JP2022530539A - Nanostructures containing cobalt porphyrin-phospholipid conjugates and polyhistidine tags - Google Patents

Abstract

The present disclosure is a cobalt-chelated porphyrin-containing nanostructure (eg, single-layer or double-layered nanostructure) in which the cobalt metal in the large-membered ring of porphyrin is in the single-layer or double-layer. Provides the nanostructures present in. The nanostructure can have a presentation molecule containing an epitope derived from a microorganism to which a histidine tag is attached, and at least one portion of the His tag is in the single layer or double layer and coordinates with a cobalt metal core. The presented molecule is exposed to the outside of the nanostructure. The nanostructures can further include cargo. The nanostructures can be used to deliver the cargo to an individual. [Selection diagram] None

Description

Cross-reference of related applications

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

The invention was completed with government support under authorization numbers DP5OD017898 and R21AI122964 granted by the National Institutes of Health. The government has certain rights to the invention.

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

In the field of functional nanoparticles, one of the challenges is to easily and reliably attach peptides and proteins to larger scaffolds. Target nanoparticles require an effective ligand and the non-conjugated peptide itself is weakly immunogenic. Bioconjugate chemistry has provided a wide range of strategies, but most nanoparticle conjugations are subject to the limitations associated with one or more of the following: 1) low conjugation rate and essential purification steps; 2) intact. Incompatibility with physiological buffers, which makes it impossible to label nanoparticles; 3) Variable labeling sites and conjugate polypeptide structures that produce heterogeneous particle populations with different efficacy; 4) The need for complex and extrinsic chemical approaches.

The standard approach for attaching ligands to aqueous nanoparticles uses maleimide, succinimidyl ester, and carbodiimide-activated carboxylic acid. They can covalently react with the amine and thiol groups of the polypeptide. The use of maleimide-lipids has been widely explored for antibody-conjugated immunoliposomes. Conjugation rates can reach 90% in overnight reactions, followed by free maleimide group quenching and further purification. Proteins may require preparatory steps for thiolation and purification prior to conjugation. Although antibody orientation is a major factor affecting the effectiveness of conjugated antibody target binding, these approaches result in numerous antibody-labeled sites and indiscriminate orientation. In recent years, biorthogonal synthesis strategies such as click reactions have been applied to preformed liposomes, which require the use of exogenous catalysts and non-conventional amino acids.

Another suitable approach 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 that induce antibody production against non-immunogenic peptides. However, when the lipopeptide was difficult to purify due to its amphipathic nature, the yield was 5-10%. It has also been shown that lipopeptides are not completely incorporated into liposomes during the formation process, resulting in aggregation.

The present disclosure provides functional nanostructures. The nanostructures can be used for cargo delivery, targeted delivery and / or delivery of presented molecules. The nanostructure may be a single layer or a bilayer that encloses the aqueous compartment therein. The bilayer structure that encloses the aqueous compartment is referred to herein as liposomes. The nanostructure may be a single layer or double layer coating on nanoparticles. The monolayer or double layer comprises a cobalt porphyrin-phospholipid conjugate, optionally a phospholipid not conjugated to porphyrin, optionally a sterol, and optionally polyethylene glycol (PEG). One or more targeting peptides or polypeptides with a polyhistidine-tag (referred to herein as the presented molecule) are incorporated into a monolayer or bilayer and a portion of the polyhistidine-tag is monolayer or It is present in the bilayer and the presentation molecule is exposed to the monolayer or the outside of the bilayer. Cobalt porphyrins can be used in place of the cobalt porphyrin-phospholipid conjugates or in addition to the cobalt porphyrin-phospholipid conjugates.

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

The data presented herein are polyhistidine-tagged (also referred to herein as "His-tagged") polys in which a double layer containing cobalt porphyrins, such as cobalt porphyrin-phospholipids (CoPoP). It is demonstrated that it can stably bind to a peptide (Fig. 1a). Other metal porphyrins such as zinc, nickel and copper cannot stably bind to His-tagged polypeptides. This represents a new binding paradigm with at least some polyhistidine embedded in the membrane phase, as porphyrins themselves form a hydrophobic portion of the bilayer and are inaccessible to the external aqueous environment (figure). 1b). This results in more stable binding, enabling a significantly simpler non-covalent post-labeling paradigm after nanoparticle formation and eliminating ambiguity regarding ligand orientation.

We show that lipid bilayers containing cobalt-chelated porphyrin-phospholipids, rather than other metals, can efficiently capture His-tagged proteins and peptides. Binding follows the transition from Co (II) to Co (III) and occurs within a protected hydrophobic bilayer, for example in serum or in a million-fold excess of competing imidazole, essentially irreversible attachment. Bring. Using this approach, we insert the homing peptide into a bilayer of preformed empty liposomes and liposomes containing cargo and site targeting (eg, tumors) without compromising the integrity of the bilayer. ) Was made possible. The peptide or synthetic peptide can be attached to a liposome containing an adjuvant (eg, lipid monophosphoryl lipid A) for producing antibodies for other non-antigenic peptides.

The present disclosure provides a single layer or double layer structure, wherein the single layer or double layer contains porphyrin and chelated cobalt thereof, and the cobalt metal is present in the single layer or double layer and the porphyrin large ring. Further, it has a molecule having a histidine tag attached to it in a non-covalent bond, and at least one part of this His tag is in a single layer or a double layer and coordinates to a cobalt metal core. The presented molecule can be used for a variety of applications, including targeting and producing an immune response. Liposomes or micelles formed in this layer may be loaded with cargo released at the desired location. The cobalt porphyrin may be a cobalt porphyrin-phospholipid (CoPoP). This layer may be used as a coating for other nanostructures such as metal nanoparticles, nanotubes and the like.

FIG. 1 shows binding of a His tag to a PoP-bilayer (a) a peptide having a His tag (green) binds to preformed CoPoP liposomes in an aqueous solution.
(B) Insertion of His-tagged polypeptide into the bilayer containing CoPoP. Only one piece of the bilayer is shown.
(C) Metal-PoP chemical structure used in this study.

FIG. 2 is a His-tagged protein that binds to Co (III) -PoP liposomes. (A) A His-tagged (7) -modified fluorescent protein containing cerulean (C) fused to Venus (V) binds to the PoP bilayer. To prove. When C is excited, FRET is generated and V fluoresces (left), which is inhibited when bound to the PoP-bilayer because FRET competes with the photonic bilayer (center). C fluorescence can be searched directly (right) even when the protein is attached to the bilayer.
(B) Multispectral fluorescence image of fusion protein electrophoresis mobility shift after incubation with the indicated metal-PoP liposomes.
(C) Binding kinetics of the fusion protein to the indicated metal-PoP liposomes based on the loss from C to VFRET.
(D) The NMR peak width of the underlined protons of the vinyl group of CoPoP demonstrates the widening of the paramagnetism of Co (II) in deuterated chloroform (CDCl ₃ ), but after the formation of CoPoP-liposomes in heavy water. Demonstrate the paramagnetic peak of Co (III). Each set of bar graphs is a bar graph of CoPoP and 2H-PoP from left to right.
(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 bar graphs is a bar graph of water and + 2M sulfites, from left to right.

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

FIG. 4: Binding of short His-tagged RGD peptide to CoPoP liposome (a) Binding of FAM-labeled short peptide to metal-PoP liposome.
(B) Effect of His tag length on the binding half-life to CoPoP liposomes. No binding (NB) was observed with peptides that were not His-tagged.
Effect of liposome composition on the binding half-life of the indicated composition to CoPoP liposomes when incubated in PBS (c) or 5 mg / mL BSA (d). Mean ± standard deviation for three measurements. In (c) and (d), each set of bar graphs is a bar graph of 50 and 0 from left to right.

FIG. 5: Release of sulforhodamine B encapsulated in POP liposomes during RGD-His targeting (a) peptide bond of liposomes containing cargo. Each set of bar charts is an 8-hour and 24-hour bar chart from left to right.
(B) Targeted uptake of liposomes containing sulforhodamine B. Cells were incubated in the indicated conditions and uptake was assessed by testing the fluorescence of sulforhodamine B. Each set of bar graphs is a bar graph of MCF7 cells and U87 cells from left to right.
(C) Confocal micrograph showing liposome uptake. Cells were incubated with the indicated liposome solution for 2 hours, washed and imaged. All images were acquired with the same settings.
(D) Internal distribution of sulforhodamine B encapsulated in CoPoP liposomes 45 minutes after injection into nude mice bearing subcutaneous U87 tumors (with or without attachment of His-tagged cyclic RGD targeting peptide). .. Mean ± standard deviation (n = 3). Each set of bar graphs, from left to right, is a non-targeted and + cRGD-His bar graph.

FIG. 6: HIV peptide vaccination with immunogenic CoPoP liposomes (a) BALB / c or thoracic gland-deficient nude mice using CoPoP liposomes containing 25 μg MPL and 25 μg His-tagged MPER peptide (derived from HIV gp41 envelope protein). Immunized. Serum titers were assessed using ELISA using a biotinylated MPER peptide lacking a His tag and probed with an anti-IgG secondary antibody. The arrow indicates the time of vaccination.
(B) Anti-MPER titers in BALB / c mice vaccinated as indicated. Mice were vaccinated at 0 and 2 weeks and serum was collected at 4 weeks.
(C) Persistent anti-MPER titers in mice vaccinated with CoPoP liposomes containing MPL. Mean ± standard deviation (n = 4 mice per group). The first two bar graphs (concatenated) on the left are CoPoP and Ni-NTA.
(D) Neutralization of HIV infection in 293 cells in the presence of the indicated antibody. IgG was purified from mouse serum using protein A. Mean ± standard deviation (n = 3).

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

FIG. 8: Stable binding of His-tagged protein to liposomes containing CoPoP The reporter protein is incubated with liposomes containing CoPoP, free Co-porphyrin or 2H-PoP, then incubated in serum and delivered to the EMSA. did. The protein was then imaged using FRET channels (ex: 430 nm, em: 525 nm). The absence of a signal in the CoPoP lane demonstrates stable binding to liposomes. Attenuated signals of Co-porphyrins in the lane demonstrate some binding of His-tagged proteins to liposomes.

FIG. 9: 90% peptide binding time of RGD-His to CoPoP liposomes of different composition 90% to CoPoP liposomes (10 mol% CoPoP) containing the indicated components when incubated in PBS with RGD-His peptides. Effect of liposome composition on peptide bond time. Each set of bar graphs is a bar graph of (+) cholesterol and (-) cholesterol, from left to right.

FIG. 10: RGD-His binding to CoPoP liposomes in the presence of serum or albumin Liposomes of the indicated composition in the presence of 50% fetal bovine serum or 50 mg / mL bovine serum albumin with the RGD-His peptide. Incubated. The luminescence of the FAM-labeled peptide was normalized by comparing the peptide luminescence when bound to CoPoP liposomes with 2H-PoP liposomes.

[FIG. 11] Membrane permeation treatment with lipopeptide Liposomes loaded with sulforhodamine B were incubated with the indicated peptide (5 μg / mL) at room temperature and emission was assessed using fluorescence. Each set of bar charts is an 8-hour and 24-hour bar chart from left to right.

FIG. 12: Binding of RGD-His peptide to liposomes containing 1 mol% CoPoP and cell targeting (a) Normalized peptide fluorescence when incubated with CoPoP liposomes containing 1 mol% CoPoP. Emissions were normalized by comparing CoPoP samples with 2H-PoP. (B) Cell uptake of sulforhodamine B liposomes containing 1 mol% CoPoP incubated with cells as indicated. Each set of bar graphs is a bar graph of MCF7 cells and U87 cells from left to right.

FIG. 13 is a photograph of coated gold nanoparticles having a CoPoP His tag binding surface (a) a coating protocol that disperses gold nanoparticles. In the absence of the POP coating, the citrate-stabilized gold aggregates after repeated centrifugation steps (arrows). (B) Size of nanoparticles before and after lipid coating. The inset shows a transmission electron micrograph of CoPoP gold with a 50 nm scale bar. (C) Absorption spectrum of citrate-stabilized gold and CoPoP gold after RGD-His binding. (D) Confocal reflection image showing uptake of target nanoparticles. Cells were incubated with the indicated gold nanoparticles for 2 hours, washed and then imaged. All images were acquired with the same settings.

