant, peptides were mixed directly in complete Freund's adjuvant (Fisher #PI-77140) and injected. Four weeks after the first injection or at the indicated time points, blood was collected from the submandibular vein and serum was obtained after clotting and centrifugation at 2000 rcf for 15 min and stored at −80°C.

Anti-MPER titers were assessed by ELISA in 96-well streptavidin-coated plates (GBiosciences #130804). 1 μg His-tag-free MPER biotin in 100 μL PBS containing 0.1% Tween 20 (PBS-T) was incubated in the wells for 2 h at 37°C. The wells were then washed five times with PBS-T and serially diluted mouse serum was added in PBS containing 0.1% casein (PBS-C) and incubated for 30 min at 37°C. The wells were then washed five times with PBS-T, after which 100 μL of goat anti-mouse IgG-HRP (GenScript #A00160) diluted in PBS-C was added to the wells to a final concentration of 1 μg/mL secondary antibody and incubated for 30 min at 37°C. Wells were washed five times with PBS-T, after which 100 μL of tetramethylbenzidine substrate solution (Amresco #J644) was added to each well and incubated at 37°C for 20 min. The reaction was stopped with 100 μL of 1 M HCl and absorbance was measured at 450 nm. Titers were defined as the reciprocal of the dilution at which the absorbance at 450 nm exceeded the same dilution of non-serum background by 0.05 absorbance units. All samples were measured in duplicate and averaged.

Viral entry experiments Viral entry experiments were performed as described above. Briefly, HIV-1 was produced by cotransfection of pHXB2-env and pNL4-3.HSA.RE into 293T cells. Two days after transfection, the cell medium was passed through a 0.45 μm filter and centrifuged. The viral pellet was dried, resuspended in 600 μL of PBS, and stored at −80°C. The infectious titer of HIV-1 stock was determined by X-Gal staining as previously described.

Sera from three mice immunized with MPER and CoPoP liposomes were pooled and IgG was isolated using immobilized protein G beads (VWR #PI20398) according to the supplier's protocol. Concentrations were determined by absorbance by Bradford assay. 2F5 was obtained from the Free NIH AIDS Reagent Program. ^1x104 TZM-bl receptor cells per well were plated in 96-well plates the day before infection. HIV (multiplicity of infection 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 at 37°C in a 5% _CO2 incubator for 2 days. Cell viability was then measured using the CellTiter-Fluor Assay (Promega) according to the manufacturer's protocol. Viral entry levels were then measured by a luciferase assay system (ONE-Glo, Promega) according to the manufacturer's protocol and normalized to virus-only samples. Data were further normalized to cell viability (all groups showed viability within 10% of control untreated cells).

result

His-tagged proteins bind to CoPoP liposomes A series of sn-1-palmitoyl sn-2-pyropheophorbide phosphatidylcholine chelates were prepared with the transition metals Co, Cu, Zn, Ni, and Mn (Fig. 1c). PoP bilayers were then formed by extrusion of 100 nm liposomes with 10 mol% metal PoPs together with 85 mol% dioleoylphosphocholine (DOPC) and 5 mol% polyethylene glycol-conjugated distearoylphosphoethanolamine (PEG-lipid). His-tagged protein binding to the PoP bilayer was assessed using a fluorescent protein receptor. As shown in Fig. 2a, the system contained a fusion protein consisting of two linked fluorescent proteins, Cerulean (blue emission) and Venus (green emission). Due to their close linkage and spectral overlap, Cerulean serves as a Förster resonance energy transfer (FRET) donor for Venus, and excitation of Cerulean results in FRET emission from Venus. Cerulean was labeled at its C-terminus with a heptahistidine tag. However, when bound to a PoP bilayer, energy transfer from Cerulean is diverted to the bilayer itself (absorbed in the Cerulean emission range) and therefore competes with FRET to Venus. On the other hand, Venus is not directly attached to the photonic bilayer and is therefore not fully quenched upon direct excitation, allowing tracking of the bound fusion protein.

A three-color electrophoretic mobility shift assay (EMSA) was developed to assess the binding of reporter fusion proteins to various PoP liposomes. 2.5 μg of protein was incubated with 50 μg of various PoP liposomes for 24 h and then subjected to agarose gel electrophoresis. As shown in the top image of Figure 2b, when PoP liposomes were imaged, only the free base (2H) liposomes and Zn-PoP were easily visualized, and to a lesser extent the Zn-PoP liposomes. This demonstrates that the metal has a quenching effect on PoP and confirms that they were stably chelated in the bilayer. As expected, the liposomes showed the smallest electrophoretic mobility due to their relatively large size. The same gel was then imaged using cerulean excitation and Venus emission to probe for inhibition of FRET (indicative of fusion protein binding to PoP liposomes). All samples showed the same amount of FRET and traveled the same distance as the free protein, with the exception of the protein incubated with CoPoP liposomes, where FRET disappeared completely (middle images). To verify the presence of the protein, Venus was directly excited and imaged. Only with CoPoP liposomes did the reporter protein colocalize with the liposomes. Together, these images demonstrate that the protein quantitatively bound to the CoPoP liposomes. Solution-based studies confirmed this finding (Fig. 2c). Of all types of PoP liposomes tested, only CoPoP caused a significant decrease in the FRET efficiency between Cerulean and Venus due to liposome binding. This binding took only three hours to reach 50% binding (t _1/2 ), but required almost a day to be fully completed. Molecular dynamics simulations of 2H-PoP bilayers showed that the porphyrin center (where metal chelation occurs) was inaccessible to the aqueous phase surrounding the bilayer. Therefore, this slow binding may be due to the His tag being partially buried by the rest of the protein and having to make its way into the protected hydrophobic bilayer.

