Nanodisks for removing lipid accumulation in the eye

Abstract

This disclosure generally relates to compositions comprising multiple particles containing an amphiphilic polymer and phospholipids, methods for preparing compositions comprising multiple particles containing an amphiphilic polymer and phospholipids, and uses of such compositions.

Description

Cross-references to related applications

[0001] This application claims the interests and priorities of U.S. Provisional Application No. 63 / 610,922, filed December 15, 2023, and International Application No. PCT / US2024 / 019239, filed March 8, 2024, the contents of which are incorporated herein by reference in their entirety. Reference to electronic sequence listing

[0002] The contents of the electronic sequence list (283912000240seqlist.xml; size: 20,955 bytes; and creation date: December 9, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure generally relates to compositions comprising multiple particles containing an amphiphilic polymer and phospholipids, methods for preparing compositions comprising multiple particles containing an amphiphilic polymer and phospholipids, and uses of such compositions. Background Technology

[0004] Particles containing amphiphilic polymers, such as amphiphilic peptides or membrane scaffold proteins (MSPs), and phospholipids provide a natural lipid-like environment for the incorporation of membrane proteins, and they have become valuable platforms for membrane biophysics research and therapeutic applications. However, most of these MSP / lipid compositions, which offer satisfactory stability and processability, are generated to form a phospholipid bilayer with a relatively high lipid content; that is, the MSP / lipid ratio produces particles that are typically fully lipidated. Summary of the Invention

[0005] In one aspect, this document provides a composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% of the plurality of particles are under-esterified.

[0006] In some embodiments of the above composition, the molar ratio of phospholipid to amphiphilic polymer in the composition is less than the phospholipid-polymer saturation ratio N. S (For example, less than the phospholipid-polymer saturation ratio N) S (approximately 95%).

[0007] In some embodiments of the composition described above, the amphiphilic polymer is an amphiphilic polypeptide. In some embodiments, the amphiphilic polypeptide comprises one or more amphiphilic helical sequences of about 9 to about 30 amino acids. In some embodiments, the amphiphilic polypeptide is selected from the group consisting of apolipoprotein AI (ApoA-I), ApoA-II, ApoC, ApoE, and variants thereof (such as variants of ApoA-I). In some embodiments, the amphiphilic polypeptide comprises one or more amphiphilic helical sequences derived from ApoA-I. In some embodiments, the amphiphilic polypeptide comprises an N-terminus truncated relative to ApoA-I. In some embodiments, the amphiphilic polypeptide is selected from the group consisting of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and variants thereof. In some embodiments, the amphiphilic polypeptide is a variant of any one of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP2N2, wherein the variant does not contain an N-terminal His-tag portion (a variant with the His-tag removed) (hereinafter also referred to as MSP1D1(-), MSP1E1D1(-), MSP1E2D1(-), MSP1E3D1(-), and MSP2N2(-)). In some embodiments, the amphiphilic polypeptide is selected from the group consisting of MSP1D1(-), MSP1E1D1(-), MSP1E2D1(-), MSP1E3D1(-), MSP2N2(-), and their variants. In some embodiments, the amphiphilic polypeptide is (i) a His-tag-removed variant of MSP1D1 containing the amino acid sequence of SEQ ID NO: 11 (i.e., MSP1D1(-)); (ii) a His-tag-removed variant of MSP1E3D1 containing the amino acid sequence of SEQ ID NO: 12 (i.e., MSP1E3D1(-)); (iii) a His-tag-removed variant of MSP1E1D1 containing the amino acid sequence of SEQ ID NO: 17 (i.e., MSP1E1D1(-)); (iv) a His-tag-removed variant of MSP1E2D1 containing the amino acid sequence of SEQ ID NO: 18 (i.e., MSP1E2D1(-)); or (v) a His-tag-removed variant of MSP2N2 containing the amino acid sequence of SEQ ID NO: 19 (i.e., MSP2N2(-)).

[0008] In some embodiments of any of the above compositions, the phospholipid comprises one or more acyl chains and a glycerol backbone. In some embodiments, one or more acyl chains have a length of about 10 to about 22 carbon units. In some embodiments, the phospholipid is cationic, anionic, zwitterionic, or any combination thereof. In some embodiments, the phospholipid comprises DMPC, DPPC, DMPS, POPC, POPs, or PiP2.

[0009] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1D1, MSP1D1(-) or a variant thereof, the phospholipid is DMPC, and N S The molar ratio of DMPC to the amphiphilic polymer in the composition is approximately 81. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer in the composition is less than approximately 78:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer in the composition is approximately 65:1 to approximately 75:1, or approximately 70:1 to approximately 75:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer in the composition is approximately 40:1 to approximately 60:1, such as approximately 46:1 to approximately 55:1, approximately 51:1, or approximately 55:1.

[0010] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1D1, MSP1D1(-) or a variant thereof, the phospholipid is DPPC, and N S The molar ratio is approximately 81. In some embodiments, the molar ratio of DPPC to the amphiphilic polymer in the composition is less than approximately 75:1.

[0011] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1D1, MSP1D1(-) or a variant thereof, the phospholipid is POPC, and N S The molar ratio is approximately 62. In some embodiments, the molar ratio of POPC to the amphiphilic polymer in the composition is less than approximately 58:1.

[0012] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1E1D1, MSP1E1D1(-) or a variant thereof, the phospholipid is DMPC, and N S The molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E1D1 or a variant thereof with the His tag removed) in the composition is less than about 100:1, such as about 60:1 to about 65:1.

[0013] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1E1D1, MSP1E1D1(-) or a variant thereof, the phospholipid is DPPC, and N SThe molar ratio is approximately 107. In some embodiments, the molar ratio of DPPC to the amphiphilic polymer (e.g., MSP1E1D1) in the composition is less than approximately 100:1.

[0014] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1E1D1, MSP1E1D1(-) or a variant thereof, the phospholipid is POPC, and N S The molar ratio of POPC to the amphiphilic polymer (e.g., MSP1E1D1 or a variant thereof with the His tag removed) in the composition is less than about 76:1.

[0015] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1E2D1, MSP1E2D1(-) or a variant thereof, the phospholipid is DMPC, and N S The molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E2D1 or a variant thereof with the His tag removed) in the composition is less than about 128:1.

[0016] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1E2D1, MSP1E2D1(-) or a variant thereof, the phospholipid is DPPC, and N S The molar ratio of DPPC to the amphiphilic polymer (e.g., MSP1E2D1 or a variant thereof with the His tag removed) in the composition is less than about 128:1.