FIG. 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 level mice in CD-1 mice were vaccinated with CoPoP / MPL or Psf25 in ISA70 after intramuscular injection. (A) before booster immunization and (B) after booster immunization (three-week antigen stimulation / three-week booster [5, 0.5, or 0.05 ug of Pfs25 per injection) CoPoP (Psf25) liposomes, or free Psf25 IgG titers were measured by ELISA on 96-well plates obtained from mice vaccinated with protein (with or without ISA70). The data show mean ± standard deviation (n = 5 mice per group).

FIG. 16 The anti-Psf25 IgG titer is defined as the reciprocal of the serum dilution that produces greater than 0.5 absorbance relative to the background.

FIG. 17 Description and Features of Coatings of NANP Peptides of Various Lengths on CoPoP Liposomes (A) Various numbers of NANP repeating peptides comprising a 7 × histidine (His) tag.
(B) The average diameter and polydispersity (PDI) of CoPoP liposomes conjugated with NANP peptides of various lengths were calculated by dynamic light scattering (n = 3). Error bar, standard deviation.
(C) Peptide bonds of NANP peptides to CoPoP liposomes and 2HCoPoP liposomes were measured by microcentrifugal filtration process and BCA analysis (n = 3).

FIG. 18: Anti-MPER IgG titer in mice pretreated with CoPoP / phosphatidylserine liposomes bound to His-tagged MPER. Mice were pretreated with MPER / CoPoP / PS liposomes 4 and 2 weeks prior to injection of MPER in CoPoP / MPLA liposomes to elicit an antibody response to MPER.

FIG. 19: Fluorescence of U87 cells after incubation with CoPoP liposomes bound to various His-tagged CPPs.

[Fig. 20]
After incubation at room temperature for 2 hours, DS-Cav1 with His tags achieved near complete binding to CoPoP nanoparticles (FIG. 20, left). The His-tag DS-Cav1 did not significantly bind to cobalt-deficient PoP particles (2HPPoP), and the His-tag-free DS-Cav1 did not achieve significant binding to any of the particles. His-tag DS-Cav1 did not induce liposome aggregation upon binding, based on liposome diameter (FIG. 20, center) or polydispersity (FIG. 20, right).

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

[Fig. 22]
Generation of OspA with His tag.

FIG. 23: characterization of Ospa-based proteoliposomes formed using CoPoP / PHAD liposomes.
(A) Optimal binding mass ratio of OspA: CoPoP / PHAD liposomes evaluated by natural PAGE (histidine-MOPS buffer system, pH 6.8).
(B) Kinetics of Ospa binding to CoPoP / PHAD liposomes incubated at room temperature at a mass ratio of 1: 4.
(C) Specific binding of His-tagged OspA to CoPoP / PHAD liposomes as measured by the microBCA assay of the supernatant obtained from high speed centrifugation of the liposomes.
(D) Fluid dynamic diameter and polydispersity index of liposomes measured using dynamic light scattering.
(E) TEM images of CoPoP / PHAD liposomes with and without binding of His-tagged protein. Obtained 3 hours after incubation of liposomes with protein. Error bars represent the standard deviation of n = 3 measurements.

FIG. 24. Evaluation of epitope utilization and stability of antigen-functionalized nanoliposomes.
(A) Stability of nanoliposomes-antigen particles in 20% (v / v) human serum based on fluorescence quenching assay.
(B) Immunoprecipitation of OspA-binding liposomes with the OspA-specific monoclonal antibody LA-2.
(C) DY-490-OspA uptake by mouse RAW 264.7 macrophage cells after 2 hours incubation with the indicated sample. Cytochalasin B was replenished with medium 1 hour prior to incubation. The error bars represent the standard deviation of the n = 3 experiment.

FIG. 25. Evaluation of immunogenicity of Ospa-based nanoliposome vaccine.
(A) CoPoP / PHAD liposome-induced anti-OspA IgG titer compared to other commercially available adjuvants. (B) Permeabilization of goat anti-mouse IgG (H + L) secondary antibody ^DyLight® 488 conjugate Detection of anti-OspA IgG antibody by indirect immunofluorescence assay of B. burgdorferi B31 (C) B. burgdorferi Detection of anti-OspA IgG antibody by immunoblotting assay with whole cell lysates of B31 and B. azfelii.
The horizontal line represents the geometric mean.

FIG. 26. Th1-biased immune response of OspA-linked CoPoP / PHAD liposomes (A) IgG isotype profiling of post-immune serum (day 42) using ELISA (B) interferon γ and 72 hours after stimulation with OspA. A splenocyte stimulation test to detect interleukin-4 secretion. Error bars represent the standard deviation from the triple stimulus experiment with n = 3.
The horizontal line shows the geometric mean.

FIG. 27. Evaluation of Borrelia killing activity of SNAP-induced OspA antibody.
(A) Serum germicidal antibody assay performed with guinea pig complement. Survival was obtained by normalizing the number of spirochete after overnight serum treatment with the number of spirochete immediately after incubation. Surviving B. burgdorferi B31-A3 was counted using a dark field microscope.
(B) An average of 50% Borrelia killing activity from 3 different mouse sera (serum dilution that effectively eliminates 50% of bacteria). The error bar indicates the standard error of the mean value. NI represents no inhibition. The statistical significance of the difference between bactericidal titers (p <0.05, indicated by an asterisk) is assessed by the Dunn's ex post Kruskal-Wallis test.

[Fig. 28]
Persistence of anti-OspA IgG levels in SNAP-immune mice. Initial and booster immunization vaccines of 100 ng of Ospa using CoPoP / PHAD liposomes were inoculated on days 0 and 21, respectively. The endpoint titer is defined as the reciprocal of serum dilution at an absorbance cutoff value of 0.5. Data points represent the geometric mean and error bars represent the 95% confidence interval.

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

[FIG. 30]
As shown in (A) to (F), 100 ng of H3 antigen Fr1478 (flu strain) was used as an adjuvant in 3 groups of mice (n = 8) using CoPoP / MPLA, 2HPoP / MPLA, or Alum (aluminum hydroxide) as an adjuvant. A / dog / from Illinois / 11613/2015) was vaccinated, followed by A / Hong Kong / 1/11968.
IgG ELISA and (B) HAI assays performed with serum samples taken throughout the (A) vaccination period show that CoPoP / MPLA + Fr1478 particles achieve the best antibody response prior to viral challenge.
(C to D) Weight is stable throughout the 8-day challenge period, and (E) viral load and (F) white blood cell count in lung tissue are both minimal in CoPoP-vaccinated mice and are effective against the virus. Defense was shown.
(GI) Follow-up challenge tests were performed to compare CoPoP / MPLA protection with MPLA-free CoPoP and another adjuvant, Montanide ISA720. Again, CoPoP / MPLA vaccinated mice show the most consistent body weight retention and the lowest viral load and white blood cell count in the lungs.

[Fig. 31]
Passive transfer of vaccinated mouse serum provides lower protection than direct vaccination, whereas serum transfer from CoPoP-vaccinated mice is (A) significantly reduced weight loss, (B) higher. Survival rate, (C) results in low clinical score. This data supports the hypothesis that the immune response resulting from CoPoP vaccination is significantly antibody-mediated.

FIG. 32
As shown in (A), His-tagged influenza antigens were obtained from various subtypes and tested for their binding affinity with CoPoP. The binding capacity of His-tagged antigens is independent of subtype, and most antigens achieve about 100% binding to CoPoP when incubated for 3 hours at room temperature. (B) Mice were vaccinated with CoPoP incubated with one of 10 selected antigens, serum was harvested and ELISA was used to quantify the binding reaction of the resulting serum antibody. Antibody responses to antigens that were homologous to the vaccine antigen resulted in the highest IgG binding, and non-specific binding of antibodies induced by other subtypes tended to show an average of 1-2 orders of magnitude lower binding. rice field.

[Fig. 33]
The structure of some examples of synthetic adjuvants.

[Fig. 34]
Liposomes are formed with DPPC: cholesterol: CoP: MPLA of 4: 2: 1: X, where MPLA is each of these types of synthetic versions, X is variable, 5, 4, 3, 2,. It was one of 1. The results for anti-Pfs25 titers for CP (PHAD), C3D6A (3D6A-PHAD), CP504 (PHAD-504), and CA (without MPLA) are shown.

The present disclosure provides nanostructures containing at least a single layer. For example, the structure can include a monolayer or bilayer, said monolayer or bilayer containing a porphyrin-phospholipid conjugate, in which cobalt is chelated so that cobalt is present within the bilayer. There is. The bilayer structure can form liposomes. The structure can include two monolayers (bilayers), with the hydrophobic groups of the two monolayers facing each other and the hydrophilic groups exposed to the surface.

The disclosures herein regarding bilayers can also be applied to monolayers. The bilayer or monolayer may also be referred to herein as a "membrane".

All ranges provided herein include all numbers contained in the range up to the tenth decimal place, unless otherwise stated.

Some or all of the cobalt porphyrin in the monolayer or bilayer can bind non-covalently to the polyhistidine-tagged molecule and at least one portion of the polyhistidine-tag is present within the bilayer. , The tagged molecule is presented on the surface of the bilayer. In the double layer or single layer of the invention, one or more histidine residues in the polyhistidine-tag are believed to coordinate to the cobalt metal core within the double layer, thereby providing structural stability. .. The histidine residue of the polyhistidine-tag may be coordinated to the cobalt metal in the porphyrin core within the membrane. The entire histidine tag may be present within the bilayer. A porphyrin-phospholipid conjugate having a cobalt metal conjugated therein is referred to herein as CoPoP. Liposomes whose bilayer contains CoPoP are referred to herein as CoPoP liposomes. CoPoP liposomes can be functionalized with histidine-tagged molecules. As used herein, the term "His-tagged molecule" means a molecule such as a peptide, polypeptide, or protein with a histidine tail, for example. For example, a peptide with a histidine tail is a His-tagged molecule. CoPoP liposomes containing such a His tag are referred to herein as His-tagged CoPoP liposomes or His-tagged CoPoP.

The CoPoP monolayer or bilayer functionalized with the His-tagged presentation molecule of the present disclosure is for presenting various molecules of interest in the circulation, or for delivery to a desired position, or their His-tag. It provides a platform for generating a specific immune response to a chemical molecule. These molecules are referred to herein as presentation molecules (PMs). The structure comprising the His-tagged CoPoP bilayer with PM attached to the histidine tag exhibits desirable stability. The His-tagged molecule is non-covalently attached (coordinated) to 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 chemistry-such as maleimide-or foreign catalysts or unnatural amino acids.

Cobalt porphyrins may be present within the bilayer of self-associating liposomes with an internal aqueous compartment. Alternatively, it may be present in a single layer or double layer coating that coats other nanoparticles. Cobalt porphyrin-phospholipids (CoPoP) behave like conventional lipids with respect to amphipathic properties. Therefore, single or double layers containing CoPoP can be used to coat nanoparticles by methods known to those of skill in the art. In one embodiment, the bilayer or monolayer of the present disclosure may be present on other nanoparticles (eg, in the form of a coating). In one embodiment, the double or single layer containing cobalt porphyrin (eg, cobalt porphyrin-phospholipid) is present as a coating on gold nanoparticles or silica nanoparticles, or other nanoparticles with a hydrophilic surface. In one embodiment, the coating may be in the form of a single layer. In one embodiment, the monolayer or double layer containing cobalt porphyrin (eg, cobalt porphyrin-phospholipids) is present as a coating on a hydrophobic surface such as carbon nanotubes. In one embodiment, the monolayer may form micelles that surround one or more hydrophobic molecules.