Coordination of Polyhistidine with CoPoP The mechanism underlying His-tag binding to immobilized metals involves metal coordination with the nitrogen-containing imidazole groups of histidine residues. The lack of His-tag binding to liposomes formed with Ni(II), Cu(II), Zn(II) and Mn(II)PoPs is likely related to the binding affinity or coordination number of the axial ligand in the porphyrin. For example, it has been proposed that Ni(II) and Cu(II) porphyrin chelates can fully coordinate to the four surrounding macrocyclic nitrogen atoms without an axial ligand. For Zn(II) and Mn(III) porphyrins, the ligand binding strength appears insufficient to provide a stable polyhistidine bond.

To determine the electronic state of CoPoP, paramagnetism was evaluated. Because Co(II) is paramagnetic, but Co(III) porphyrin is low spin and diamagnetic, NMR was used to examine the peak broadening induced by paramagnetic species. As shown in Figure 2d, peak broadening was observed only for CoPoP and only in organic solvents, based on the hydrogens attached to each carbon of the vinyl group in PoP. When CoPoP was formed in aqueous liposomes, the peak narrowed, indicative of oxidation to diamagnetic Co(III) within the bilayer. To further verify this mechanism, a reducing agent, sodium sulfite, was added to CoPoP liposomes after they quantitatively bound fluorescently labeled His-tagged peptide. As shown in Figure 2e, 2 M sulfite induced peptide release from CoPoP liposomes. Liposomes were also formed using commercially available Ni-NTA lipids. His-tagged peptides did not bind strongly to Ni-NTA liposomes. As expected from the non-reducible Ni(II), no peptide release was observed upon addition of sulfite to the system. Together, these data suggest that CoPoP undergoes a Co(II) to Co(III) transition during the formation of CoPoP liposomes, and that the polyhistidine imidazole group coordinates with the chelated Co(III) in PoP within the bilayer.

Stable His-tag binding to CoPoP liposomes We then used fluorescent reporter proteins to compare the binding of His-tagged proteins to liposomes containing either CoPoP or Ni-NTA-lipids (Fig. 3a). To allow for FRET-based protein binding measurements, Ni-NTA liposomes contained 10 mol% 2H-PoP. By EMSA, the protein migrated unimpeded in both the FRET and protein channels when incubated without liposomes or with 2H-PoP liposomes. When incubated with Ni-NTA liposomes, protein migration was only slightly inhibited, suggesting that protein binding was not resistant to the electrophoretic conditions. The FRET channel was not quenched, confirming the absence of binding to Ni-NTA liposomes. In contrast, when incubated with CoPoP liposomes, the protein was stably bound, the FRET channel was completely abolished, and the electrophoretic mobility was reduced (consistent with the protein remaining bound to the liposomes).

A challenging obstacle to using Ni-NTA-lipid for biomedical applications is that it does not maintain stable His-tag binding in biological media such as serum. To test whether liposomes can maintain binding in the presence of serum, fetal bovine serum was added at a 1:1 volume ratio to a solution of liposomes conjugated with His-tagged proteins. As shown in Figure 3b, Ni-NTA liposomes did not completely capture all proteins, which is consistent with the weak binding shown in the EMSA results. Moreover, after the addition of serum, all binding disappeared over a period of 24 h. Under the same conditions, CoPoP liposomes stably captured His-tagged reporter proteins without substantial protein release.

Because histidine side chains contain imidazole groups, we used an imidazole competition assay to compare the binding stability of Ni-NTA and CoPoP liposomes to His-tagged polypeptides. As shown in Figure 3c, CoPoP liposomes maintained over 75% binding to the reporter protein even at imidazole concentrations approaching 1 M. This represents a roughly 10-million-fold excess of imidazole over the 100 nM protein concentration used in the binding studies. In contrast, Ni-NTA liposomes released over 90% of the His-tag in the presence of only 30 mM imidazole. The dramatically stronger binding of CoPoP liposomes to the His-tag can be attributed to at least two factors: the excellent and stable chelation of Co(III) to the imidazole groups and the protected hydrophobic environment of the CoPoP bilayer, which limits access to competing external molecules.

Liposomes formed with Ni-NTA-lipid, cobalt-chelating Co-NTA-lipid, and CoPoP were able to bind the fluorescent peptide in solution (Fig. 7a). However, the binding of Co-NTA and Ni-NTA was not maintained during gel filtration chromatography (Fig. 7b). Liposomes formed with Co-NTA and Ni-NTA released the peptide when incubated in serum, whereas CoPoP did not (Fig. 7c). This demonstrates the significance of the polyhistidine bond trapped in the bilayer. We next tested whether CoPoP was required for stable binding in serum or whether a simple cobalt porphyrin (Co-pyro) inserted into the liposome was sufficient. After initial binding, incubation with serum displaced the polypeptide from the liposome (Fig. 8). This result is consistent with recent demonstration that membrane-inserted porphyrins, but not PoP, were readily exchanged with serum components and moved out of the liposome. Taken together, these results suggest an important role for CoPoP in stably binding His-tagged polypeptides.