[0017] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1E2D1, MSP1E2D1(-) or a variant thereof, the phospholipid is POPC, and N S The molar ratio of POPC to the amphiphilic polymer (e.g., MSP1E2D1 or a variant thereof with the His tag removed) in the composition is less than about 96:1.

[0018] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1E3D1, MSP1E3D1(-) or a variant thereof, the phospholipid is DMPC, and N SThe molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the composition is less than about 158:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the composition is about 50:1 to about 100:1, such as about 87:1 to about 98:1, about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1.

[0019] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1E3D1, MSP1E3D1(-) or a variant thereof, the phospholipid is DPPC, and N S The molar ratio of DPPC to the amphiphilic polymer (e.g., MSP1E3D1 or a variant thereof with the His tag removed) in the composition is less than about 158:1.

[0020] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP1E3D1, MSP1E3D1(-) or a variant thereof, the phospholipid is POPC, and N S The molar ratio of POPC to the amphiphilic polymer (e.g., MSP1E3D1 or a variant thereof with the His tag removed) in the composition is less than about 118:1.

[0021] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP2N2, MSP2N2(-) or a variant thereof, the phospholipid is DMPC, and N S The molar ratio is approximately 387. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer in the composition is less than approximately 367:1.

[0022] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP2N2, MSP2N2(-) or a variant thereof, the phospholipid is DPPC, and N S The molar ratio is approximately 387. In some embodiments, the molar ratio of DPPC to the amphiphilic polymer in the composition is less than approximately 367:1.

[0023] In some embodiments of any of the above compositions, the amphiphilic polymer is MSP2N2, MSP2N2(-) or a variant thereof, the phospholipid is POPC, and N S The molar ratio is approximately 291. In some embodiments, the molar ratio of POPC to the amphiphilic polymer in the composition is less than approximately 276:1.

[0024] In some embodiments of any of the above compositions, the average size of the particles in the composition is from about 2 nm to about 20 nm in diameter. In some embodiments, the polydispersity index of the particle size in the composition is less than about 0.2.

[0025] In some embodiments of any of the above compositions, the polydispersity index of the phospholipid content of the particles in the composition is less than about 0.2.

[0026] In some embodiments of any of the above compositions, the composition is a pharmaceutical composition.

[0027] In another aspect, this document provides a method for preparing a composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% of the plurality of particles are under-esterified, the method comprising: (1) incubating the amphiphilic polymer and phospholipids to obtain a preparation mixture, wherein the phospholipids are dissolved with a detergent or organic compound prior to incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain particles.

[0028] In some embodiments of the preparation method described above, the molar ratio of phospholipid to amphiphilic polymer in the composition is less than the polymer-phospholipid saturation ratio N. S .

[0029] In some embodiments according to any of the above preparation methods, the molar ratio of phospholipids to amphiphilic polymers in the prepared mixture is less than or equal to the molar ratio of phospholipids to amphiphilic polymers in the composition. In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the prepared mixture is less than about 95% of the molar ratio of phospholipids to amphiphilic polymers in the composition.

[0030] In some embodiments according to any of the above preparation methods, the detergent or organic compound comprises a bile salt.

[0031] In some embodiments according to any of the above preparation methods, the amphiphilic polymer is provided in a solution containing a buffer.

[0032] In some embodiments according to any of the above preparation methods, the concentration of the amphiphilic polymer in the solution is at least about 0.5 mg / mL, such as about 1 mg / mL to about 5 mg / mL, or about 2.5 mg / mL to about 4 mg / mL.

[0033] In some embodiments of any of the preparation methods described above, the method further includes separating the particles. In some embodiments, the particles are separated by chromatography.

[0034] In some embodiments of any of the above preparation methods, the method further includes subjecting the particles to one or more freeze-thaw cycles with or without a cryoprotectant.

[0035] In some embodiments according to any of the above preparation methods, the amphiphilic polymer is an amphiphilic polypeptide. In some embodiments, the amphiphilic polypeptide comprises one or more amphiphilic helical sequences of about 9 to about 30 amino acids. In some embodiments, the amphiphilic polypeptide is selected from the group consisting of apolipoprotein AI (ApoA-I), ApoA-II, ApoC, ApoE, and variants thereof (such as variants of ApoA-I). In some embodiments, the amphiphilic polypeptide comprises one or more amphiphilic helical sequences derived from ApoA-I. In some embodiments, the amphiphilic polypeptide comprises an N-terminus truncated relative to ApoA-I. In some embodiments, the amphiphilic polypeptide is selected from the group consisting of MSP1D1, MSPE1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and variants thereof. In some embodiments, the amphiphilic polypeptide is a variant of any of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP2N2, wherein the variant does not contain an N-terminal His-tag portion (a variant with the His-tag removed). In some embodiments, the amphiphilic polypeptide is selected from the group consisting of MSP1D1(-), MSP1E1D1(-), MSP1E2D1(-), MSP1E3D1(-), MSP2N2(-), and their variants. In some embodiments, the amphiphilic polypeptide is (i) a His-tag-removed variant of MSP1D1 containing the amino acid sequence of SEQ ID NO: 11 (i.e., MSP1D1(-)); (ii) a His-tag-removed variant of MSP1E3D1 containing the amino acid sequence of SEQ ID NO: 12 (i.e., MSP1E3D1(-)); (iii) a His-tag-removed variant of MSP1E1D1 containing the amino acid sequence of SEQ ID NO: 17 (i.e., MSP1E1D1(-)); (iv) a His-tag-removed variant of MSP1E2D1 containing the amino acid sequence of SEQ ID NO: 18 (i.e., MSP1E2D1(-)); or (v) a His-tag-removed variant of MSP2N2 containing the amino acid sequence of SEQ ID NO: 19 (i.e., MSP2N2(-)).

[0036] In some embodiments according to any of the above preparation methods, the phospholipid comprises one or more acyl chains, such as those having a length of about 10 to about 22 carbon units. In some embodiments, the phospholipid has a glycerol backbone. In some embodiments, the phospholipid is cationic, anionic, zwitterionic, or any combination thereof. In some embodiments, the phospholipid comprises DMPC, DPPC, DMPS, POPC, POPS, or PiP2.