This disclosure provides nanostructures comprising monolayers or bilayers, wherein the monolayer or bilayer optionally coordinates i) phospholipids and ii) cobalt to form cobalt porphyrins. Including porphyrin. Optionally, the nanostructures also have one or more polyhistidine-tagged presentation molecules. At least a portion of the polyhistidine-tag is present in the hydrophobic moiety 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 to the outside of the nanostructure. The nanostructures can take the form of liposomes surrounding the aqueous compartment. However, the nanostructures may be coated with a hydrophilic or hydrophobic substance 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 can constitute 1-100 mol% of the monolayer or bilayer, the range comprising a value and range of 0.1 mol% in between. For example, the cobalt porphyrin can constitute 1-20 mol%, or 5-10 mol%, of the monolayer or bilayer. When cobalt porphyrin constitutes 100% of the monolayer or bilayer, there are no phospholipids unconjugated to cobalt porphyrin. The bilayer or monolayer may contain sterols and / or polyethylene glycol. The sterol may be cholesterol.

The number of histidines in the polyhistidine-tag in the monolayer or bilayer may be 2-20. For example, the number of histidines in the polyhistidine-tag may be 6-10. 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 liposome may be spherical or non-spherical. The size of the liposome can be 50 to 1000 nm or more. In one embodiment, the liposome has a size (eg, maximum dimension, eg diameter, etc.) of 50-1000 nm (including all integer nm values and ranges in between). For example, the size may be 50 to 200 nm, or 20 to 1000 nm. If the liposome is not spherical, its maximum size may be 50-1000 nm. These dimensions can be achieved while preserving the nanostructure width of the bilayer single layer. The liposome can carry cargo in its aqueous compartment. The cargo or a portion thereof may be further or instead incorporated into the monolayer or bilayer.

In one embodiment, the present disclosure provides liposomes comprising a monolayer or bilayer, where the monolayer or bilayer is a cobalt porphyrin-phospholipid conjugate, optionally phosphorus not conjugated to cobalt porphyrin. It comprises a lipid and a polyhistidine-tagged presentation molecule, wherein at least a portion of the polyhistidine-tag is present in the hydrophobic moiety of the monolayer or bilayer and is one or more of the polyhistidine-tags. Histidine coordinates with cobalt in the cobalt porphyrin-phospholipid conjugate. At least a portion of the polyhistidine-tagged presentation molecule is exposed to the outside of the nanostructure. The nanostructures (eg, liposomes) can enclose an aqueous compartment. The monolayer or double layer need not contain phospholipids that are not conjugated to cobalt porphyrins, in this case having only cobalt porphyrin-phospholipid conjugates. Cargo can be present in the aquatic compartment. The cargo need not be present only in the aqueous compartment, and a part thereof may be present in a single layer or a double layer.

The present disclosure also provides a monolayer or bilayer, which comprises a phospholipid monomer and a porphyrin coordinated to cobalt (forming cobalt porphyrin). The monolayer or bilayer has one or more polyhistidine-tagged presentation molecules associated therein, and at least a portion of the polyhistidine-tag is present in the 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 presenting molecule is present outside the bilayer or monolayer. In various examples, the single layer or double layer encloses an aqueous compartment or forms a coating on nanoparticles (eg, gold nanoparticles or silica nanoparticles).

The present disclosure provides a core and a nanostructure comprising a single layer or double layer coating on the core, wherein the single layer or double layer is coordinated with a phospholipid and cobalt to provide cobalt porphyrin. Contains forming porphyrins. The nanostructures can have one or more polyhistidine-tagged presentation molecules, where at least a portion of the polyhistidine-tag is present in the hydrophobic moiety of a 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 to the outside of the nanoparticles. The core of the nanostructure may be nanoparticles such as gold nanoparticles or silica nanoparticles.

The liposomes, or nanoparticles having a coating or monolayer or bilayer, can have a presentation molecule on it, as described herein, which is an antigen molecule and / or a targeting molecule ( It may be a targeting molecule). The presented molecule can also provide targeting ability and / or imaging function or other function.

Liposomes or other nanostructures containing His-tagged polypeptides and CoPoP compositions exhibit high serum stability with respect to binding of His-tagged polypeptides to liposomes. In one embodiment, when incubated with serum (eg, diluted serum) at room temperature, more than 60% of the His-tagged peptide remains bound to the CoPoP-containing bilayer after 24 hours of incubation. In one embodiment, more than 85% of the His-tagged peptide remains bound to the CoPoP layer after incubation with serum for 24 hours.

CoPoP liposomes or His-tagged CoPoP liposomes can be loaded with cargo. The cargo is typically present in the aqueous compartment, but may be present completely or partially embedded in the bilayer if it is hydrophobic or has a hydrophobic component. In addition to having presentation molecules on the surface, these structures can be used to load cargo in aqueous compartments within the structure or in bilayers. Cargo release from CoPoP liposomes can be triggered by near infrared (NIR) light. The cargo can be released at the desired location (eg, by internal translocation into target cells or by light stimulated emission).

Single-layer or double-layer cobalt porphyrins are porphyrins with cobalt (Co) cations conjugated to porphyrins. The porphyrin can be conjugated to a phospholipid (referred to herein as a cobalt porphyrin-phospholipid or a cobalt porphyrin-phospholipid conjugate).

The porphyrin moiety of a cobalt porphyrin or cobalt porphyrin conjugate that constitutes at least a portion of the liposome bilayer or other structure comprises a porphyrin, a porphyrin derivative, a porphyrin analog, or a combination thereof. Representative porphyrins include hematoporphyrins, protoporphyrins, and tetraphenylporphyrins. Typical porphyrin derivatives include pyrofeophorbids, bacteriochlorophylls, chlorophyll A, benzoporphyrin derivatives, tetrahydroxyphenylchlorins, purpurins, benzochlorins, naphthochlorins, verdins, rhodins. ), Ketochlorins, Azachlorins, Bacteriochlorophylls, Tripolphyrins, and Benzobacteriochlorophylls. Representative porphyrin analogs include extended porphyrin family members (eg, texaphyrins, sapphirines and hexaphyrins) and porphyrin isomers (eg, porphyrins, inverted porphyrins, phthalocyanines, and naphthalocyanines). included. For example, cobalt porphyrin may be vitamin B ₁₂ (cobalamin) or a derivative thereof.

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

In one embodiment, the layer (single or bilayer) has only CoPoP with His-tagged presenting molecules incorporated therein. In this embodiment, the only phospholipid in the layer is CoPoP (ie, 100 mol% CoPoP). In one embodiment, the layer (single or bilayer) comprises only CoPoP and a porphyrin-conjugated phospholipid (POP), where CoPoP has histidine embedded therein, wherein the histidine is. It has a peptide or other presentation molecule attached thereto. In certain embodiments, there are no other phospholipids, but the layer (single or bilayer) may optionally contain sterols and / or PEG-lipids.

In one embodiment, in addition to the CoPoP, the bilayer or monolayer also comprises a phospholipid that is not conjugated to porphyrin and therefore not coordinated to Co. Such phospholipids are also referred to herein as "further phospholipids." The bilayer or monolayer may contain sterols and PEG-lipids. In one embodiment, the bilayer or monolayer consists essentially of CoPoP, phospholipids not conjugated to porphyrin, and optionally sterols and / or PEG alone, or is conjugated to CoPoP, porphyrin. It consists only of phospholipids that are not, and optionally sterols and / or PEG, where the PEG may be conjugated to the lipid. In one embodiment, the only metal-PoP in the bilayer is CoPoP, which has a His-tagged presentation molecule embedded therein. In one embodiment, the only metal in the bilayer is Co.

In one embodiment, the liposome bilayer comprises CoPoP and PoP. In addition to CoPoP and PoP, the bilayer can contain additional phospholipids. The bilayer or monolayer may further contain sterols 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 sterols and / or PEG alone, or CoPoP, PoP, additional phospholipids, and optionally sterols and /. Alternatively, it consists only of PEG, in which case the 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 nanoparticles in an amount of 0.1-10 mol% and the remaining 99.9-90 mol% is composed of additional lipids (% is the total lipid in the bilayer). Is for). For example, a combination of CoPoP can be present in 0.1-10 mol%, sterol can be present in 0.1-50 mol%, and optionally an attenuated lipid A derivative (eg, monophospholipid A, or 3- Deacylated monophosphoryl lipid A) or related analogs can be present in 0-20 mol% or 0.1-20 mol% and the rest can be composed of additional phospholipids. The phospholipids are DOPC, DSPC, DMPC or combinations thereof, and sterols (if sterols are present; sterols may be cholesterol).

In one embodiment, the combination of CoPoP and PoP may be present in the nanoparticles in an amount of 0.1-10 mol%, the remaining 99.9-90 mol% being composed of additional phospholipids. For example, a combination of CoPoP and PoP can be present in 0.1-10 mol%, sterol can be present in 0-50 mol% or 0.1-50 mol%, and optionally PEG can be present in 0-20 mol% or 0.1-. It can be present in 20 mol% and the rest can be composed of phospholipids. The phospholipid may be DOPC, DSPC, DMPC or a combination thereof, and sterols (if sterols are present; sterols may be cholesterol).

As used herein, a "phospholipid" is a lipid with a hydrophilic head that has a phosphate group linked to a hydrophobic lipid tail via a glycerol skeleton. The phospholipid contains an acyl side chain having 6 to 22 carbon atoms, and the numerical value includes all integer carbon values in between and a range in between. In certain embodiments, the phospholipid in the porphyrin conjugate is 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine. Phospholipids of porphyrin conjugates may contain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and / or phosphatidylinositol, or may consist essentially of them alone, or may consist solely of them.

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 carbons in between).

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 an appropriate number of carbon atoms to form a bilayer. For example, the fatty acid chain may contain 12, 14, 16, 18 or 20 carbon atoms. In various embodiments, the bilayer comprises phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and / or phosphatidylinositol.

The bilayer and monolayer of the present invention may contain sterols. The sterol may be an animal sterol or a vegetable sterol. Examples of sterols include cholesterol, sitosterol, stigmasterol, and cholesterol. In one embodiment, cholesterol may be 0 mol% -50 mol%, or 0.1-50 mol%. In other embodiments, cholesterol may be present in 1-50 mol%, 5-45 mol%, 10-30 mol%.

In certain embodiments, the bilayer or monolayer further comprises a PEG-lipid. The PEG-lipid may be DSPE-PEG (eg DSPE-PEG-2000, DSPE-PEG-5000 or other size DSPE-PEG). The PEG-lipid is present in an amount of 0-20 mol%, including all% amounts in between to the 10th decimal place. The average molecular weight of the PEG component may be between 500 and 5000 daltons and all integer values and ranges in between.

In certain embodiments, the bilayer or monolayer further comprises an adjuvant, eg, an attenuated Lipid A derivative such as monophosphoryl lipid A or 3-deacylated monophosphoryl lipid A.

The histidine tag (His tag) may carry a variety of presentation molecules of interest for a variety of uses. At least one end 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 2-20 histidine chain. In one embodiment, the His tag is a chain consisting of 4-12 histidines and all integer values of histidines in between. In one embodiment, it consists of 6-10 histidine. In one embodiment, it is 6,7,8,9 or 10 histidine. In one embodiment, one end of the His tag is free and the other end has a peptide or other molecule attached. At least a portion of the His tag is believed to be located within the bilayer so as to coordinate to the cobalt metal core.

The liposomes of the present disclosure (without His-tagged molecules) may be substantially spherical in size (eg, including all integer values and ranges up to nm in between) from 30 nm to 250 nm. , Maximum dimensions, eg diameter, etc.). In one embodiment, the liposomes are 100-175 nm in size. In one embodiment, at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% of the liposomes in the composition are 30-250 nm or 100-175 nm in size. Have. The liposome or nanostructure may be larger than 200 nm. In one embodiment, the nanostructure is larger than 1000 nm. In one embodiment, the nanostructure is 200-1000 nm. The liposome or nanostructure may be spherical or non-spherical. In one embodiment, the maximum size of the nanostructure is less than 200 nm while maintaining the nanostructure width of the single layer or bilayer. In one embodiment, the size of the nanostructure exceeds 200 nm in certain dimensions while maintaining the nanostructure width of the single layer or bilayer. In one embodiment, the size of the nanostructure exceeds 1000 nm in certain dimensions while maintaining the nanostructure width of the single layer or bilayer.