Binding of peptides to CoPoP liposomes

Peptide targeting has attracted interest for use as disease- and tissue-specific "zip codes." The short RGD tripeptide found in fibronectin and vibronectin is a promising targeting ligand due to its efficient binding to integrin _αvβ3 expressed on tumor vascular endothelial cells. CoPoP liposomes were tested to verify whether they could be delivered to molecular receptors on target cells by a His-tagged ligand approach using the linear single amino acid sequence GRGDSPKGAGAKG-HHHHHHH (SEQ ID NO: 1). Carboxyfluorescein (FAM) was labeled at the N-terminus, allowing detection of binding to PoP-liposomes by FRET. It was demonstrated that the linear RGD peptide can be labeled with a fluorophore without interfering with integrin binding. When the peptide was incubated with various metal-PoP liposomes, only CoPoP bound to the peptide, as shown in Figure 4a. Compared to protein binding, peptide binding was about 5 times faster. Presumably, the smaller size, faster molecular motion, and reduced steric hindrance of the peptides allowed them to fit faster into the bilayer, interact with CoPoP, and bind irreversibly. Based on previous estimates that each ∼100 nm liposome contains about 80,000 lipids, this equates to 8000 CoPoPs and 750 peptides per liposome. Since each peptide contains seven histidine residues, the ratio of CoPoP to histidine in the bilayer was 1:0.66. In conventional His-tag binding to Ni-NTA, only the i-th and i+2 or i+5-th histidine residues of all residues of the His-tag are thought to be involved in coordination with the metal. The porphyrin and polyhistidine density in the CoPoP bilayer is likely to be higher, and therefore the coordination mechanism may be different.

The effect of His-tag length on peptide binding to CoPoP liposomes was examined. A series of N-terminally FAM-labeled peptides were synthesized with varying lengths of His-tag attached to the C-terminus. As demonstrated in Fig. 4b, no peptide binding was observed when the His-tag was removed from the peptide. With two histidine residues, binding was slow, with an association t _1/2 of approximately 10 h. As the His-tag was lengthened, the binding rate increased rapidly. With six residues, equivalent to a common hexahistidine tag, the binding t _1/2 was less than 1 h. Increasing the length of the His-tag to 10 residues reduced the binding t _1/2 to 20 min.

Next, the lipid composition was varied to determine the effect of membrane fluidity on His-tag binding. Liposomes were formed with either 90 mol% DSPC, DMPC or DOPC and 10 mol% CoPoP. Alternatively, 50 mol% cholesterol was incorporated into the bilayer and the amount of standard lipid used was reduced accordingly. DSPC forms a rigid gel-phase bilayer at room temperature, whereas DMPC and DOPC have a lower transition temperature and are in the liquid crystalline phase. Cholesterol occupies space in the bilayer and may have a moderating effect on membrane fluidity. Interestingly, no significant differences in peptide binding kinetics were observed for membranes of different compositions with or without cholesterol (Figure 4c). The peptide binding process may occur in a multi-step process and once the peptide begins to insert into the bilayer, polyhistidine cooperativity is not affected by lipid composition. However, in 5 mg/mL bovine serum albumin (BSA), significant differences were observed whether the membrane had cholesterol or not (Figure 4d). The slower binding in cholesterol-free liposomes was likely due to greater interaction of BSA with the membrane, which interfered with peptide binding. The binding half-life was not reached at 50 mg/mL BSA, and serum completely inhibited binding (Figures 9 and 10).

Biotargeting of Cargo-Laden Liposomes Given the binding efficacy of His-tagged peptides 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 a self-quenching concentration and membrane permeabilization was examined. As shown in Figure 5a, dye-loaded liposomes did not release substantial amounts of dye over 8 hours, and the peptide was fully bound to the liposomes. At the 24 hour time point, CoPoP liposomes with bound peptide released less than 10% of the dye. Thus, the His-tag insertion and binding process was gentle and non-destructive enough to keep the bilayer integrity and encapsulated cargo intact. A similar palmitoylated lipopeptide resulted in permeabilization of cargo-loaded liposomes upon incubation (Figure 11), further demonstrating the robustness of the His-tag approach.

Next, we evaluated whether RGD-decorated liposomes could bind to their molecular targets using an established cell line pair, U87 glioblastoma cells (RGD-conjugated) and MCF7 breast cancer cells (RGD-unconjugated). After encapsulation of sulforhodamine B, liposomes were first incubated with His-tagged RGD peptides and then with both cell lines. Approximately 550 peptides were attached to each liposome. Liposome uptake was assessed by examining intracellular fluorescence after washing and lysis (to remove cargo self-quenching effects). As shown in Figure 5b, high liposome uptake was observed in U87 cells incubated with targeted CoPoP liposomes, whereas negligible binding occurred in MCF7 cells. As expected, in the absence of RGD targeting ligand, no uptake occurred in any of the cell lines. Inclusion of PEG-lipids in the liposome formulation resulted in liposomes that did not target any of the cell lines. The presence of PEG appears to have an interfering effect on the peptide being tethered directly to the bilayer surface. The presence of excess free RGD peptide was able to inhibit binding, confirming the targeting specificity of this approach. Confocal microscopy verified these binding results (Fig. 5c). The FAM-labeled peptide was quenched by the PoP liposomes, but sufficient signal remained to verify binding of both the targeting peptide and the liposomal cargo. Both cargo and peptide were internalized and colocalized in U87 cells. When CoPoP liposomes and targeting peptide were added and 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 and remained untargeted. The same results were observed with 2H-PoP liposomes without peptide conjugation. When liposomes were formed with only 1 mol% CoPoP, peptide binding was maintained (Fig. 12a) and selective binding to U87 cells was maintained (Fig. 12b). Cargo-loaded liposomes incubated with His-tagged cRGD moieties were injected intravenously into nude mice bearing U87 tumors. As shown in Figure 5d, 45 min after intravenous injection, the targeted liposomes accumulated in the tumor and had 2.5-fold higher activity than nontargeted liposomes. These data indicate that CoPoP liposomes can be loaded with cargo in the liposomal core, can be labeled with His-tagged targeting peptides without causing cargo leakage, and can be directed to molecular receptors expressed on cells expressing specific surface proteins in vitro and in vivo.