[0037] In some embodiments according to any of the above preparation methods, the amphiphilic polymer is MSP1D1, MSP1D1(-), or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1D1 or a His-label-removed variant thereof) in the composition is from about 65:1 to about 75:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1D1 or a His-label-removed variant thereof) in the preparation mixture is less than about 65:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1D1 or a His-label-removed variant thereof) in the composition is from about 65:1 to about 75:1 (e.g., from about 70:1 to about 75:1), and the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1D1 or a His-label-removed variant thereof) in the preparation mixture is about 55:1.

[0038] In some embodiments according to any of the above preparation methods, the amphiphilic polymer is MSP1D1, MSP1D1(-), or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1D1 or a His-label-removed variant thereof) in the composition is from about 46:1 to about 55:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1D1 or a His-label-removed variant thereof) in the preparation mixture is less than about 45:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1D1 or a His-label-removed variant thereof) in the composition is from about 46:1 to about 55:1 (e.g., about 51:1 or about 55:1), and the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1D1 or a His-label-removed variant thereof) in the preparation mixture is about 40:1 or about 30:1.

[0039] In some embodiments according to any of the above preparation methods, the amphiphilic polymer is MSP1E3D1, MSP1E3D1(-), or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the composition is from about 85:1 to about 100:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the preparation mixture is less than or equal to about 90:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the composition is from about 87:1 to about 98:1 or from about 87:1 to about 96:1 (e.g., about 94:1 or about 93:1), and the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the preparation mixture is about 90:1.

[0040] In some embodiments according to any of the above preparation methods, the amphiphilic polymer is MSP1E3D1, MSP1E3D1(-), or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the composition is from about 55:1 to about 80:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the preparation mixture is less than or equal to about 60:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the composition is from about 58:1 to about 68:1 (e.g., about 63:1), and the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a His-label-removed variant thereof) in the preparation mixture is about 60:1. In some embodiments, the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a variant thereof with its His tag removed) in the composition is from about 58:1 to about 68:1 (e.g., about 63:1 or about 62:1), and the molar ratio of DMPC to the amphiphilic polymer (e.g., MSP1E3D1 or a variant thereof with its His tag removed) in the mixture is about 50:1.

[0041] On the other hand, this document provides compositions comprising particles prepared according to any of the above-described preparation methods.

[0042] On the other hand, this document provides a method for treating or preventing a disease associated with lipid accumulation in an individual (e.g., a person), the method comprising administering an effective amount of any of the above-described compositions to the individual. In some embodiments, the disease is associated with lipid accumulation in the eye. In some embodiments, the disease is age-related macular degeneration (AMD), photoreceptor neurodegeneration, optic atrophy, vision loss, hemianopsia, visual agnosia, strabismus, retinal neurovascular disease, lipid keratopathy, corneal liposis, or any combination thereof. In some embodiments, the disease is AMD, such as dry AMD. In some embodiments, dry AMD is geographic atrophy (GA). In some embodiments, the composition reduces lipids (e.g., cholesterol) in the individual by at least about 5% or prevents lipid accumulation. Attached Figure Description

[0043] The accompanying drawings illustrate some embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any way.

[0044] Figure 1A A preparative size exclusion chromatography (SEC) chromatogram of an exemplary DMPC / MSP1D1(-) particulate composition prepared at a 30:1 ratio is shown. (-) indicates the removal of the His tag from MSP1D1. The dark gray shaded area of ​​the peak (top inset) corresponds to an under-esterified particulate composition containing approximately 51 lipids per MSP1D1(-) (bottom inset). Figure 1B A preparative SEC chromatogram of an exemplary DMPC / MSP1E3D1(-) particulate composition prepared at a 50:1 ratio is shown. A single peak is collected (top inset), which corresponds to an under-esterified particulate composition with approximately 62 lipids per MSP1E3D1(-).

[0045] Figure 2 Gas chromatography (GC) results are shown for determining the phospholipid to MSP ratio of an exemplary DMPC / MSP1D1(-) particle composition prepared at a ratio of 30:1.

[0046] Figure 3A Cholesterol efflux assays using various under-esterified particle compositions are shown. The lipid to MSP ratio was measured from the final particle composition (not the preparation mixture). 2.5 μg of sample was used. Controls included a fully esterified particle composition with DMPC:MSP1D1(-) = 81:1, a fully esterified particle composition with DMPC:MSP1D1 = 83:1, lipid-free MSP1D1(-), lipid-free Apo-AI, lipid-free HDL, a positive control (from the assay kit), and a PBS buffer blank (not shown). Figure 3BCholesterol efflux dose-dependent effects are shown for fully esterified particulate compositions DMPC:MSP1D1(-)=81:1 and exemplary under-esterified particulate compositions DMPC:MSP1D1(-)=51:1 at doses of 2.5 μg and 10 μg. (-) indicates removal of the N-terminal His tag.

[0047] Figure 4 The SEC chromatogram of an exemplary under-esterified DMPC / MSP1D1(-) particulate composition prepared at a ratio of 30:1 (final composition ratio of about 51:1) after freezing and thawing without cryoprotectant, compared to before freezing, is shown.

[0048] Figure 5 Cholesterol efflux results are shown for an exemplary under-esterified DMPC:MSP1D1(-) particle composition prepared at a ratio of 30:1 (final composition ratio of about 51:1) before and after freezing and thawing, compared to a control comprising a fully esterified DMPC:MSP1D1 particle composition prepared at a ratio of 85:1 (final composition ratio of 83:1), HDL, and PBS solution.

[0049] Figures 6A to 6D The particulate composition with a DMPC / MSP1D1(-) ratio of approximately 81:1 is shown (preparation mixture 80:1; final composition 81±3:1). Figure 6A A particulate composition with a DMPC / MSP1D1(-) ratio of approximately 75:1 (preparation mixture 55:1; final composition 75±1.5:1) Figure 6B A particulate composition with DMPC / MSP1D1(-) ratio of approximately 51:1 (preparation mixture 40:1; final composition 51±1:1) Figure 6C A particulate composition with a DMPC / MSP1D1(-) ratio of approximately 51:1 (preparation mixture 30:1; final composition 51±1:1) Figure 6D The correspondence between the phospholipid / MSP1D1(-) ratio in the prepared mixture and the final particulate composition.