On the one hand, the present disclosure provides compositions comprising the liposomes or other structures of the present disclosure, or mixtures of different liposomes or other structures. The composition is a suitable sterile carrier for administration to an individual, including humans, eg, physiological buffers such as sucrose, dextrose, physiological saline, pH buffering components (eg pH 5-9, pH). (For example, to make the pH 7 to 8 and the pH to 7.2 to 7.6 (for example, 7.4)) (for example, histidine, citrate, or phosphate) and the like can also be included. In one embodiment, the composition comprises at least 0.1% (w / v) of CoPoP liposomes or His-tagged-CoPoP liposomes or other structures. In various embodiments, the composition comprises 0.1-100 mol% of CoPoP liposomes or His-tagged-CoPoP liposomes or other structures (eg, nanoparticles coated with a bilayer). In one embodiment, the composition comprises 0.1-99 mol% of CoPoP liposomes (having His-tagged presentation molecules associated with it).

In one embodiment, the compositions of the present disclosure do not have maleimide or succinimidyl ester reactive groups. In one embodiment, the tagged molecule attached to the membrane is free of unnatural amino acids.

The presentation molecule having a His tag may be a small molecule or a polymer. In one embodiment, the molecule is a peptide or peptide derivative. In one embodiment, the molecule is a polypeptide, polynucleotide, carbohydrate or polymer. The His tag may be chemically conjugated to the molecule of interest. The His tag may be incorporated into the primary amino acid sequence of the polypeptide. In one embodiment, the molecule is an antigen, eg, a peptide (any peptide consisting of 2 to 50 amino acids and an amino acid length between 2 and 50) or a polypeptide (50 to 1000 amino acids and between 50 and 1000). An amino acid length (all polypeptides) or proteins (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 amino acids and non-naturally occurring amino acids, or only non-naturally occurring amino acids. You may have.

The presentation molecule attached to the His tag may be an antigen molecule, a targeting molecule, a therapeutic molecule, a diagnostic molecule, or a molecule that provides other types of function. The tagged molecule may be used for targeting, i.e., guiding a structure having a single layer or a bilayer to its target site. For example, a peptide ligand may be attached to a His tag to direct a liposome (or other structure) to a cell that has a receptor or recognition molecule for that ligand. In one embodiment, the adherent peptide can provide an alternative or additional function. For example, adherent peptides can provide therapeutic, diagnostic, or immunogenicity-related functions.

In certain embodiments, the presented molecule may be a targeting molecule (eg, another molecule such as an antibody, peptide, aptamer, or folic acid). The term "target-directed molecule" is used to refer to a molecule that is capable of directing a bilayered structure, such as a liposome, to a particular target (eg, a receptor on the surface of a target cell or other). By binding to the molecule). The targeting molecule may be a protein, peptide, nucleic acid molecule, saccharide or polysaccharide, receptor ligand or other small molecule. The degree of specificity can be regulated through the selection of target-directed molecules. For example, antibodies usually show high specificity. These may be polyclonal, monoclonal, fragment, recombinant, single chain, or Nanobodies, many of which are commercially available or readily available using standard techniques.

The presentation molecule may be an antigen molecule (ie, a molecule having an antigen epitope). In one embodiment, the molecule is a peptide. In one embodiment, the peptide is an RGD-carrying peptide sequence. Such sequences can be 7-20 amino acids with epitopes or longer amino acids. The peptide may be a fragment of a polypeptide or protein that is part of a microorganism (eg, a virus, bacterium, parasite, or fungus) such as a pathogenic microorganism, or an epitope of such a polypeptide or protein. May include. Examples include respiratory syncytial virus (RS virus), 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 (eg, a viral protein that is not known to be virtually immunogenic). The peptide may be a fragment of the HIV antigen, for example an HIV outer envelope protein, or may contain an epitope of the HIV antigen. In one embodiment, the HIV antigen is gp41. For example, the peptide may be the proximal outer region of the membrane (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 comprises a structure having a double layer, wherein the antigen having a histidine tail is non-covalently conjugated to cobalt porphyrin (or cobalt porphyrin phospholipid) and the His tag is double layered. Implanted in, one or more epitopes of the antigen are exposed on the surface. The composition may include an adjuvant 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. Includes an adjuvant system (SAS) or saponin. Other carriers such as wetting agents, emulsifiers and fillers can also be used. Includes AddaVax (Invitrogen), or saponin. Other carriers such as wetting agents, emulsifiers, fillers, etc. can also be used.

A wide variety of cargoes can be loaded into the liposomes or other structures of the present disclosure. The cargo can be delivered to the desired location using near infrared light. For example, a physiologically active substance or a therapeutic substance, a pharmaceutical substance, or a drug can be encapsulated inside a CoPoP liposome. 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 liposome comprises an active substance encapsulated therein (eg, a therapeutic substance and / or a diagnostic substance, which may be a chemotherapeutic agent such as doxorubicin). include. The chemotherapeutic agent doxorubicin can be actively loaded and released by NIR irradiation, providing a strong and direct photoinduced release using CoPoP liposomes.

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

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

In one embodiment, the cargo loaded on the liposome is a diagnostic agent. A "diagnostic agent" or "diagnostic agent" is any chemical component that can be used for diagnosis. For example, diagnostic agents include contrast agents such as those containing radioisotopes such as indium and technetium; contrast agents containing iodine and gadolinium; enzymes such as western wasabi peroxidase, GFP, alkaline phosphatase, or β-galactosidase. Etc .; fluorescent substances such as europium derivatives; luminescent substances such as N-methylacrydium derivatives; etc. are included.

The cargo may be two or more substances. For example, the cargo may contain a combination of a diagnostic agent, a therapeutic agent, an immunogenic substance, and / or a contrast agent, and / or other types of substances. It is also possible that the same substance has multiple functions. For example, the substance may be diagnostic and therapeutic, or the substance may be image processing and immunogenic.

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

The present disclosure also provides a method of using the structure having the bilayer described herein. In one embodiment, the disclosure provides a method of inducing an immune response in a host. The immune response can produce antibodies. The method comprises 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 may be administered in a single dose or in multiple doses, including booster shots. The antibody titer 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, if it is desired to reduce immunogenicity, nanostructures in which phospholipids containing PS (or otherwise) are present can be used. Compositions containing these nanostructures can be administered to reduce immunogenicity.

On the one hand, the present disclosure provides a method of delivering a substance contained as a cargo in a liposome or other nanostructure to a desired position. The substance, in whole or in part, may be contained within or in CoPoP liposomes, which may be present in the aqueous compartment, bilayer or both. The method 1) prepares a composition comprising a liposome or other structure having a bilayer of the present disclosure optionally containing cargo (such as an activator); 2) said liposome in a selected or desired location. 3) Irradiating the liposomes with radiation having a near-infrared wavelength under conditions such that at least some of the cargo is released from the liposomes; Alternatively, or in addition, the cargo may reach the inside of the cell by intracellular migration of the liposomes and then by the action of intracellular processes to release the cargo.

The liposome has a wavelength of 650 to 1000 nm (including all integer values and ranges up to nm in between) and an output of 50 to 1000 mW / cm ² (including all integer values and ranges up to mW / cm ² in between). It may be irradiated with near-infrared light from a laser. In another embodiment, the wavelength is 650-800 nm, which includes all integer values up to nm in between and all ranges. The liposome may be irradiated for up to 30 minutes or less. In various embodiments, the liposomes may be irradiated in vitro or in vivo for 0.5-30 minutes and all numerical values up to 10 decimal places in between. In one embodiment, the liposome is irradiated with a 658 nm laser diode for up to 10 minutes. In another embodiment, the liposome is irradiated with a wavelength of 665 nm or 671 nm. Infrared rays may be delivered directly to the desired area by directing a laser beam to that area, or fiber optic probes may be used. In the case of a tumor, a fiber optic probe can be inserted into the tumor for local irradiation (ie, by catheter or endoscopic device).

On the one hand, the present disclosure provides a method of preparing a bilayer containing CoPoP. The free base PoP is a monocarboxylic acid porphyrin such as pyrofeohorbido-a by 2-palmitoyle-2-hydroxy-sn-glycero-3-phosphocholine (lyso-C16-PC, Avanti # 855675P). A molar ratio of lyso-C16-PC: Pyro: EDC: DMAP = 1: 1: 2: 2 in chloroform using -ethyl-3- (3-dimethylaminopropyl) carbodiimide and 4-dimethylaminopyridine. It can be esterified and produced by stirring overnight at room temperature. PoP is then purified by silica gel chromatography. CoPoP is a porphyrin-phospholipid conjugation with a molar excess (eg, 10-fold molar excess) of cobalt salt (eg, cobalt acetate (II) tetrahydrate) in a solvent (eg, methanol) in the dark. It can be made by contacting the gate.

In one embodiment, the present disclosure provides a method of coating nanoparticles with a cobalt porphyrin (eg, CoPoP) double layer or single layer. The method generally involves hydrating the nanoparticles with a solution of lipid to disperse the particles in water.

The composition comprising the liposome in a suitable carrier can be administered to the individual by any suitable route to deliver the cargo to the desired location or for normal administration. 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 during a blood supply to a particular organ or tissue or tumor. When irradiated with NIR, the contents of PoP liposomes may be released within the circulatory system and then into surrounding tissues.

In the following description, various examples of the nanostructures, compositions and methods of the present disclosure are disclosed.
1. 1. Nanostructures (eg, liposomes) that
A) monolayer or double layer with i) optionally phospholiposomes and ii) porphyrins coordinated to cobalt to form cobalt porphyrin; and b) optionally , Polyhistidine-tagged presentation molecule, where at least a portion of the polyhistidine-tag is present in the hydrophobic moiety of a monolayer or bilayer, and one or more histidines of the polyhistidine-tag are said cobalt. If coordinated to cobalt in porphyrin, at least a portion of the polyhistidine-tagged presentation molecule is exposed to the outside of the nanostructure (eg, liposome), and if it is a liposome, the liposome is an aqueous compartment. Surrounding;
Nanostructures containing.
2. 2. The nanostructure (eg, liposome) according to 1 above, wherein the cobalt porphyrin is conjugated to a phospholipid to form a cobalt porphyrin-phospholipid conjugate.
3. 3. 2. The nanostructure (eg, liposome) according to 2 above, wherein the cobalt porphyrin-phospholipid conjugate comprises 1 to 25 mol% of a monolayer or bilayer.
4. 3. The nanostructure (eg, liposome) according to 3 above, wherein the cobalt porphyrin-phospholipid conjugate comprises 5-10 mol% of a monolayer or bilayer.
5. The nanostructure (eg, liposome) according to any one of 1 to 4 above, wherein the bilayer further comprises a sterol (eg, cholesterol).
6. The nanostructure (eg, liposome) according to any one of 1 to 4 above, wherein the bilayer further comprises phosphatidylserine and optionally cholesterol.
7. The nanostructure (eg, liposome) according to any one of 1 to 4 above, wherein the polyhistidine-tag contains 6 to 10 histidine residues.
8. The nanostructure (eg, liposome) according to any one of 1 to 4 above, wherein the liposome has a size of 50 nm to 200 nm.
9. The nano according to any one of 1 to 4 above, wherein when the nanostructure (for example, a liposome) contains a cargo and is a liposome, at least a part of the cargo is present in the aqueous compartment of the liposome. Structures (eg, liposomes).
10. The nanostructure 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 histidine of His tag). , Liposomes).
11. The nanostructure (eg, liposome) according to any one of 1 to 10 above, wherein the presented molecule is a protein of 4 to 500 kDa.
12. The nanostructure (eg, liposome) according to any one of 1 to 11 above, wherein the presented molecule is an antigen molecule and the monolayer or bilayer further comprises an adjuvant incorporated therein. ..
13. 12. The nanostructure (eg, liposome) according to 12 above, wherein the adjuvant is an attenuated Lipid A derivative.
14. 13. The nanostructure (eg, liposome) according to 13 above, wherein the attenuated lipid A derivative is monophosphoryl lipid A or 3-deacylated monophosphoryl lipid A.
15. It ’s a nanostructure,
a) core; and b) a single or double layer on the core, i) optionally coordinated to a phospholipid monomer, and ii) cobalt to form cobalt porphyrin (eg, CoPoP). Monolayer or bilayer with porphyrin, and c) optionally a polyhistidine-tagged presenting molecule, where at least a portion of the polyhistidine-tag is present in the hydrophobic moiety of the monolayer or bilayer. , One or more histidines of the polyhistidine-tag are coordinated to the cobalt of said cobalt porphyrin, and at least a portion of the polyhistidine-tagged presenting molecule is exposed to the outside of the nanostructure;
Nanostructures containing.
16. The nanostructure according to 15 above, wherein the core is gold nanoparticles.
17. A targeted delivery method for cargo
a) In the pharmaceutical carrier, the nanostructure according to any one of 9 to 16 (for example, liposome) or the nanostructure according to any one of 9 to 16 (for example, liposome). The composition comprising the combination is administered to the individual; and b) the liposomes are subjected to wavelength 650 after the appropriate time has elapsed to allow the nanostructures (eg, liposomes) to reach the desired position of the individual. A method comprising exposing the liposomes to cargo by exposure to near-infrared rays of ~ 1000 nm.
18. 17. The method according to 17 above, wherein the individual is a human or non-human animal.
19. A method for generating an immune response in an individual host, wherein the nanostructure (eg, liposome) according to any one of the above 1 to 16 or any one of the above 1 to 16 is described in a pharmaceutical carrier. A method comprising administering to an individual a composition comprising a combination of nanostructures (eg, liposomes), wherein the presented molecule comprises an immunogenic epitope.
20. 19. The method of 19 above, wherein the presented molecule is a peptide, polypeptide or protein derived from a pathogenic microorganism.
21. 19 or 20 above, wherein the individual is a human or non-human animal.