Liposomes represent only a small fraction of the total class of nanomaterials used in biomedical applications. CoPoP was evaluated as a generalized surface coating with selective adhesion to His-tags. Gold nanoparticles were used as model nanoparticles because they are used in numerous biological applications. Using an established protocol for lipid-coated gold nanoparticles, a citrate-stabilized 60 nm gold dispersion was used to hydrate a thin film of PoP-lipids. Repeated centrifugation and resuspension removed the citrate and caused aggregation of the nanoparticles (Fig. 13a). However, in the presence of PoP-lipids, the nanoparticles remained coated and dispersible. Compared to citrate-stabilized gold, PoP-coated nanoparticles had a slightly larger hydrodynamic diameter, corresponding to a bilayer coating over the gold (Fig. 13b). The presence of a coating after His-tag attachment did not affect the gold plasmon peak at 540 nm, demonstrating the mild nature of ligand binding (Fig. 13c). As shown in Figure 13d, only RGD-His CoPoP-coated gold nanoparticles targeted U87 cells, and free RGD inhibited binding as measured by backscattering microscopy. CoPoP gold alone and 2H-PoP-coated gold with RGD-His peptide had no effect on targeting U87 cells.

Development of antigenic liposomes
Many of the monoclonal antibodies that broadly neutralize HIV viral entry (e.g., 2F5, Z13, and 4E10) target conserved linear epitopes in the membrane-proximal external region (MPER) of the gp41 envelope protein, making the MPER a primary target for HIV peptide vaccines. However, it is only exposed 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 interactions with the lipid bilayer in which MPER resides. We used 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 technique based on the attachment of MPER His-tagged polypeptides to Ni-NTA liposomes generated low antibody titers. We tested whether the same approach could be enhanced using CoPoP liposomes.

A liposome-peptide vaccination system was used with the MPER-His sequence NEQELLELDKWASLWNGGKGG-HHHHHHH (SEQ ID NO: 11). The MPER-His peptide was conjugated to CoPoP liposomes containing MPL. A single injection containing 25 μg of MPER-His and 25 μg of MPL was administered to BALB/c and athymic nude mice. This induced titers on the order of ¹⁰⁴ in both BALB/c and nude mice, demonstrating a strong humoral immune response (Figure 6a). This may be important since HIV infects helper T cell populations, making B cell-mediated responses important. After booster injections, anti-MPER titers in athymic nude mice were unaffected, whereas an order of magnitude increase in titers was observed in healthy mice, indicating a T cell-mediated memory effect. Therefore, the vaccination protocol generated both B cell- and T cell-mediated immunity.

Next, various vaccine components were tested to better determine the specificity of the immune response (Fig. 6b). The MPER-His peptide did not elicit antibodies when injected by itself, in Freund's complete adjuvant, or together with 2H-PoP liposomes containing MPL. Interestingly, when the peptide was administered together with CoPoP liposomes without MPL, no antibodies were produced. Another lipid adjuvant, trehalose dibehenate (TDB), also did not elicit antibody production. TDB does not act on TLR-4, emphasizing the importance of MPL in immune activation in the liposomal vaccine system. When MPER-His bound to Ni-NTA liposomes was used, weak antibody titers of less than 10 ³ were observed, consistent with previous reports. However, when CoPoP liposomes were used, a two-order-of-magnitude stronger response was observed. Presumably, the stable binding of the peptide to the liposomes in vivo is directly responsible for this effect. This CoPoP immunization strategy was effective, with antibody titers sustained for at least 3 months, whereas Ni-NTA liposomes produced no detectable antibodies beyond one month (FIG. 6c).

Post-vaccination sera from mice were pooled and purified using protein G agarose to obtain purified IgG, which was then used to evaluate the inhibition of viral entry by HIV (Fig. 6d). When 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 inhibitory effect was greater than that of the broadly neutralizing monoclonal antibody 2F5 incubated at a concentration of 2 μg/mL, but less than that of 2F5 incubated at a concentration of 20 μg/mL. These data demonstrate the potential of a vaccination approach using CoPoP liposomes together with HIV-derived peptides to induce antibody production that can prevent viral entry.

In this example, a vaccine was developed using His-tagged Pfs25 (a recombinant protein derived from Plasmodium falciparum) and liposomes containing CoPoP and MPLA.