[0050] Figures 7A to 7C The particulate composition with a DMPC / MSP1E3D1(-) ratio of approximately 94:1 is shown (preparation mixture 90:1; final composition 94±4:1). Figure 7A A particulate composition with DMPC / MSP1E3D1(-) ratio of approximately 63:1 (preparation mixture 60:1; final composition 63±4:1) Figure 7B A particulate composition with a DMPC / MSP1E3D1(-) ratio of approximately 62:1 (preparation mixture 50:1; final composition 62±0.5:1) Figure 7CThe correspondence between the phospholipid / MSP1E3D1(-) ratio in the prepared mixture and the final particulate composition.

[0051] Figure 8 The cholesterol:MSP1D1(-) protein ratio over time is shown, which was tested by mixing a mixture of under-lipidemic DMPC:MSP1D1(-) = 51:1 particles and LUV containing 70% POPC: 30% cholesterol.

[0052] Figure 9A Chromatograms of POPC MSP1D1 particle compositions formed at different POPC to MSP1D1 ratios are shown. The results indicate that a 65:1 ratio results in a fully esterified particle composition. Figure 9B Chromatograms of DMPC / MSP1D1 particulate compositions formed at different DMPC / MSP1D1 ratios are shown. The results indicate that fully esterified particulate compositions are formed at a DMPC / MSP1D1 ratio of 85:1.

[0053] Figure 10 The percentage of cholesterol efflux from macrophages in vitro using various particle compositions with different lipid-to-MSP ratios is shown. Lipid A refers to DMPC, and lipid B refers to POPC.

[0054] Figure 11 It was shown that, after 4 hours of incubation, the fully lipotropic DMPC MSP1D1 particle composition with a lipid-to-MSP ratio of 85:1 had the ability to remove almost twice the amount of cholesterol as naturally occurring ApoA-I from macrophages.

[0055] Figures 12A to 12C The fully lipotropic particle composition (MSP1D1 lipid B) with a POPC to MSP1D1 ratio of 65:1, the fully lipotropic particle composition (MSP1D1 lipid A) with a DMPC to MSP1D1 ratio of 85:1, and the MSP1D1 protein alone are shown after incubation for 2 hours. Figure 12A ), 4 hours ( Figure 12B ) and 6 hours ( Figure 12C The percentage of cholesterol effluxed from human retinal pigment epithelial (RPE) cells outside the body after [the event].

[0056] Figures 13A to 13B The image shows fluorescence images of an exemplary fully lipid-modified GFP-tagged particle composition delivered in vivo into a mouse eye. From left to right, Figure 13A Fluorescence images are shown at 15 minutes, 30 minutes, 60 minutes, 6 hours, and 24 hours after delivery of an exemplary fully esterified GFP-labeled particle composition into the eyes of mice. Figure 13BThe first row in the image, from left to right, shows fluorescence images at 7, 7, 10, 15, and 20 minutes after the exemplary fully esterified GFP-labeled particle composition was delivered into the eyes of mice. Figure 13B The second row, from left to right, shows fluorescence images at 30, 30, 33, and 47 minutes after the exemplary fully esterified GFP-labeled particle composition was delivered into the eyes of mice. Figure 13B The third row in the image, from left to right, shows fluorescence images at 6 hours, 6 hours, and 24 hours after the exemplary fully esterified GFP-labeled particle composition was delivered into the eyes of mice.

[0057] Figure 14A is a schematic diagram depicting the timeline of intravitreal injection and tissue harvesting of the exemplary fully liposomed particulate composition labeled with fluorescence. Figure 14B shows representative images of immunostained retinal sections taken from injected mice, depicting retinal neurons (solid arrows) and the exemplary fully liposomed particulate composition labeled with fluorescence (blank arrows). Figure 14B shows fluorescence images from left to right at 0 min, 15 min, 30 min, 1 hour, 6 hours, and 24 hours after delivery of the exemplary fully liposomed particulate composition labeled with fluorescence into the mouse eye. In Figure 14B, from top to bottom, the image shows fluorescence signals at the retinal pigment epithelial cells (RPE), outer nuclear layer (ONL), and inner nuclear layer (INL).

[0058] Figure 15A is a schematic timeline depicting laser-induced choroidal neovascularization (CNV), intravitreal injection of an exemplary fully lipotropic particulate composition, intravenous injection of FITC-glucan, and tissue harvest. Figure 15B shows a representative confocal image of CNV regions in mice labeled with FITC-glucan and outlined in circles after laser injury. Figure 15C shows a dot plot of CNV areas in mice receiving an exemplary fully lipotropic particulate composition via intravitreal injection at 0 (“Cntl”), 1 (“particulate composition”), or 2 (“particulate composition + d3”). Data (n = 8 to 10 mice / group) are presented as mean (±SEM).

[0059] Figure 16A illustrates a timeline depicting the administration of a high-fat diet, intravitreal injection, baseline (T0), and follow-up (T1) imaging and tissue harvesting in Abca1 / g1-rod / -rod mice. Figures 16B through 16C show line graphs illustrating the amplitude and ratio of full-field dark-vision (Figure 16B) and mild bleaching recovery (Figure 16C) responses in the media (“media”) and exemplary particulate composition-treated (“disc”) mouse groups. Data (media, n=3; disc, n=4) are presented as mean (±SEM). Figure 16DRepresentative images of hematoxylin and eosin stained (H&E) sections of the entire eye (left) and retina (right) are shown. Figure 16E Representative images of immunostained retinal sections are shown, illustrating the expression of retinal neurons (solid arrows) and complement-3 (blank arrows) in the central (left) and intermediate-peripheral (right) retina. Figure 16F Representative baseline (top) and follow-up (bottom) optical coherence tomography (OCT) images of cross-sections of the retina of live mice are shown, which show the incidence of (medial) inner segment / outer segment abnormalities and the absence of (disc) inner segment / outer segment abnormalities (arrows in the lower left image) and retinal pigment epithelium detachment (arrows in the lower right image) at the reference baseline (asterisks and arrows in the top image).

[0060] Figure 17A shows a schematic timeline depicting the administration of a high-fat diet, intravitreal injection, baseline (T0) and follow-up (T1) imaging, blood collection, and tissue harvesting in Abca1 / g1-rod / -rod mice. Figures 17B through 17C show line graphs illustrating the amplitude and ratio of full-field scotopic response (Figure 17B) and mild bleaching recovery (Figure 17C) in the mediator (“media”) and exemplary particulate composition-treated (“disc”) mouse groups. The y-axis represents amplitude (μV). Data (mediator, n=3; disc, n=4) are presented as mean (±SEM). Figure 17D shows a box plot illustrating the proportion of cholesterol esters (CE) in retinal lipids measured in mediator-treated and particulate composition-treated mice. Data (mediator, n=3; disc, n=4) are presented as medians, with interquartile range, minimum, and maximum values ​​indicated.