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

This example illustrates the synthesis and functionalization of a cobalt porphyrin-phospholipid (CoPoP) bilayer using a histidine-tagged ligand and an antigen.

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

Recombinant heptahistidine-tagged Cerulean-Venus fusion reporter protein for protein binding was produced and purified in E. coli and characterized as described above. Stoichiometric estimates are based on the assumption that liposomes of ~ 100 nm each contain 80,000 lipids.

Production of PoP- lipids, PoP-liposomes and PoP-gold Free base (2H) PoP sn-1-palmitoyle sn-2-pyrofeohorbid phosphatidylcholine was synthesized as described above. CoPoP was produced by stirring 100 mg of 2H-PoP and a 10-fold molar excess of cobalt (II) acetate tetrahydrate in 4 mL of methanol in the dark for 17 hours. Reaction completion and product purity 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 was 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) (yield 77%). Identity was confirmed by mass spectrometry). Other metals-PoP were synthesized using the same method. For Ni-PoP, Ni (II) acetate tetrahydrate was used and incubated for 17 hours. For Zn-PoP, acetic anhydride Zn (II) 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 on a 1 mg scale. After dissolving the lipids in chloroform in vitro, the solvent was evaporated and the film was further dried under vacuum overnight. Lipids were rehydrated in 1 mL of phosphate buffered saline (PBS), sonicated, subjected to 10 freezing-thaw cycles, and then using a small extruder (Avanti # 610000), 100 nm. Extruded through a polycarbonate film (VWR # 28157-790). Liposomes were formed using 10 mol% PoP, 85 mol% DOPC (Avanti # 850375P), and 5 mol% PEG-lipid (Avanti # 880120P) for protein and peptide binding analysis. Ni-NTA liposomes contained 10 mol% Ni-NTA lipid diole oil-glycero-Ni-NTA (Avanti # 790404P) and 10 mol% 2H-PoP. Liposomes incorporating free Co-porphyrins contained 10 mol% Co-pyrofeophorbide with 85 mol% DOPC and 5 mol% PEG-lipids. Co-NTA-liposomes were prepared using liposomes containing 10 mol% Dioreoil-glycero-NTA (Avanti # 790528P). Liposomes were incubated with 20 mg / mL cobalt (II) chloride for 2 hours and then dialyzed against PBS. Liposomes loaded with sulforhodamine B contained 10 mol% PoP, 35 mol% cholesterol (Avanti # 700000P), 55 mol% DOPC and PEG-lipids, as indicated. The lipid film was hydrated with a solution of sulforhodamine B (VWR # 89139-502), then frozen-thawed and then sonicated. Unencapsulated dye was removed by 10 mL Sephadex G-75 (VWR # 95016-784) column followed by dialysis in PBS. A 50 mM dye was used for bilayer integrity and quantitative cell junction studies, and a 10 mM dye was used for microscopic studies.

45 mol% distearoylphosphocholine (Avanti # 850365P), 45 mol% di, using 60 nm citrate-stabilized gold nanoparticles (Ted Pella # 15709-20) for gold coating. A 1 mg lipid film consisting of stearoylphosphoglycerol (Avanti # 840465X) and 10 mol% PoP was hydrated. After a short vortex and sonication, the sample was repeatedly centrifuged for 15 minutes at a relative centrifugal force of 1500 (rcf). The supernatant was discarded and the pellet was resuspended and recentrifugated more than once. PoP gold was resuspended in water for further analysis.

Polypeptide bond
1 μg of fluorescent reporter protein was incubated in 96-well plates with 20 μg of liposomes in 200 μL PBS. The 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 relative to the FRET signal in the protein without liposomes. Incubate 2.5 μg of protein with 50 μg liposomes, followed by electrophoresis in a 0.75% agarose gel (50 V applied for 90 minutes) and image processing using the indicated excitation and luminescence filters by the IVIS Lumina II system. The EMSA experiment was conducted. For serum stability testing, 3 mg of protein was preincubated with 60 mg of liposomes in 40 ul PBS. After 24 hours of incubation, 40 uL of FBS was added and incubated for an additional 8 hours. For imidazole substitution experiments, 1 μg of reporter protein was bound to 20 μg of liposomes in PBS. Then imidazole was titrated and the binding was evaluated by fluorescence. For serum stability, 1 μg of reporter protein was bound to 20 μg liposomes in 100 μL PBS, followed by addition of equal volume fetal bovine serum (VWR # 82013-602) and fluorescence monitoring of binding. did. After incubating 500 ng of peptide with 20 μg liposomes, peptide bonds were evaluated by RGD-His FAM fluorophore quenching.

Target-oriented experiment
U-87 and MCF-7 cell lines were obtained from ATCC and cultured according to the supplier's protocol. 2 × 10 ⁴ cells were seeded overnight in wells in 96-well plates. 500 ng of RGD-His peptide was bound to 20 μg of liposomes loaded with sulforhodamine B and the liposomes were incubated with the cells for 2 hours. The medium was removed, the cells were washed 3 times with PBS, then the cells and liposomes were lysed with 1% Triton X-100 solution. Liposomal uptake was assessed by measuring the fluorescence of sulforhodamine B.

For confocal image analysis, 104 cells were seeded overnight in Nunc chamber slides (Nunc # ¹⁵⁵⁴¹¹ ) in DMEM containing 10% fetal bovine serum (FBS). 20 μg of liposomes were added to the medium containing serum and incubated for 2 hours. The medium was removed and the cells were washed 3 times with PBS. New 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 binding, gold was centrifuged to remove the unbound RGD peptide.

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

Vaccination Unless otherwise noted, 8-week-old female BALB / c mice (Harlan Laboratories) were injected posteriorly on days 0 and 14 with 25 μg of MPER peptide in 50 μL sterile PBS. It was injected into the footpad on the side. Where indicated, the injection also contains 25 μg MPL (Avanti # 699800P) or TDB (Avanti # 890808P) in the liposomes, which are DOPC: cholesterol: MPL: PoP, 50:30: 5: 5. Included in molar ratio. For Freund's adjuvant, the peptides were mixed directly in Freund's complete adjuvant (Fisher # PI-77140) and injected. Blood was taken from the submandibular vein 4 weeks after the first injection, or after the indicated period, serum was obtained after blood coagulation and centrifugation at 2000 rcf for 15 minutes and stored at -80 ° C.

Anti-MPER titers were assessed by ELISA in 96-well streptavidin-coated plates (GBiosciences # 130804). 1 μg His-tagged-free MPER biotin in 100 μL PBS (PBS-T) containing 0.1% Tween 20 was incubated in a well at 37 ° C. for 2 hours. Wells were then washed 5 times with PBS-T, mouse serum was serially diluted with PBS containing 0.1% casein (PBS-C) and incubated at 37 ° C. for 30 minutes. The wells were washed 5 times with PBS-T, then 100 μL of goat anti-mouse IgG-HRP (GenScript # A00160) diluted with PBS-C was added to the wells to a final concentration containing 1 μg / mL secondary antibody. And incubated at 37 ° C for 30 minutes. The wells were washed 5 times with PBS-T, then 100 μL of tetramethylbenzidine substrate solution (Amresco # J644) was added to each well and incubated at 37 ° C. for 20 minutes. The reaction was stopped with 100 μL of 1M HCl and absorption was measured at 450 nm. Titer was defined as the reciprocal of the dilution when the absorbance at 450 nm exceeded the same dilution of non-serum background by 0.05 absorbance units. All samples were measured twice and averaged.

Virus Penetration Test As described above, a virus penetration test was carried out. Briefly, HIV-1 was produced by co-transfection of 293T cells with pHXB2-env and pNL4-3.HSA.RE. Two days after transfection, cell medium was passed through a 0.45 μm filter and centrifuged. The virus pellet was dried, resuspended in 600 μL PBS and stored at −80 ° C. The infectious titer of the HIV-1 stock was determined by X-Gal staining, as previously reported.

Serum from three mice immunized with MPER and CoPoP liposomes was pooled and IgG was isolated using immobilized protein G beads (VWR # PI20398) according to the supplier's protocol. The concentration was determined by absorption by Bradford analysis. 2F5 was obtained from the Free NIH AIDS Reagent Program. 1 × 10 ⁴ TZM-bl receptor cells per well were sown on 96-well plates the day before infection. HIV (multiplicity of infection 0.1) was incubated with the antibody at 37 ° C for 30 minutes, added to the cells, spinocurated at 25 ° C for 1 hour at 1000 rcf, followed by an additional 5% CO ₂ incubator, 37. Incubated at ° C 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 analysis system (ONE-Glo, Promega) according to the manufacturer's protocol and normalized relative to virus-only samples. The data were further normalized to cell viability (all groups showed viability within 10% of control untreated cells).

result

The His-tagged protein used the transition metals Co, Cu, Zn, Ni and Mn that bind to CoPoP liposomes to generate a series of sn-1-palmitoyle sn-2-pyrofeohorbid phosphatidylcholine chelates (Figure). 1c). Then, using 10 mol% metal PoP, with 85 mol% dioleoil phosphocholine (DOPC) and 5 mol% polyethylene glycol-conjugated distearoylphosphoethanolamine (PEG-lipid), 100 by extrusion. PoP bilayers were formed by forming liposomes at nm. His-tagged proteins bound to the PoP bilayer were evaluated using fluorescent protein receptors. As shown in FIG. 2a, the system included 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 acts as a Venus Förster Resonance Energy Transfer (FRET) donor, and Cerulean excitation results in FRET emission from Venus. The C-terminus of Cerulean was labeled with a heptahistidine tag. However, when bound to the PoP bilayer, the energy transfer from the cerulean is diverted to the bilayer itself (absorbed into the cerulean emission range) and therefore competes with FRET to Venus. Venus, on the other hand, does not adhere directly to the photonic bilayer, so it does not completely quench when directly excited, allowing tracking of bound fusion proteins.