Preparation of liposomes
To generate 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 molar ratios indicated (Table 2). After overnight _N2 flow and vacuum, a dry lipid film was formed, which was rehydrated in PBS to a final lipid concentration of 3 mg/mL. The liposome suspension was subjected to 11 freeze/thaw cycles using ice-cold _CO2 /acetone and water baths, followed by 10 extrusions at 60°C through 200 nm polycarbonate membranes.

Psf25 insect protein (recombinant subunit Pfs25 purified from super sf9 cells, concentration 1 mg/mL as determined by BCA analysis) was combined with liposomes overnight (20 h) at 4°C. Psf25 incubated in _H2O served as a control. As a negative control, BSA was combined with liposomes. Binding of Psf25 protein to liposomes was measured by microcentrifuge filtration.

Microcentrifugal filtration for binding
CoPoP and Psf25 protein, reference PoP (without cobalt) and Psf25 protein, and water and Psf25 protein were placed in a Nanosep centrifuge device (100K, OMEGA). Before use, the device was rinsed with _QH2O and centrifuged at 1100 rpm for 3 min. Each sample was added and spun at 1400 rpm for 3 min. The device was washed twice with _QH2O and centrifuged at 1400 rpm for 3 min. Samples were collected from the bottom filtrate and analyzed for protein concentration by BCA assay (Thermo cat.23235) measuring absorbance at 562 nm.

Vaccination
CD-1 mice (8 week old female, Envigo) were injected intramuscularly (im) with 5, 0.5, or 0.05 μg of Psf25 protein on days 0 and 21. Where indicated, the injections also contained 20 μg of MPLA incorporated into liposomes (Avanti No. 699800P). ISA720 was also used as an adjuvant incubated with 5 μg of Psf25 protein. Treatment groups and flow chart are shown in Table 3.

Serum anti-Psf25 IgG levels by ELISA Anti-Psf25 titers were assessed by enzyme-linked immunosorbent assay (ELISA) in 96-well plates (Thermo, Maxisorp). His-tagged Psf25 (0.1 μg) in 100 μL of coating buffer ( _3.03 g _Na2CO3 and 6.0 g _NaHCO3 /1 L distilled water, pH 9.6) was incubated in the wells overnight at 4°C. The wells were washed three times with PBS containing 0.1% Tween (PBS-T) and blocked with PBS containing 0.1% casein (PBS-C) and then incubated for 2 h. The wells were then washed three times with PBS-T and goat anti-mouse IgG-HRP (GenScript No. A00160) was diluted in PBS-C to 1 μg/mL and added to each well. The wells were again washed six times with PBS-T, after which tetramethylbenzidine (Amresco No. J644) was added.

result

Characterization of Liposomes and Protein Binding Ability after Psf25 Protein Coating The average diameters of CoPoP liposomes before and after Psf25 protein binding were 122.9 and 139 nm, respectively, as measured by dynamic light scattering (Figure 14A), with similar polydispersity indices of about 0.05, indicating desirable liposome sizes with high monodispersity after binding. To further examine whether Psf25 protein efficiently and specifically binds to CoPoP liposomes, Pfs25 was incubated with CoPoP or 2H-PoP liposomes overnight at 4°C, and then protein binding to liposomes was measured by microcentrifuge filtration. The amount of Pfs25 in the filtrate detected by BCA analysis showed that the majority of Psf25 bound to CoPoP liposomes (99.27%) but not to 2H-PoP liposomes (12.33%). Binding of Pfs25 to CoPoP liposomes was ∼99% (Fig. 14A).

Anti-Pfs25 IgG levels

CD-1 mice were vaccinated i.m. with CoPoP/MPLA/Pfs25 liposomes conjugated to Psf25 (5, 0.5 and 0.05 μg concentrations), unconjugated to Psf25, or free Psf25 (with or without ISA70) on days 0 (prime) and 21 (boost). On days 20 and 42, serum was collected and anti-Psf25 IgG titers were measured by ELISA (Figures 15A and 15B). Mice prior to boosting with CoPoP/MPLA/Psf25 liposomes showed similar serum IgG levels in both 5 μg and 0.5 μg Psf25 conjugated groups. For each treatment group, different dilutions (starting at 1/1000) of anti-Psf25 IgG-serum samples were tested by Psf25(plant) ELISA (Table 3). Here, the cut-off point was set at an O.D. equal to 0.5. The data show that in G1 and G2 (representing CoPoP/MPLA/Pfs25 liposomes conjugated with 5 or 0.5 μg of Psf25 protein, Table 3), the IgG titers changed from 1/5000 to 1/45000 after boosting. On the other hand, the IgG titers of CoPoP/MPLA/Pfs25 liposomes conjugated with 0.05 μg of Psf25 protein switched from 1/1000 to 1/15000 after boosting, and similar results were shown in G6 (representing 5 μg of Psf25 protein incubated with ISA70). This data is also reflected in Figure 16, which shows that 0.05 ug of Pfs25 in CoPoP/MPLA liposomes produced a higher titer than 5 μg of Pfs25 in ISA720.