[0061] Figure 18 An exemplary chromatogram of a fully esterified particulate composition formulation is shown. Detailed Implementation

[0062] This invention provides compositions comprising a plurality of particles containing an amphiphilic polymer (e.g., membrane scaffold protein (MSP)) and phospholipids, wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidized, i.e., the particles contain less phospholipids than would be required to saturate the particles. Such compositions are also referred to hereinafter as “under-lipidized particle compositions”. A method for preparing such under-lipidized particle compositions is also provided.

[0063] Particle compositions containing MSP and lipids are typically produced with relatively high lipid content to meet stability and processability requirements. Such particle compositions are referred to below as “fully liposomed particle compositions,” meaning the particles contain the amount of phospholipids required to saturate the particles. Fully liposomed particle compositions have been shown to remove lipids, such as cholesterol and cholesterol esters. See, for example, U.S. Provisional Application No. 63 / 450,945, filed March 8, 2023, the entire contents of which are incorporated herein by reference. In some embodiments, fully liposomed particles may have a certain capacity to adsorb cholesterol and cholesterol esters. The ability of particles to take up cholesterol and cholesterol esters is an important parameter for overall efficacy and for minimizing the recipient’s xenobiotic burden in any therapeutic application.

[0064] The inventors of this invention have surprisingly discovered that hypolipidemic particle compositions can be successfully prepared and can be homogeneous. This was unknown at the time of this invention. These stable hypolipidemic particles exhibit several unexpectedly superior effects. The inventors have discovered that, for the hypolipidemic particles described herein, the ability of the particles to absorb cholesterol and / or cholesterol esters may be related to the lipid saturation of the particles. The hypolipidemic particle compositions described herein possess novel physical and chemical properties and can lead to improved therapeutic solutions. First, compared to an equivalent dose of a fully lipidemic particle composition, or even at much lower doses, the hypolipidemic particle compositions exhibit a greater lipid (e.g., cholesterol) efflux capacity, demonstrating promising applications in the removal of lipids (e.g., cholesterol and cholesterol esters) from cells and tissues and in the treatment of related diseases. The cholesterol efflux capacity of the hypolipidemic particle compositions was found to be significantly better than that of high-density lipoprotein (HDL), lipid-free MSP protein, and lipid-free Apo-AI. Secondly, the under-lipidemic particulate compositions exhibit excellent stability, such as excellent stability after freeze-thaw cycles with or without a cryoprotectant. The cholesterol efflux capacity of the under-lipidemic particulate compositions remains unaffected after freeze-thaw cycles. Thirdly, the methods provided herein for producing under-lipidemic particulate compositions not only yield remarkably stable under-lipidemic particulate compositions with excellent cholesterol efflux activity, but are also simple and require very few purification steps, sometimes only a single purification. The resulting under-lipidemic particulate compositions exhibit a remarkably narrow size distribution and a narrow molar ratio distribution between phospholipids and amphiphilic polymers. Fourthly, when mixed with large monolayer vesicles (LUVs) containing lipids (e.g., cholesterol), the under-lipidemic particulate compositions exhibit high lipid (e.g., cholesterol) binding capacity at equilibrium. For example, exemplary under-lipidemic particulate compositions show binding of 7 mol of cholesterol per mol of particulate composition at equilibrium. The maximum lipid (e.g., cholesterol) binding capacity of the under-lipidemic particulate compositions may be even higher.

[0065] In one aspect, this document provides a composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-esterified. In some embodiments, the molar ratio of phospholipids to the amphiphilic polymer in the composition is less than the polymer-phospholipid saturation ratio N. S In some embodiments, the molar ratio of phospholipid to amphiphilic polymer in the composition is less than the polymer-phospholipid saturation ratio N described herein. S In some embodiments, the molar ratio of phospholipid to amphiphilic polymer in the composition is less than the polymer-phospholipid saturation ratio N.S The content is approximately 95% (e.g., less than any of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower). The inventors of this invention have further discovered that certain specific ratios of lipids to amphiphilic polypeptides can produce homogeneous particles with maximal uptake capacity for cholesterol and cholesterol esters. This enhanced capacity is crucial for the therapeutic applications described herein. Several exemplary under-lipidized particle compositions with different amphiphilic polymer components and different phospholipid-to-amphiphilic polymer ratios are provided herein.

[0066] In another aspect, a method is provided for preparing a composition comprising a plurality of particles, wherein each of the plurality of particles contains an amphiphilic polymer and a phospholipid, wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified, the method comprising: (1) incubating the amphiphilic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is dissolved with a detergent or an organic compound prior to incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain particles. In some embodiments, the molar ratio of phospholipid to amphiphilic polymer in the composition is less than the polymer-phospholipid saturation ratio N. S In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture is equal to or less than the molar ratio of phospholipids to amphiphilic polymers in the composition (e.g., less than any one or lower of about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%). In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture is approximately equal to about any one of the molar ratios of phospholipids to amphiphilic polymers in the composition. For example, the deviation between the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture and the molar ratio of phospholipids to amphiphilic polymers in the composition is less than any one of about 20%, 15%, 10%, 5%, 2%, 1%, or 0.1%. In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture described herein produces a homogeneous, under-esterified particulate composition free of foreign matter.

[0067] The compositions and treatments described herein are particularly helpful in treating a wide range of diseases characterized by lipid accumulation, such as eye diseases (including macular degeneration and atrophy in the eye).

[0068] I. Definition

[0069] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the / that” include multiple indicators.

[0070] The term "about" indicates and covers the indicated value as well as the range above and below that value. In some embodiments, the term "about" indicates a specified value of ±20%, ±15%, ±10%, ±5%, or ±1%. In some embodiments, the term "about" indicates a specified value ± one standard deviation of that value.

[0071] The terms "effective amount" and "pharmaceutically effective amount" refer to the amount of reagent sufficient to provide a desired biological outcome. This outcome can be a reduction (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and / or ablation of the signs, symptoms, or cause of a disease or condition, or any other desired alteration of a biological system. Those skilled in the art can determine the appropriate effective amount in any individual case using routine laboratory methods.

[0072] The terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and refer to any animal with a complement system, which in some embodiments is a mammal and in some embodiments is a human, including those who require treatment or are susceptible to a disease or its sequelae. An individual may include, for example, dogs, cats, pigs, cattle, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans. In some embodiments, the individual is a human.