Tricolor electrophoretic mobility shift analysis (EMSA) was developed to evaluate 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 hours and then subjected to agarose gel electrophoresis. As shown in the upper image of FIG. 2b, when PoP liposomes were imaged, only free base (2H) liposomes and Zn-PoP were easily visualized, and the degree of Zn-PoP liposomes was lower. This demonstrates that the metals have a quenching effect on PoP and confirms that they are stably chelated in the bilayer. As expected, liposomes showed the lowest electrophoretic mobility due to their relatively large size. The same gel was then imaged using Cerulean excitation and Venus luminescence to probe for FRET inhibition (indicators 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 proteins incubated with CoPoP liposomes, in which case the FRET disappeared completely (middle image). Venus was directly excited and imaged to verify the presence of the protein. Only when CoPoP liposomes were used did the reporter protein co-localize with the liposomes. Together, these images demonstrate that the protein was quantitatively bound to CoPoP liposomes. Solution-based studies confirmed this finding (Fig. 2c). Of all types of PoP liposomes tested, only CoPoP resulted in a significant reduction in FRET efficiency between Cerulean and Venus due to liposome binding. This binding took only three hours to reach 50% binding (t _1/2 ), but took almost a day to complete. Molecular dynamics simulations of the 2H-PoP bilayer showed that the center of porphyrin (where metal chelation occurs) does not have access to the aqueous phase surrounding the bilayer. Therefore, this slow binding may be due to the His tag, which is partially obscured by the rest of the protein, and the need to make a path into the protected hydrophobic bilayer.

Coordination of polyhistidine with CoPoP The underlying mechanism of His-tag binding to the immobilized metal involves the metal coordination of the histidine residue with the nitrogen-containing imidazole group. Lack of His-tag binding to liposomes formed with Ni (II), Cu (II), Zn (II) and Mn (II) PoP is the binding affinity or coordination of axial ligands within porphyrins. It seems to be related to the number. For example, it has been proposed that Ni (II) and Cu (II) porphyrin chelates can be completely coordinated to the surrounding four macrocyclic nitrogen atoms without axial ligands. For Zn (II) and Mn (III) porphyrins, the ligand binding strength appears to be insufficient to provide a stable polyhistidine bond.

Paramagnetism was evaluated to determine the electronic state of CoPoP. Since Co (II) is paramagnetic, but Co (III) porphyrin has low spin and diamagnetism, NMR was used to investigate the spread of peaks induced by paramagnetic species. As shown in FIG. 2d, peak widening was observed only for CoPoP and only in organic solvents, based on the hydrogen attached to each carbon of the vinyl group in PoP. When CoPoP was formed in aqueous liposomes, the peak narrowed and was an indicator of diamagnetic Co (III) oxidation in the bilayer. To further verify this mechanism, CoPoP liposomes were quantitatively bound to the fluorescently labeled His-tagged peptide, and then a reducing agent, sodium sulfite, was added to the CoPoP liposomes. As shown in FIG. 2e, 2 M sulfites induced peptide release from CoPoP liposomes. Liposomes were also formed using commercially available Ni-NTA lipids. The His-tagged peptide did not bind strongly to Ni-NTA liposomes. No peptide release was observed with the addition of sulfites to the system, as would be expected from Ni (II), which cannot be easily reduced. Together, these data show that during the formation of CoPoP liposomes, CoPoP transitions from Co (II) to Co (III) and the polyhistidine imidazole group is chelated in PoP in the bilayer Co (III). It is suggested to coordinate with.

Stable His-Tag Binding to CoPoP Liposomes Next, fluorescent reporter proteins were used to compare the binding of His-tagged proteins to liposomes containing either CoPoP or Ni-NTA-lipids (FIG. 3a). To enable FRET-based protein binding measurements, Ni-NTA liposomes contained 10 mol% 2H-PoP. According to EMSA, proteins migrated unimpeded in both 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 did not resist the conditions of electrophoresis. The FRET channel was not quenched and it was confirmed that there was no binding to Ni-NTA liposomes. In contrast, when incubated with CoPoP liposomes, the protein bound stably, the FRET channel disappeared completely, and the electrophoretic mobility decreased (which is consistent with the protein that remained bound to the liposome).

A difficult barrier to using Ni-NTA-lipids for biomedical applications is that they do 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 to a solution of liposomes bound to His-tagged protein in a 1: 1 volume ratio. As shown in FIG. 3b, Ni-NTA liposomes do not completely capture all proteins, which is consistent with the weak binding shown in the EMSA results. Furthermore, after the addition of serum, all bonds disappeared over 24 hours. Under the same conditions, CoPoP liposomes stably captured the His-tagged reporter protein without substantial protein release.

Since the histidine side chain contains an imidazole group, an imidazole competitive assay was used to compare the binding stability of Ni-NTA to the His-tagged polypeptide of CoPoP liposomes. As shown in FIG. 3c, CoPoP liposomes maintained more than 75% binding to the reporter protein even at imidazole concentrations close to 1 M. This means about 10 million times more imidazole excess than the 100 nM protein concentration used in the binding study. In contrast, Ni-NTA liposomes released more than 90% His tags in the presence of only 30 mM imidazole. Dramatically strong binding of CoPoP liposomes to His tags is due to at least two factors: excellent stable chelation of Co (III) to the imidazole group, and the protected hydrophobic environment of the CoPoP bilayer (competitive external). It may be due to (restricting access to molecules).

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

Peptide binding to CoPoP liposomes

Targeting of peptides is of interest for use as disease and tissue-specific "zip codes". The short RGD tripeptides found in fibronectin and vibronectin are promising target-directed ligands due to their efficient binding to integrin αvβ ₃ expressed on tumor vascular endothelial cells. CoPoP liposomes were tested to verify whether CoPoP liposomes could be delivered to molecular receptors on target cells by the His-tagged ligand approach using the linear single-chain amino acid sequence GRGDSPKGAGAKG-HHHHHHH (SEQ ID NO: 1). Carboxyfluorescein (FAM) was labeled at the N-terminus and FRET allowed detection of binding to PoP-liposomes. It has been demonstrated that the linear RGD peptide can be labeled with a fluorophore without interfering with integrin binding. As shown in FIG. 4a, when this peptide was incubated with various metal-PoP liposomes, only CoPoP bound to the peptide. Peptide binding was about 5 times faster than protein binding. Presumably, the smaller size of the peptide, faster molecular motion, and reduced steric hindrance allow faster mating to the bilayer, interacting with CoPoP and irreversibly binding. Based on previous estimates that each ~ 100 nm liposome contains about 80,000 lipids, this is equivalent to 8000 CoPoP and 750 peptides per liposome. The ratio of CoPoP to histidine in the bilayer was 1: 0.66 because each peptide contained 7 histidine residues. In conventional His tag binding to Ni-NTA, of all the residues of the His tag, only the i-th and the i + 2 or i + 5 histidine residues are thought to be involved in the coordination with the metal. .. Porphyrin and polyhistidine densities within the CoPoP bilayer appear to be higher, and therefore the coordination mechanism may be different.

The effect of His tag length on peptide binding to CoPoP liposomes was investigated. A series of N-terminal FAM-labeled peptides were synthesized with varying lengths of His tags attached to the C-terminus. No peptide bonds were observed when the His tag was removed from the peptide, as demonstrated in FIG. 4b. With two histidine residues, binding was slow and binding t _1/2 was approximately 10 hours. As the His tag became longer, the binding rate increased rapidly. With six residues corresponding to a common hexahistidine tag, binding t _1/2 was less than 1 hour. Increasing the length of the His tag to 10 residues reduced the binding t _1/2 to 20 minutes.

Next, the lipid composition was changed 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 lipids used was reduced accordingly. Whereas DSPC forms a hard gel phase bilayer at room temperature, DMPC and DOPC have lower transition temperatures and are in the liquid crystal phase. Cholesterol occupies space in the bilayer and may have a palliative effect on membrane fluidity. Interestingly, no significant difference in peptide binding rates was observed for membranes of different composition with or without cholesterol (Fig. 4c). The peptide binding process can occur in a multi-step process, and once the peptide has begun to be inserted into the bilayer, the synergistic action of polyhistidine is unaffected by the lipid composition. However, in 5 mg / mL bovine serum albumin (BSA), a significant difference was observed depending on whether the membrane had cholesterol (Fig. 4d). The slower binding in cholesterol-free liposomes was probably due to the greater interaction of BSA with the membrane that interfered with peptide binding. BSA at 50 mg / mL did not reach the binding half-life and serum completely inhibited binding (FIGS. 9 and 10).

Considering the binding potency of His-tagged peptides to biotargeting liposomes of cargo-laden liposomes, the integrity of the bilayer was evaluated to determine if peptide binding induces membrane destabilization. Fluorophoresulforhodamine B (water-soluble dye) was loaded into the aqueous core of the liposome at a self-quenching concentration, and the membrane permeability was examined. As shown in FIG. 5a, the dye-loaded liposomes did not release a substantial amount of dye over 8 hours and the peptide was completely bound to the liposome. At 24 hours, CoPoP liposomes with bound peptides 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 the enclosed cargo intact. Similar palmitoylated lipopeptides, when incubated, resulted in permeabilization of cargo-carrying liposomes (Fig. 11), further demonstrating the robustness of the His-tagged approach.

Next, using an established cell line pair of U87 glioblastoma cells (RGD-bound) and MCF7 breast cancer cells (RGD unbound), evaluate whether RGD-decorative liposomes can bind to their molecular targets. did. After encapsulation of sulforhodamine B, the liposomes were first incubated with His-tagged RGD peptide and then with both cell lines. About 550 peptides were attached to each liposome. Liposomal uptake was assessed by washing and lysis (to remove cargo self-quenching) and then testing intracellular fluorescence. As shown in FIG. 5b, high liposome uptake was observed in U87 cells incubated with targeted CoPoP liposomes, whereas the binding that occurred in MCF7 cells was negligible. As expected, no uptake occurred in any cell line without the RGD targeting ligand. When the liposome preparation contained PEG-lipid, liposomes that did not target any cell line were obtained. The presence of PEG appears to have the effect of interfering with peptides directly attached to the surface of the bilayer. The presence of excess free RGD peptide could inhibit binding, confirming the target specificity of this approach. Confocal microscopy demonstrated these binding results (Fig. 5c). The FAM-labeled peptide was quenched by PoP liposomes, but sufficient signal remained to verify the binding of both the target-directed peptide and the liposome cargo. Both the cargo and the peptide internalized into U87 cells and became coexistent. When the CoPoP liposome and the targeting directional peptide were added and mixed immediately before incubation with the U87 cells, the targeting directional peptide itself bound to the U87 cells but did not have time to attach to the liposomes and remained untargeted. The same results were observed with 2H-PoP liposomes that were not bound to the peptide. 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 components were intravenously injected into nude mice bearing U87 tumors. As shown in FIG. 5d, 45 minutes after intravenous injection, targeted liposomes accumulated in the tumor and were 2.5 times more active than non-targeted liposomes. These data indicate that CoPoP liposomes can load cargo into the core of the liposome, can be labeled with His-tagged target-directed peptides without causing cargo leakage, and are specific surface proteins in vitro and in vivo. It is shown that it is possible to go to the molecular receptor expressed in the expressing cell.