In this example, a synthetic His-tagged peptide containing the repeat sequence of NANP derived from the circumsporozoite protein of Plasmodium falciparum was used to develop a liposomal vaccine. The liposomes had the same composition as in Example 1, but peptides were used instead of proteins. The peptides tested are shown in Figure 17A. The mean diameters of CoPoP liposomes before and after conjugation of different NANPs, measured by dynamic light scattering, were 122.9 and 139 nm, respectively (Figure 17B), and the polydispersity index was about 0.05, which was comparable, indicating the desired liposome size after conjugation along with high monodispersity. To further examine whether the NANP peptide efficiently and specifically binds to CoPoP liposomes, the NANP peptide was incubated with CoPoP liposomes or 2H-PoP liposomes overnight at 4°C, and peptide binding to liposomes was subsequently measured by microcentrifuge filtration. The amount of peptides in the filtrate detected by BCA analysis showed that the majority of NANP bound to CoPoP liposomes (~80% for various peptide lengths) but not to 2H-PoP liposomes (<20%). NANP binding to CoPoP liposomes was ~80% for all different peptide lengths (Figure 17C).

In this example, CoPoP liposomes containing phosphatidylserine (PS) were used to reduce antibody responses to His-tagged presentation molecules. Liposomes were formed by thin film hydration and extrusion with a CoPoP:phosphatidylserine:DOPC:cholesterol ratio of 10:30:30:30. His-tagged MPER was then conjugated to the liposomes. These PS liposomes were injected into the footpad of mice 4 and 2 weeks prior to vaccination with MPER-decorated CoPoP/MPLA liposomes. As shown in Figure 18, pretreatment with PS liposomes resulted in a reduction in IgG titers against MPER.

In this example, CoPoP liposomes were targeted to cells by His-tagged cell-penetrating peptides. The following His-tagged cell-penetrating peptides were obtained:
HHHHHHHGRKKRRQRRRPPQ (SEQ ID NO: 13) (TAT peptide);
HHHHHHHRRRRRRRR (SEQ ID NO:14) (R8 peptide):
HHHHHHHRQIKIWFQNRRMKWKK (SEQ ID NO: 15) (PEN peptide).
As shown in Figure 19, after direct aqueous incubation with CoPoP liposomes containing [PoP:CoP:CHOL:DMPC][2:5:30:63], these liposomes were able to bind and be internalized after 1 hour of incubation with U87 cells.

In this example, CoPoP liposomes were used with a pre-fusion RSV antigen (His-tagged antigen, DS-Cav1 for RSV). His-tagged antigens can be used with the CoPoP/PHAD system to generate functional antibodies.

The antigen DS-Cav1 is the pre-fusion form of the F protein on the surface of the RSV virus. His-tagged DS-Cav1 has been shown to induce neutralizing antibodies against RSV A2.

CoPoP liposomes bind His-tagged proteins by simple mixing of antigen with liposomes, such that the resulting liposomes become decorated with conformationally intact antigens that are presented in a uniform orientation. Antigen binding is non-covalently stable in biological environments. CoPoP liposomes have been shown to provide improved antigen delivery to antigen-presenting cells in vitro and in vivo (using another His-tagged antigen, Pfs25, a malaria transmission-blocking antigen).

His-tagged DS-Cav1 was shown to bind efficiently to CoPoP liposomes. A 50 μL volume of antigen-liposome solution was prepared using CoPoP and PoP (also called 2H) and diluted to 200 μL. Liposomes without antigen and antigen without liposomes reference samples were also prepared. Samples were centrifuged at 20,000 rcf for 90 min and the supernatant was extracted from the resulting liposome pellet. An equal volume of BCA was added to a total volume of 400 μL. Samples were then incubated at 60°C in a water bath for 10 min. A 100 μL volume of each sample was transferred to each of three wells of a 96-well plate and the absorbance was measured at 562 nm. The difference in absorbance between the antigen-liposome supernatant and the liposome-only reference sample was used to calculate the percent binding, where higher absorbance corresponds to higher protein concentration in the supernatant and therefore less binding.

Mice were then injected with RSV antigen along 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 booster vaccination (Figure 21A). In contrast, only a total of two mice from the remaining five groups exhibited detectable IgG titers. Two weeks after booster vaccination, IgG titers in mice vaccinated with CoPoP, 2HPoP, and SAS all showed significant increases, but CoPoP with PHAD remained the most effective formulation for inducing antibody production (Figure 21B). The mean antibody titer in the CoPoP/PHAD group exceeded that of the other five groups, and CoPoP/PHAD was the only group that did not contain any mice that were unresponsive to the vaccine after the booster vaccination.

An RSV neutralization assay was performed using serum collected from mice 35 days after the first injection (Figure 21C, D). The results of this assay indicate that antibodies generated in response to the CoPoP/PHAD vaccine were significantly more effective at neutralizing RSV types A and B than any other vaccine group. From the data, it was concluded that CoPoP/PHAD performed better than alternative adjuvants such as Alum, SAS, and AddaVax. Furthermore, neither CoPoP liposomes nor the PHAD adjuvant component of CoPoP alone were sufficient to increase efficacy, and these results can only be attributed to the combined effect of the novel CoPoP/PHAD formulation.

In this example, CoPoP liposomes were used with the Lyme disease antigen, OspA. His-tagged antigens can be used with the CoPoP/PHAD system to generate functional antibodies.

OspA is a Lyme disease antigen. His-tagged OspA was generated based on the B31 strain protein sequence without the lipid tail and purified as shown in Figure 22.