[0073] The term "treatment" generally refers to a clinical intervention designed to alter the natural course of the treated individual or cells during a clinicopathological process. Desired therapeutic effects include slowing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a disease or condition are reduced or eliminated, including but not limited to reducing the frequency and / or severity of signs and / or symptoms caused by the disease, improving the quality of life of a person with the disease, reducing the dosage of other medications required to treat the disease, and / or prolonging the individual's survival. Treatment can be preventative (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms of the disease) or therapeutic suppression or reduction of symptoms after the onset of the disease.

[0074] As used herein, “percentage of amino acid sequence identity (%)” and “homology” for a polypeptide sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular polypeptide sequence after sequence alignment and cleavage (if necessary) to achieve the maximum percentage of sequence identity, without regard to any conserved substitutions as part of sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN.™ (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0075] As used herein, "amphiphilic" refers to a molecule (e.g., a polypeptide) having both a polar, water-soluble (hydrophilic) group and a water-insoluble, nonpolar (hydrophobic) group. Amphiphilic molecules possess both hydrophilic and hydrophobic properties. Examples include phospholipids, cholesterol, amphiphilic peptides, glycolipids, and fatty acids. In some embodiments, amphiphilic peptides include, but are not limited to, membrane scaffold proteins (MSPs), such as MSP1, MSP1D1, MSP1E1, MSP1E1D1, MSP1E2, MSP1E2D1, MSP1E3, and MSPE3D1 as described herein.

[0076] It should be understood that the embodiments of the present invention described herein include embodiments that are "composed of" and / or "composed primarily of".

[0077] II. Particulate Composition

[0078] In one aspect, this document provides a composition (e.g., a pharmaceutical composition) comprising a plurality of particles (hereinafter also referred to as a "particle composition") containing a polymer (e.g., an amphiphilic polymer) and phospholipids, wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-esterified. In some embodiments, the molar ratio of phospholipids to the amphiphilic polymer in the composition is less than the polymer-phospholipid saturation ratio N. S (For example, less than any of about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower). In some embodiments, the amphiphilic polymer is an amphiphilic polypeptide, such as ApoA-I, or a membrane scaffold protein (MSP), such as an ApoAI-derived MSP.

[0079] In some embodiments, the amphiphilic polymer is ApoA-I, ApoA-II, ApoC, ApoE, or variants thereof. In some embodiments, the amphiphilic polypeptide is a variant of ApoA-I. In some embodiments, the amphiphilic polypeptide comprises one or more amphiphilic helical sequences derived from ApoA-I. In some embodiments, the amphiphilic polypeptide comprises an N-terminus truncated relative to ApoA-I. In some embodiments, the amphiphilic polypeptide is selected from the group consisting of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and variants thereof (e.g., variants with the N-terminal His tag portion removed). In some embodiments, the phospholipid comprises one or more acyl chains, optionally having a length of about 10 to about 22 carbon units. In some embodiments, the phospholipid has a glycerol backbone. In some embodiments, the phospholipid is cationic, anionic, zwitterionic, or any combination thereof. In some embodiments, the phospholipid comprises one or more acyl chains, which may be unsaturated, partially saturated, or fully saturated. In some embodiments, the phospholipid is selected from the group consisting of DMPC, DPPC, and DMPS. In some embodiments, the phospholipid is selected from the group consisting of POPC, POPS, and PiP2.

[0080] In some embodiments, all particles within the composition are under-lipidized. In some embodiments, at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99% or more) of the particles within the composition are under-lipidized. In some embodiments, the composition further comprises a lipid-free amphiphilic polymer (e.g., a lipid-free MSP) not assembled into particles. In some embodiments, no more than about 15% (e.g., no more than about 10%, 5%, 2%, 1% or less) of the particles within the composition are fully lipidized. In some embodiments, the under-lipidized particle composition is further purified (e.g., by SEC), for example, purified once, twice, three times or more. In some embodiments, the under-lipidized particle composition is not purified. In some embodiments, the under-esterified particulate composition comprises a plurality of particles having a narrow molar ratio distribution between phospholipids and polymers (e.g., amphiphilic peptides), for example, wherein the polydispersity index of the phospholipid content in the particles of the composition is less than any one of about 0.5, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. In some embodiments, the under-esterified particulate composition is stable when subjected to freezing or thawing (e.g., one, two, three, or more cycles), for example, with or without a cryoprotectant, after at least one (e.g., one, two, three, four, or five) freeze-thaw cycles (compared to before freezing), the change in the molar ratio between phospholipids and polymers (e.g., amphiphilic peptides) in the particulate composition is less than any one of about 20%, 10%, 5%, 1%, or lower. In some embodiments, the change in lipid (e.g., cholesterol) efflux capacity of the under-esterified particulate composition subjected to freezing or thawing (e.g., one, two, three, or more cycles) compared to before freezing with or without a cryoprotectant is less than any of about 20%, 10%, 5%, 1%, or lower. In some embodiments, the particles may be in the form of a nanocomposite material, comprising a lipid bilayer containing phospholipids surrounded by a stabilizing band containing a polymer (e.g., an amphiphilic polypeptide). In some embodiments, the average size of the particles in the composition is from about 2 nm to about 20 nm in diameter. In some embodiments, the polydispersity index of the particle size in the composition is less than about 0.2, such as less than any of about 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. In some embodiments, the polydispersity index of the lipid content of the particles in the composition is less than any one of about 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01.

[0081] In some embodiments, a composition is provided comprising particles containing MSP1D1 (or a variant thereof with its His tag removed, such as SEQ ID NO: 11) and DMPC, wherein the molar ratio of DMPC to MSP1D1 (or a variant thereof with its His tag removed (or MSP1D1(-)), such as SEQ ID NO: 11) in the composition is from about 40:1 to about 81:1, such as from about 65:1 to about 75:1, from about 70:1 to about 75:1, or about 75:1. In some embodiments, a composition is provided comprising a plurality of particles containing MSP1D1 (or a variant thereof with its His tag removed (or MSP1D1(-)), such as SEQ ID NO: 11) and DMPC, wherein the molar ratio of DMPC to MSP1D1 (or a variant thereof with its His tag removed, such as SEQ ID NO: 11) in the composition is from about 40:1 to about 60:1, such as from about 46:1 to about 55:1, about 51:1, about 55:1, or about 50:1. In some embodiments, a composition is provided comprising a plurality of particles containing MSP1E3D1 (or a His-tagged variant thereof (or MSP1E3D1(-)), such as SEQ ID NO: 12) and DMPC, wherein the molar ratio of DMPC to MSP1E3D1 (or a His-tagged variant thereof, such as SEQ ID NO: 12) in the composition is from about 50:1 to about 100:1, such as from about 87:1 to about 98:1, from about 87:1 to about 96:1, from about 58:1 to about 68:1, from about 63:1, or from about 62:1. In some embodiments, the average size of the particles in the composition is from about 2 nm to about 20 nm in diameter. In some embodiments, the polydispersity index of the particle size in the composition is less than about 0.2, such as less than any one of about 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. In some embodiments, the polydispersity index of the lipid content of the particles in the composition is less than about 0.2, such as less than any one of about 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.01.