Liposomes represent only some of all types 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. This is because gold nanoparticles are used in many biological applications. A thin film of PoP-lipid was hydrated using a citrate-stabilized 60 nm gold dispersion using a probabilistic protocol for lipid-coated gold nanoparticles. Repeated centrifugation and resuspension removed the citrate and resulted in agglomeration of nanoparticles (Fig. 13a). However, in the presence of PoP-lipids, the nanoparticles were coated and remained dispersible. Compared to citrate-stabilized gold, PoP-coated nanoparticles had a slightly larger hydrodynamic diameter, corresponding to the double layer coating over gold (FIG. 13b). The presence of the coating after His tag binding did not affect the gold plasmon peak at 540 nm, demonstrating the mild nature of ligand binding (Fig. 13c). As shown in FIG. 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 virus invasion (eg, 2F5, Z13 and 4E10) target conserved linear epitopes in the proximal outer region of the membrane (MPER) of the gp41 envelope protein, making MPER an HIV peptide. It is the main target for vaccines. However, it is exposed only during viral entry, and attempts to use MPER peptides to produce neutralizing antibodies face challenges. This has created a paradigm that vaccination strategies should consider the interaction of the antibody with the lipid bilayer present in MPER. We used liposomes containing the Toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid A (MPL) in combination with the liposome-bound MPER peptide sequence. However, the use of a simple anchor technique based on the binding of the MPER His tagged polypeptide to Ni-NTA liposomes resulted in low antibody titers. We tested whether the same approach could be enhanced with 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 bound to CoPoP liposomes containing MPL. A single injection containing 25 μg of MPER-His and 25 μg of MPL was administered to BALB / c mice and thymic-deficient nude mice. This elicited a titer ^on the order of 104 in both BALB / c and nude mice, demonstrating a strong humoral immune response (Fig. 6a). This can be important because HIV infects the helper T cell population and makes B cell-mediated responses important. After booster injection, the anti-MPER titers of athymic nude mice were unaffected, but healthy mice showed a single digit increase in titers, indicating a T cell-mediated memory effect. Therefore, the vaccination protocol resulted in both B-cell and T-cell immunity.

Various vaccine components were then tested to better determine the specificity of the immune response (FIG. 6b). The MPER-His peptide itself did not induce antibody when injected in Freund's complete adjuvant or with 2H-PoP liposomes containing MPL. Interestingly, no antibody was produced when the peptide was administered with MPL-free CoPoP liposomes. Another lipid adjuvant, trehalose gibberellinate (TDB), also did not induce antibody production. Since TDB does not act on TLR-4, the importance of MPL in immune activation of the liposome vaccine system is emphasized. When MPER-His bound to Ni-NTA liposomes was used, a weak antibody titer of less than 103 was confirmed ^, consistent with previous reports. However, when using CoPoP liposomes, a double-digit stronger response was observed. Presumably, the stable binding of the peptide to liposomes in vivo is a direct cause of this effect. This CoPoP immunization strategy was effective and antibody titers persisted for at least 3 months, but no antibodies were detected in Ni-NTA liposomes beyond 1 month (Fig. 6c).

Post-vaccinated sera of mice were pooled and purified using Protein G agarose to give purified IgG. It 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 broad 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 show the potential of a vaccination approach using CoPoP liposomes with HIV-derived peptides to induce antibody production that can prevent viral invasion.

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
1,2-Dimyristyl-sn-glycero-3-phosphocholine (DMPC), cholesterol (CHOL), monophosphoryl lipid A (MPLA), and CoPoP (or 2H-PoP) to generate CoPoP and 2H-PoP liposomes. ) Was dissolved in chloroform at the indicated molar ratio (Table 2). After overnight _N2 flow and reduced pressure, 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 freezing / thawing cycles using ice-cold CO ₂ / acetone and a water bath, followed by extruding 10 times at 60 ° C. through a 200 nm polycarbonate membrane.

Psf25 insect protein (recombinant subunit Pfs25 purified from super sf9 cells, concentration measured by BCA analysis was 1 mg / mL) was combined with liposomes overnight (20 hours) at 4 ° C. Psf25 incubated in H ₂ O was used as the control. As a negative control, BSA was combined with liposomes. Binding of Psf25 protein to liposomes was measured by microcentrifugal 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 (100K, OMEGA). The device was rinsed with QH ₂ O before use and centrifuged at 1100 rpm for 3 minutes. Each sample was added and rotated at 1400 rpm for 3 minutes. The device was washed twice with QH ₂ O and centrifuged at 1400 rpm for 3 minutes. Samples were collected from the bottom filtrate and the protein concentration was analyzed by BCA analysis (Thermo cat. 23235), which measures the absorbance at 562 nm.

Vaccination
On days 0 and 21, CD-1 mice (8-week-old female, Envigo) were injected intramuscularly (im) with 5, 0.5, 0.05 μg of Psf25 protein. If indicated, this injection also included 20 μg MPLA incorporated into liposomes (Avanti No. 699800P). ISA720 was also used as an adjuvant to be incubated with 5 μg of Psf25 protein. The treatment groups and flowcharts are shown in Table 3.

Serum anti-Psf25 IgG levels by ELISA Anti-Psf25 titers were evaluated on a 96-well plate (Thermo, Maxisorp) by an enzyme-linked immunosorbent assay (ELISA). His-tagged Psf25 (0.1 μg) in 100 μL coating buffer (3.03 g Na ₂ CO ₃ and 6.0 g NaHCO _3/1 L distilled water, pH 9.6) was incubated overnight at 4 ° C. in wells. Wells were washed 3 times with PBS containing 0.1% Tween (PBS-T), blocked with PBS containing 0.1% casein (PBS-C), and then incubated for 2 hours. The wells were then washed 3 times with PBS-T and goat anti-mouse IgG-HRP (GenScript No. A00160) was diluted with PBS-C to 1 μg / mL and added to each well. The wells were washed again with PBS-T six times, after which tetramethylbenzidine (Amresco No. J644) was added.

result

Determining the ability of liposomes to bind to proteins after coating with Psf25 proteins The average diameters of CoPoP liposomes before and after binding to Psf25 proteins, measured by dynamic light scattering, were 122.9 and 139 nm, respectively (Fig. 14A), and were polydisperse. The index was about 0.05, which was similar, and showed the desired liposome size after binding with a high degree of monodisperse. In addition, to determine if the Psf25 protein efficiently and specifically binds to CoPoP liposomes, Pfs25 is incubated overnight with CoPoP or 2H-PoP liposomes at 4 ° C., followed by protein binding to liposomes. Was measured by microcentrifugal filtration. According to the amount of Pfs25 in the filtrate detected by BCA analysis, 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 immunized with CoPoP / MPLA / Pfs25 liposomes conjugated with Psf25 (concentrations 5, 0.5 and 0.05 μg) or not conjugated with Psf25, or free Psf25 (with or without ISA70) by i.m. , Vaccinated on day 0 (initial stimulation) and day 21 (liposome). Serum was collected on days 20 and 42 and anti-Psf25 IgG titers were measured by ELISA (FIGS. 15A and 15B). Mice prior to booster immunization with CoPoP / MPLA / Psf25 liposomes showed comparable serum IgG levels in both the 5 μg and 0.5 μg Psf25 conjugated groups. For each treatment group, anti-Psf25 IgG-serum samples with different dilutions (starting at 1/1000) were tested in Psf25 (plant) ELISA (Table 3). Here, the cutoff point was set to O.D., which is equal to 0.5. The data show that in G1 and G2 (representing CoPoP / MPLA / Pfs25 liposomes conjugated to 5 or 0.5 μg of Psf25 protein; Table 3), the IgG titer changed from 1/5000 to 1/45000 after booster immunization. show. On the other hand, the IgG titer of CoPoP / MPLA / Pfs25 liposomes conjugated with 0.05 μg of Psf25 protein switched from 1/1000 to 1/15000 after booster immunization, with similar results for G6 (5 μg of Psf25 incubated with ISA70). Represents a protein). This data is also reflected in FIG. 16, which shows that the titer of 0.05 ug of Pfs25 in CoPoP / MPLA liposomes yielded a higher titer of 5 μg of Pfs25 in ISA720.

In this example, a liposome vaccine was developed using a synthetic His-tagged peptide containing repetitive sequences of NANPs derived from the peri-sporozoite protein of Plasmodium falciparum. Liposomes had the same composition as in Example 1, but peptides were used instead of proteins. The peptides tested are shown in FIG. 17A. The average diameters of CoPoP liposomes before and after binding of different NANPs, measured by dynamic light scattering, were 122.9 and 139 nm, respectively (Fig. 17B), and the polydispersity index was about 0.05, which is similar and desirable after binding. The size of the liposomes is shown with a high degree of monodispersity. In addition, to determine if the NANP peptide efficiently and specifically binds to CoPoP liposomes, the NANP peptide is incubated with CoPoP or 2H-PoP liposomes overnight at 4 ° C., followed by peptide binding to the liposomes. Was measured by microcentrifugal filtration. According to the amount of peptide in the filtrate detected by BCA analysis, the majority of NANP bound to CoPoP liposomes (~ 80% at various peptide lengths) but not to 2H-PoP liposomes (20). %Less than). Binding of NANP to CoPoP liposomes was -80% for all different peptide lengths (Fig. 17C).

In this example, CoPoP liposomes containing phosphatidylserine (PS) were used to reduce the antibody response to His-tagged presentation molecules. Liposomes were formed by thin film hydration and extrusion at CoPoP: phosphatidylserine: DOPC: cholesterol = 10:30: 30:30. The His-tagged MPER was then attached to the liposome. These PS liposomes were injected through the soles of mice 4 and 2 weeks prior to vaccination with MPER-decorated CoPoP / MPLA liposomes. As shown in FIG. 18, pretreatment with PS liposomes resulted in a decrease in IgG titer to MPER.

In this example, CoPoP liposomes targeted cells with a His-tagged cell-permeable peptide. The following His-tagged cell-permeable peptides were obtained:
HHHHHHHGRKKRRQRRRPPQ (SEQ ID NO: 13) (TAT peptide);
HHHHHHHRRRRRRRRR (SEQ ID NO: 14) (R8 peptide):
HHHHHHHRQIKIWFQNRRMKWKK (SEQ ID NO: 15) (PEN peptide).
As shown in FIG. 19, after direct aqueous incubation with CoPoP liposomes containing [PoP: CoP: CHOL: DMPC] [2: 5:30:63], these liposomes bind to U87 cells for 1 hour. After incubation, it could be taken up.

In this example, CoPoP liposomes were used with 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-labeled DS-Cav1 has been shown to induce neutralizing antibodies to RSV A2.

CoPoP liposomes bind to His-tagged proteins in a simple mixture of antigen and liposomes, resulting in the resulting liposomes being decorated with a conformationally presented conformationally intact antigen. .. Antigen binding is non-covalently stable in the biological environment. 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, malaria transmission blocking antigen).

His-tagged DS-Cav1 was shown to efficiently bind to CoPoP liposomes. A 50 μL volume of antigen-liposome solution is prepared using CoPoP and PoP (also called 2H) and diluted to 200 μL. Antigen-free liposomes and reference samples of the liposome-free antigen were also prepared. The sample was centrifuged at 20,000 rcf for 90 minutes and the supernatant was extracted from the resulting liposome pellet. Equal volumes of BCA were added to give a total volume of 400 μL. The sample was then incubated in a water bath at 60 ° C. for 10 minutes. Each sample in 100 μL volume was transferred to each of the 3 wells of a 96-well plate and the absorbance was measured at 562 nm. The difference in absorbance between the antigen-liposomal supernatant and the liposome-only reference sample was used to calculate the percentage of binding. Here, higher absorbance corresponds to higher protein concentration in the supernatant and therefore less binding.

Mice were then injected with RSV antigen with one of six adjuvants (CoPoP with PHAD, CoPoP without PHAD, 2HPoP with PHAD, aluminum hydroxide (Alum), Sigma Adjuvant System (SAS), and AddaVax). I injected it. When mice were vaccinated with RSV, all mice injected with the RSV vaccine containing CoPoP / PHAD particles showed detectable IgG antibody titers prior to booster immunization (FIG. 21A). In contrast, only a total of 2 mice from the remaining 5 groups showed detectable IgG titers. Two weeks after the booster inoculation, the IgG titers of mice inoculated with CoPoP, 2HPoP, and SAS all showed a significant increase, but CoPoP with PHAD was still the most effective preparation for inducing antibody production. (FIG. 21B). The mean antibody titer of the CoPoP / PHAD group was higher than that of the other 5 groups, and CoPoP / PHAD was the only group that did not include mice that were unresponsive to the vaccine after additional immunization injection.