Spontaneous formation of proteoliposomes occurs via insertion of a histidine tag into the hydrophobic bilayer and subsequent coordination of the imidazole moiety to the metal center. Binding conditions were evaluated using native electrophoresis, where the limited pore size of the acrylamide gel allows for physical separation of liposome-bound and free proteins. The observed optimal binding mass ratio of 4:1 PHAD:OspA (Figure 23A) is consistent with previous studies with the malaria antigen Pfs25. Incubation of 80 μg/mL OspA with an equal amount of 320 μg/mL CoPoP liposomes requires 3 h of incubation at room temperature (Figure 23B). Visual inspection of the acrylamide gel showed that the binding kinetics began to plateau after 15 min, likely due to steric hindrance to subsequent binding to nearby non-binding sites. Based on the relative band intensities at 0 and 15 min, it is estimated that the initial stage of binding occurred at a fast rate. In particulate conditions, specific binding is estimated to be about 80% based on microBCA analysis of the supernatant obtained from high-speed centrifugation (Figure 23C). Using the same method, low nonspecific binding was observed for PoP/PHAD liposomes lacking chelated metals. Lysozyme, a non-histidine tagged protein, did not bind to CoPoP/PHAD or PoP/PHAD liposomes. Based on DLS measurements, liposome size after incubation remains essentially the same for both CoPoP/PHAD and PoP/PHAD liposomes (Figure 23D). Furthermore, transmission electron micrographs revealed that CoPoP/PHAD liposomes retain their shape after antigen binding (Figure 23E).

Serum stability studies (Figure 24A) showed that incubation of proteoliposomes with human at 37°C did not significantly increase the fluorescence signal after 12 days. This essentially indicates that OspA remains predominantly associated with the metal-chelated liposomes during the study period. CoPoP liposomes showed more than 100-fold increase in stability compared to nickel-chelated liposomes due to the strategic spatial location of the metal chelate bond within the hydrophobic bilayer. Serum stable antigen binding ensures the integrity of the proteoliposomes during transport to the draining lymph nodes.

Previous studies with similar nanoparticle systems have demonstrated variability in immunogenicity and protective efficacy depending on the point of attachment to the nanoparticle scaffold. This highlights the importance of proper antigen epitope presentation on the particle surface. In this study, a polyhistidine tag was added to the N-terminus opposite the localization of the protective epitope to ensure epitope accessibility on the liposomal surface and avoid possible blockage of the important C-terminal epitope. This configuration essentially mimics the incorporation of lipoproteins into the outer membrane of Borrelia burgdorferi. Assessment of epitope availability using immunoprecipitation showed exposure of the LA-2 epitope on surface-bound OspA (Figure 24B). A negative control, a monoclonal antibody against a non-homologous protein, showed low PoP fluorescence signal. These results indirectly supported the findings in CD spectroscopy elucidating the secondary structure of recombinant OspA. Furthermore, LA-2 antibody-OspA recognition suggested that the structural integrity of the bound antigen was maintained after binding to liposomes.

The utility of liposomes as antigen delivery vesicles is due to their enhanced uptake by antigen-presenting cells. Nanoparticle internalization studies using mouse macrophage-like cells (Figure 24C) showed high antigen uptake only with surface-functionalized liposomes (CoPoP/PoP liposomes). Minimal uptake was observed with free non-adjuvanted or mixed non-associated (i.e., PoP/PHAD) forms of antigen. These results support studies demonstrating enhanced antigen internalization in nanoparticle formulations. Macrophage-selective uptake of PoP/PHAD liposomes in mixed form further implies that physical association of antigen to liposomes may be necessary for liposomal adjuvant action. Simultaneous delivery of surface-exposed antigen and immunostimulants to immune cells is an important aspect of CoPoP/PHAD liposomes. A phagocytosis-mediated cellular uptake mechanism is supported by the decreased uptake of both antigen and liposomes in the presence of cytochalasin B.

Immunogenicity assessment using ELISA indicates that this novel vaccine platform can stimulate a strong immune response at low antigen doses. Homologous prime-boost vaccination with CoPoP/PHAD liposomes elicited higher OspA-specific IgG antibody titers than other commercially available adjuvants (Figure 25A). Furthermore, surface labeling of B. burgdorferi B31 cells (Figure 25B) suggested that the generated OspA-specific antibodies could recognize epitopes on the cell surface, supporting the results obtained from the immunoprecipitation assay. Fluorescence microscopy further reveals the low functionality of anti-OspA antibodies induced from immunization with alum. Immunoblots using antisera derived from CoPoP/PHAD immunization showed that OspA-specific antibodies recognized different strains of Borrelia burgdorferi but to different extents (Figure 25C). Different band intensities indicate the antigenic heterogeneity of OspA across different genotypes. No obvious bands were observed in the negative control B. hermsii (lacking the ospa gene). Minimal non-specific bands were observed for the E. coli lysate with histidine-tagged OspA. The lower molecular weight observed for the OspA band is due to the replacement of the lipidation signal sequence with a shorter polyhistidine segment.

Inclusion of PHAD (a TLR-4 agonist), a synthetic version of monophosphoryl lipid A , in the liposomal formulation biased the immune response toward Th1 and switched the isotype to IgG2a. CoPoP/PHAD liposomes produced higher levels of OspA-specific IgG2a antibodies than IgG1 (Figure 26A). Alum, on the other hand, induced higher levels of the IgG1 isotype. The dominance of the IgG2a isotype is important because this isotype exhibits higher bactericidal activity and greater complement activation capacity than the IgG1 isotype. The Th1-biased immune response observed with CoPoP/PHAD liposomes correlates with a higher stimulation of interferon-γ than interleukin-4 in splenocyte studies (Figure 26B).