[0082] a. polymers

[0083] In some embodiments, the particles described herein contain amphiphilic polymers. In some embodiments, each particle contains one, two, or three amphiphilic polymers. In some embodiments, each particle contains two amphiphilic polymers, such as any amphiphilic polypeptides described herein. As used herein, "polymer" refers to a composition of individual structural units of an organic or inorganic compound that produces an amphiphilic material with extended amphiphilic properties. In some embodiments, the polymer is a polypeptide. In some embodiments, in the case of amino acid-based polymers (e.g., polypeptides), the number of individual structural units may be in the range of approximately 2 to 1000, such as approximately 5-75, 5-50, 5-25, 25-500, 25-250, 25-200, 25-150, 25-100, or 25-50. In some embodiments, the amphiphilic polypeptide may be any amphiphilic polypeptide capable of supporting a lipid bilayer structure, such as membrane scaffold proteins (MSPs) described herein. In some embodiments, the amphiphilic peptide may include, but is not limited to, MSP1D1, MSP1E1D1, MSP1E2D1, MSPE3D1, and MSP2N2 as described herein. In some embodiments, the amphiphilic peptide is MSP1, MSP1E1, MSP1E2, MSP1E3, or variants thereof (e.g., variants with the His tag removed). In some embodiments, the peptide is an apolipoprotein or a variant thereof. In some embodiments, the peptide is a membrane scaffold protein (MSP). In some embodiments, the polymer is an amphiphilic polymer. In some embodiments, the amphiphilic polymer is an amphiphilic peptide. In some embodiments, about 80% (w / w), 85% (w / w), 90% (w / w), 92% (w / w), 94% (w / w), 96% (w / w), 99% (w / w), or 99.5% (w / w) of the total amount of peptide in the particle is an amphiphilic peptide as described herein. In some embodiments, the amphiphilic polypeptide comprises one or more amphiphilic helical sequences of about 9 to about 30 amino acids, such as about 10 to about 25 amino acids, about 11 to about 22 amino acids, about 11 amino acids, or about 22 amino acids.

[0084] Apolipoproteins may include, but are not limited to, apolipoprotein A (Apo-A), such as Apo-A1, Apo-A2, Apo-A4, and Apo-A5; apolipoprotein B (Apo-B), such as Apo-B48 and Apo B-100; apolipoprotein C (Apo-C), such as ApoC-I, apoApoC-II, apoApoC-III, and ApoC-IV; apolipoprotein D (Apo-D); apolipoprotein E (Apo-E); and apolipoprotein M (ApoM). In some embodiments, the apolipoproteins may be derived from any organism, including but not limited to humans, cattle, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, mice, rats, cats, guinea pigs, hamsters, ducks, chickens, salmon, and eels. In some embodiments, the apolipoproteins are human apolipoproteins.

[0085] In some embodiments, the polymer (e.g., an amphiphilic polymer) comprises a variant of an apolipoprotein selected from the group consisting of ApoA-I, ApoA-II, Apo-C, Apo-E, Apo-M, and any combination thereof. In some embodiments, the polymer comprises a fragment of a native apolipoprotein derived from the group consisting of ApoA-I, ApoA-II, Apo-C, Apo-E, and Apo-M. In some embodiments, the polymer contains a mutation (e.g., insertion, deletion, substitution, or any combination thereof) compared to a naturally occurring apolipoprotein (e.g., ApoA-I). In some embodiments, the polymer contains one or more substitutions for the amino acid cysteine ​​(Cys). In some embodiments, the mutation (e.g., Cys substitution) is located at the N-terminus, C-terminus, and / or between the amphiphilic helices of the polymer. In some embodiments, the Cys substitution is in one helix of the polymer. In some embodiments, the cysteine ​​may be linked to an imaging group, optionally a fluorescent group (e.g., fluorescein, rhodamine, or other common small luminescent molecules). In some embodiments, the polymer (e.g., an amphiphilic polymer) is fused to a fluorescent protein (such as GFP, RFP, or YFP). In some embodiments, the polymer (e.g., an amphiphilic polymer) contains a tag, such as a tag commonly used for protein expression and / or purification. In some embodiments, the tag is polyhistidine, c-Myc, FLAG, biotin, or any combination thereof. In some embodiments, the polymer (e.g., an amphiphilic polymer) contains an enzymatic cleavage site, such as a tobacco etch virus protease (TEV) recognition site (boxed in Table 1).

[0086] In some embodiments, the polymer (e.g., an amphiphilic polymer, such as an amphiphilic polypeptide) is an MSP. In some embodiments, the MSP contains a His tag. In some embodiments, the MSP does not contain a His tag. As used herein, the symbol "(-)" refers to an amphiphilic polymer (e.g., an MSP) with the His tag removed. In these constructs, the His tag is followed by a TEV protease recognition site. TEV cleavage leaves an additional glycine (G) residue at the amino terminus of the MSP. In some embodiments, the MSP is derived from naturally occurring ApoA-I, such as human ApoA-I. The TEV protease recognition site sequence is boxed in Table 1.

[0087] ApoA-I is a major protein component of high-density lipoprotein (HDL) particles in human plasma. Human ApoA-I contains an N-terminal globular domain (GLOB) of approximately 43 residues, followed by ten amphiphilic helices (H1-H10) of 11, 22, or 24 residues each. Seven of the helices are 22 amino acids long, one helix (H10) is 24 amino acids long, and the two helices (H3, H9) are 11 amino acids long. The helices are separated by glycine or proline residues. Table 1 indicates the sequences of the different domains of human ApoA-I. The full-length human ApoA-I from N' to C' comprises: GLOB-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10. Human ApoA-I contains the amino acid sequence of SEQ ID NO: 1. In some embodiments, the GLOB domain contains the amino acid sequence DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN (SEQ ID NO: 14).