An RSV neutralization assay was performed using serum collected from mice 35 days after the first injection (FIGS. 21C, D). The results of this assay show that antibodies produced in response to the CoPoP / PHAD vaccine are significantly more effective in neutralizing RSV types A and B than any other vaccine group. The data conclude that CoPoP / PHAD works better compared to alternative adjuvants such as Alum, SAS, and AddaVax. Moreover, neither CoPoP liposomes nor the PHAD adjuvant component of CoPoP alone is sufficient to increase efficacy, and these results may be due solely 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 lipid tailless B31 strain protein sequence and purified as shown in FIG.

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, which allows for the physical separation of liposome-binding and free proteins due to the limited pore size of the acrylamide gel. The observed optimal binding mass ratio of PHAD: OspA of 4: 1 (FIG. 23A) is consistent with previous studies with the malaria antigen Pfs25. Incubation of 80 μg / mL OspA with an equal volume of 320 μg / mL CoPoP liposomes requires 3 hours of incubation at room temperature (FIG. 23B). Visual inspection of the acrylamide gel showed that after 15 minutes the binding kinetics began to plateau, probably due to steric hindrance to subsequent binding to nearby non-binding sites. Based on the relative band intensities at 0 and 15 minutes, it is estimated that the early stages of binding occurred at a high rate. Under particleization conditions, specific binding is estimated to be approximately 80% based on micro-BCA analysis of supernatants obtained from high speed centrifugation (FIG. 23C). Using the same method, low non-specific binding was observed for PoP / PHAD liposomes lacking the chelated metal. Lysozyme, a non-histidine-tagged protein, did not bind to CoPoP / PHAD or PoP / PHAD liposomes. Based on DLS measurements, the liposome size after incubation remains essentially the same for both CoPoP / PHAD and PoP / PHAD liposomes (FIG. 23D). Furthermore, transmission electron micrographs revealed that CoPoP / PHAD liposomes retain their shape after antigen binding (FIG. 23E).

A serum stability test (FIG. 24A) showed that incubation of proteoliposomes with humans at 37 ° C did not significantly increase the fluorescent signal after 12 days. This basically indicates that OspA remains predominantly associated with the metal chelated liposomes during the study period. CoPoP liposomes showed a> 100-fold increase in stability compared to nickel chelated liposomes due to the strategic spatial location of metal chelate bonds within the hydrophobic bilayer. Serum-stable antigen binding ensures the integrity of proteoliposomes during transport to the influx region lymph nodes.

Previous studies using similar nanoparticle systems have demonstrated a variety of immunogenicity and protective effects depending on the point of attachment to the nanoparticle scaffold. This underscores the importance of presenting the appropriate antigenic epitopes on the particle surface. In this study, a polyhistidine tag was added to the N-terminus opposite the locality of the protective epitope to ensure epitope reachability on the liposome surface and avoid possible occlusion of important C-terminal epitopes. This configuration basically mimics the integration of lipoproteins into the outer membrane of Lyme disease borrelia. Assessment of epitope utilization using immunoprecipitation showed exposure of the LA-2 epitope on surface-bound OspA (FIG. 24B). Negative controls, which are monoclonal antibodies to non-homologous proteins, showed a low PoP fluorescence signal. These results indirectly support the findings in CD spectroscopy to elucidate the secondary structure of recombinant OspA. Furthermore, LA-2 antibody-OspA recognition suggested that the structural integrity of the binding antigen was maintained after binding to liposomes.

The utility of liposomes as antigen-delivering vesicles is due to their enhanced uptake by antigen-presenting cells. Nanoparticle internalization studies using mouse macrophage-like cells (FIG. 24C) showed high antigen uptake only in surface-functionalized liposomes (CoPoP / PoP liposomes). Minimal uptake was observed in the free, non-aggregate or mixed, non-associative (ie, PoP / PHAD) form of the antigen. These results support studies demonstrating enhanced antigen internal translocation in nanoparticle formation. Macrophage-selective uptake of PoP / PHAD liposomes in mixed form further means that physical association of the antigen to the liposomes may be required for liposome adjuvant action. Simultaneous delivery of surface-exposed antigens and immunostimulators to immune cells is an important aspect of CoPoP / PHAD liposomes. The phagocytotic cell uptake mechanism is supported by reduced uptake of both antigens and liposomes in the presence of cytochalasin B.

Assessment of immunogenicity using ELISA shows that this novel vaccine platform can stimulate a strong immune response at low antigen levels. Homological prime-boost vaccination with CoPoP / PHAD liposomes induced higher OspA-specific IgG antibody titers than other commercially available adjuvants (FIG. 25A). In addition, surface labeling of B. burgdorferi B31 cells (FIG. 25B) suggested that the OspA-specific antibodies produced could recognize epitopes on the cell surface, supporting the results obtained from the immunoprecipitation assay. Fluorescence micrographs further reveal the low functionality of anti-OspA antibodies induced by immunization with alum. Immunoblots with antisera derived from CoPoP / PHAD immunity showed that OspA-specific antibodies recognize different strains of Lyme disease boleria, but to varying degrees (FIG. 25C). Different band intensities indicate the antigenic heterogeneity of OspA across different gene species. No obvious band is found in the negative control B. hermsii (lacking the ospa gene). A minimal non-specific band was observed for E. coli lysates with histidine-tagged OspA. The lower molecular weight observed for the OspA band is due to the replacement of the lipid addition signal sequence with a shorter polyhistidine segment.

The inclusion of a synthetic version of monophosphoryl lipid A, PHAD (TLR-4 agonist) in the liposome formulation distorted the immune response to Th1 and switched the isotype to IgG2a. CoPoP / PHAD liposomes produced higher levels of OspA-specific IgG2a antibody than IgG1 (FIG. 26A). On the other hand, Alam induced higher levels of IgG1 isotypes. 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 for CoPoP / PHAD liposomes correlates with higher stimulation of interferon-γ than interleukin-4 in splenocyte studies (FIG. 26B).

Sterilization assays were used to demonstrate the ability of OspA-specific antibodies to eradicate spirochete. Antibodies generated from CoPoP / PHAD-OspA immunization showed higher bactericidal activity compared to Alam or PoP / PHAD liposomes (FIG. 27). The calculated 50% Borrelia killing titer for CoPoP / PHAD liposomes is significantly higher than that for Alam.

OspA-based transmission-blocking vaccines rely heavily on high levels of circulating antibodies. The ELISA titers calculated at different time points remain almost the same after one year, indicating the high endurance of the antibody response (Fig. 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 a major surface antigen on influenza virus and a major vaccine target. We obtained His-labeled H3 strain HA. As shown in FIG. 29, His-tagged HA efficiently bound to CoPoP liposomes.

A 50 μL volume of antigen-liposome solution is prepared using CoPoP and PoP (also called 2H) and diluted to 200 μL. Liposomes without liposomes and antigens without liposomes (reference sample) were also prepared. The sample was centrifuged at 20,000 rcf for 90 minutes and the supernatant was extracted from the resulting liposome pellet. Equal volumes of BCA were added to bring the total volume to 400 μL. The sample was then incubated in a water bath at 60 ° C. for 10 minutes. A 100 μL volume of each sample was transferred to each of the 3 wells of a 96-well plate and the absorbance was measured at 562 nm. The difference in absorbance between the antigen-liposomal supernatant and the liposome-only reference sample was used to calculate the percentage of binding. Here, higher absorbance correlates with higher protein concentration (ie, less binding) in the supernatant.

As shown in FIG. 30, immunization with CoPoP liposomes and His-tagged HA was very strong.

Passive serum transfer experiments have established that vaccine protection is at least partially due to antibody production (Fig. 31).

The ability of multiplexing was then evaluated using multiple His-tagged HAs. As shown in FIG. 32A, various HA and NA proteins bound to CoPoP liposomes. When immunized, all HAs can produce specific antibodies with minimal cross-reactivity (FIG. 32B).

This example demonstrates that different types of synthetic MPLAs, including PHAD, PHAD-504, and 3D6A-PHAD, can be used in the compositions and methods. The structure of these adjuvants is shown in FIG. Liposomes are formed with DPPC: cholesterol: CoPoP: MPLA of 4: 2: 1: X, where MPLA is each of these types of synthetic versions, X is variable, 5, 4, 3, 2, It was one of 1.

As shown in FIG. 34, all types of MPLA can be included to generate liposomes, resulting in generally higher antibody production than MPLA shedding (“CA”). His-tagged Pfs25 was mixed at an antigen: CoPoP mass ratio of 1: 4 and the mice were immunized on days 0 and 21. Serum was collected on day 42 and evaluated for Pfs25-bound IgG ELISA.

Although the present invention has been described through specific embodiments, ordinary modifications are obvious to those of skill in the art and such modifications are intended to be within the scope of the present disclosure.

Claims (20)

It ’s a liposome,
a) A double layer having the following i) and ii);
i) Phospholipids,
ii) Porphyrins coordinated to cobalt to form cobalt porphyrins and b) polyhistidine-tagged presentation molecules, where at least a portion of the polyhistidine-tag is monolayer or bilayer hydrophobic. One or more histidines present in the polyhistidine-tag are coordinated to the cobalt of the cobalt porphyrin;
Including
At least a portion of the polyhistidine-tagged presenting molecule is exposed to the outside of the liposome, the polyhistidine-tagged presenting molecule contains an epitope of microbial origin, and the liposome surrounds the aqueous compartment. Liposomes. The liposome according to claim 1, wherein the microorganism is a virus, a bacterium, or a parasite. The liposome according to claim 2, wherein the epitope is derived from respiratory syncytial virus (RSV), Lyme disease boleria, or influenza virus. The liposome according to claim 3, wherein the epitope is derived from DS-Cav1, Ospa, hemagglutinin, or neuraminidase. The liposome according to claim 1, wherein the cobalt porphyrin is conjugated to a phospholipid to form a cobalt porphyrin-phospholipid conjugate. The liposome according to claim 5, wherein the cobalt porphyrin-phospholipid conjugate constitutes 1 to 25 mol% of a monolayer or a bilayer. The liposome according to claim 6, wherein the cobalt porphyrin-phospholipid conjugate constitutes 5 to 10 mol% of a monolayer or a bilayer. The liposome according to claim 1, wherein the bilayer further comprises cholesterol and / or phosphatidylserine. The liposome according to claim 1, wherein the polyhistidine-tag contains 6 to 10 histidine residues. The liposome according to claim 1, wherein the liposome has a size of 50 nm to 200 nm. The liposome according to claim 18, wherein the adjuvant is an attenuated Lipid A derivative or a phosphorylated hexaacyl disaccharide. It ’s a nanostructure,
A) core; and b) single or bilayer on said core with i) and ii) below;
i) Phospholipid monomers,
ii) A polyhistidine-tagged presentation molecule containing a porphyrin coordinated to cobalt to form cobalt porphyrin and c) an epitope derived from a microorganism, wherein at least a portion of the polyhistidine-tag is monolayered. Alternatively, present in the hydrophobic portion of the 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 a nanostructure. Exposed to the outside of
Nanostructures containing. The nanostructure according to claim 12, wherein the core is gold nanoparticles. 12. The nanostructure of claim 12, wherein the microorganism is a virus, bacterium, or parasite. 12. The nanostructure of claim 12, wherein the epitope is derived from respiratory syncytial virus (RSV), Lyme disease boleria, or influenza virus. The nanostructure according to claim 12, wherein the epitope is derived from DS-Cav1, Ospa, hemagglutinin, or neuraminidase. A method of eliciting an immune response in a host individual comprising administering to the individual the composition comprising the liposome according to claim 1 during a pharmaceutical carrier, wherein the presented molecule is a virus, bacterium, or parasite. A method comprising an immunogenic epitope of origin. 17. The method of claim 17, wherein the epitope is derived from respiratory syncytial virus (RSV), Lyme disease boleria, or influenza virus. 18. The method of claim 18, wherein the epitope is derived from DS-Cav1, OspA, hemagglutinin, or neuraminidase. 17. The method of claim 17, wherein the individual is a human or non-human animal.

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