Bactericidal assays were used to demonstrate the ability of OspA-specific antibodies to eradicate spirochetes. Antibodies generated from CoPoP/PHAD-OspA immunization showed higher bactericidal activity compared to alum or PoP/PHAD liposomes (Figure 27). The calculated 50% borreliacidal titer for CoPoP/PHAD liposomes was significantly higher than alum.

The OspA-based transmission blocking vaccine relies heavily on high levels of circulating antibodies. ELISA titers calculated at different time points remained almost the same after one year, indicating a high durability of the antibody response (Figure 28).

In this example, CoPoP liposomes were used with hemagglutinin antigen and neuraminidase for influenza. His-tagged antigens can be used with the CoPoP/PHAD system to generate functional antibodies.

Influenza hemagglutinin (HA) is the major surface antigen on influenza viruses and is a major vaccine target. We obtained His-tagged H3 strain HA. As shown in Figure 29, the His-tagged HA bound efficiently to CoPoP liposomes.

A 50 μL volume of antigen-liposome solution was prepared using CoPoP and PoP (also called 2H) and diluted to 200 μL. Liposomes without antigen and antigen without liposomes (reference sample) 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 μL. Samples were then incubated at 60°C in a water bath for 10 minutes. A 100 μL volume of each sample was transferred to each of three wells of a 96-well plate and absorbance was measured at 562 nm. The difference in absorbance between the antigen-liposome supernatant and the liposome-only reference sample was used to calculate the percent binding, where higher absorbance correlates with higher protein concentration in the supernatant (i.e., less binding).

As shown in Figure 30, immunization with CoPoP liposomes and His-tagged HA was highly potent.

Passive serotransfer experiments established that vaccine protection was due, at least in part, to antibody production (Figure 31).

The ability to multiplex was then evaluated using multiple His-tagged HAs. As shown in Figure 32A, various HA and NA proteins were bound to CoPoP liposomes. When immunized, all HAs were able to generate specific antibodies with minimal cross-reactivity (Figure 32B).

This example demonstrates that different types of synthetic MPLA, including PHAD, PHAD-504, and 3D6A-PHAD, can be used in the present compositions and methods. The structures of 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 was varied to be 5, 4, 3, 2, or 1.

As shown in Figure 34, liposomes can be produced containing all types of MPLA, which generally result in higher antibody production than MPLA shedding ("CA"). His-tagged Pfs25 was mixed at a 1:4 mass ratio of antigen:CoPoP and mice were immunized on days 0 and 21. Serum was collected on day 42 and evaluated for Pfs25-binding IgG ELISA.

Although the present invention has been described through specific examples, routine modifications will be apparent to those skilled in the art, and such modifications are intended to be within the scope of this disclosure.

Claims (9)

A liposome comprising:
a) a bilayer having the following i) and ii)
i) phospholipids,
ii) Porphyrins that coordinate with cobalt to form cobalt porphyrins
b) a polyhistidine-tagged presentation molecule, where at least a portion of the polyhistidine tag is present in a hydrophobic portion of the bilayer and one or more histidines of the polyhistidine tag are coordinated to the cobalt of the cobalt porphyrin; and
c) phosphorylated hexaacyl disaccharides (PHADs);
where
the cobalt porphyrin is conjugated to a phospholipid to form a cobalt porphyrin-phospholipid conjugate;
at least a portion of the polyhistidine-tagged presentation molecule is exposed on the exterior of the liposome;
the polyhistidine-tagged presentation molecule comprises an epitope derived from a microorganism ;
the epitope is derived from DS-Cav1, OspA, hemagglutinin (HA), or neuraminidase; and
The liposome surrounds an aqueous compartment.
Liposomes. The liposome of claim 1 , wherein the cobalt porphyrin-phospholipid conjugate constitutes 1 to 25 mole percent of the bilayer . The liposome of claim 2 , wherein the cobalt porphyrin-phospholipid conjugate constitutes 5-10 mol % of the bilayer . The liposome of claim 1, wherein the bilayer further comprises cholesterol and/or phosphatidylserine. The liposome of claim 1, wherein the polyhistidine tag contains 6 to 10 histidine residues. The liposome according to claim 1, wherein the size of the liposome is 50 nm to 200 nm. A nanostructure comprising:
a) core ;
b) a monolayer or bilayer on said core having: i) and ii) a polyimide layer;
i) phospholipid monomers,
ii) Porphyrins that coordinate with cobalt to form cobalt porphyrins
c) a polyhistidine-tagged presentation molecule that contains an epitope derived from a microorganism ; and
d) phosphorylated hexaacyl disaccharides (PHADs);
where
the cobalt porphyrin is conjugated to a phospholipid to form a cobalt porphyrin-phospholipid conjugate;
at least a portion of said polyhistidine-tag is present in a hydrophobic portion of the monolayer or bilayer,
one or more histidines of the polyhistidine tag are coordinated to the cobalt of the cobalt porphyrin;
At least a portion of the polyhistidine- tagged presentation molecule is exposed on the exterior of the nanostructure ; and
the epitope is derived from DS-Cav1, OspA, hemagglutinin (HA), or neuraminidase;
Nanostructures. The nanostructure of claim 7 , wherein the core is a gold nanoparticle. A composition for generating an immune response in a host individual, comprising a liposome according to any one of claims 1 to 6 in a pharmaceutical carrier.

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