[0088] MSPs containing alternative amphiphilic helical polypeptide sequences can be generated by adding or removing one or more helical segments (e.g., H1, H2, etc.) or partial helical segments (e.g., removing part of the H1 sequence of ApoA-I to achieve H(0.5)), or by adding, removing, or substituting amino acid residues based on ApoA-I (e.g., human ApoA-I) proteins. In some embodiments, MSPs can be derived from ApoA-I (e.g., human ApoA-I) by removing the GLOB domain. In some embodiments, MSPs can be derived from ApoA-I (e.g., human ApoA-I) by adding one (e.g., H4), two (e.g., H4 and H5), three (e.g., H4-H6), or more helical segments.

[0089] Table 1 indicates exemplary MSPs and their contained domains and sequences. Other MSPs with variations relative to those in Table 1 may also be used in this paper. For example, by adding or removing one or more helical segments or portions thereof, and / or by adding, removing and / or substituting one or more amino acid residues at the N-terminus, interior and / or C-terminus (e.g., adding peptide linkers between helical segments), and / or by adding, removing and / or substituting tag sequences.

[0090] Table 1. Sequences of ApoA-I and exemplary membrane scaffold proteins (MSPs)

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] ApoA-1 can originate from any organism. In some embodiments, ApoA-1 is derived from animals selected from the group consisting of humans, cattle, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, mice, rats, cats, guinea pigs, hamsters, ducks, chickens, salmon, eels, and any combination thereof. In some embodiments, ApoA-1 is human ApoA-1. In some embodiments, human ApoA-1 comprises the amino acid sequence of SEQ ID NO:1 (or is substantially composed of or consists of this amino acid sequence).

[0097] In some embodiments, the amphiphilic polypeptide is a variant of ApoA-I. In some embodiments, the amphiphilic polypeptide has at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or higher) sequence identity with SEQ ID NO: 1. In some embodiments, the amphiphilic polypeptide is selected from the group consisting of: MSP1D1 (SEQ ID NO: 2), MSP1E1D1 (SEQ ID NO: 3), MSP1E2D1 (SEQ ID NO: 4), MSP1E3D1 (SEQ ID NO: 5), and MSP1N2 (SEQ ID NO: 6). In some embodiments, the amphiphilic polypeptide is a variant of any of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP1N2, such as variants with insertions, deletions, and / or substitutions (e.g., conserved substitutions). Variants of any of the amphiphilic polypeptides described herein may have at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99% or higher) sequence identity with their corresponding reference amphiphilic polypeptide.

[0098] In some embodiments, the amphiphilic polypeptide comprises an N-terminal truncation relative to ApoA-I. In some embodiments, the truncation comprises the deletion of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 100 or more consecutive amino acids from the N-terminus of ApoA-I (e.g., relative to SEQ ID NO: 1). In some embodiments, the truncation comprises the deletion of about 10 to 70 amino acids from the N-terminus of ApoA-I. In some embodiments, the N-terminal GLOB domain of ApoA-I is removed from the amphiphilic polypeptide. In some embodiments, the first 11 amino acids of H1 of ApoA-I are removed from the amphiphilic polypeptide, thereby producing the H(0.5) helical portion of STFSKLREQLG (SEQ ID NO: 16) (see H(0.5) in Table 1). In some embodiments, both the N-terminal GLOB domain of ApoA-I and the first 11 amino acids of H1 are removed from the amphiphilic polypeptide.

[0099] In some embodiments, the amphiphilic polypeptide includes the amino acid sequence MGHHHHHHHHDYDIPTTENLYFQG (SEQ ID NO: 15) at its N-terminus, also referred to below as the “His tag” or “His tag moiety”. The TEV protease recognition site is indicated by a box. In some embodiments, the amphiphilic polypeptide does not include the N-terminal His tag moiety (his tag removed), indicated herein by “(-)” at the end of the amphiphilic polypeptide name. In some embodiments, the N-terminal His tag (SEQ ID NO: 15) is removed from the amphiphilic polypeptide by cleavage with the TEV protease. It should be understood that cleavage at the TEV protease recognition site may produce a residual glycine (G) residue at the N-terminus of the resulting MSP sequence. For example, MSP1D1(-) (SEQ ID NO: 11) was obtained by cleaving MSP1D1 (SEQ ID NO: 2) with TEV protease; MSP1E1D1(-) (SEQ ID NO: 17) was obtained by cleaving MSP1E1D1 (SEQ ID NO: 3) with TEV protease; MSP1E2D1(-) (SEQ ID NO: 18) was obtained by cleaving MSP1E2D1 (SEQ ID NO: 4) with TEV protease; and MSP1E3D1(-) (SEQ ID NO: 12) was obtained by cleaving MSP1E3D1 (SEQ ID NO: 5) with TEV protease. It should also be understood that for MSPs in which the indicated His tag (SEQ ID NO: 15) is not removed, the N-terminal methionine residue will generally be removed during heterologous expression (e.g., if expressed in bacteria such as E. coli).

[0100] In some embodiments, the amphiphilic polypeptide is MSP1. In some embodiments, MSP1 comprises the amino acid sequence of SEQ ID NO:13. MSP1 is derived from the sequence of ApoA-1 but lacks the globular N-terminal domain of native ApoA-1. In some embodiments, the amphiphilic polypeptide has at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity with SEQ ID NO:13.

[0101] In some embodiments, the amphiphilic polypeptide is MSP1D1 and comprises the amino acid sequence of SEQ ID NO: 2. MSP1D1 is derived from the sequence of ApoA-1 but lacks the globular N-terminal domain of native ApoA-1 and removes 11 amino acids from the first helix labeled H1 in the parent ApoA-1 to generate a half-helix labeled H(0.5). In some embodiments, the amphiphilic polypeptide is a variant of MSP1D1 having at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity with SEQ ID NO: 2. In some embodiments, the amphiphilic polypeptide is a variant of MSP1D1 without the N-terminal His-tag portion (the his-tag removed variant) (hereinafter referred to as MSP1D1(-)) comprising the amino acid sequence of SEQ ID NO: 11.

[0102] In some embodiments, the amphiphilic polypeptide comprises one or more amphiphilic helical sequences, such as one or more amphiphilic helical sequences derived from MSP or apolipoproteins (e.g., ApoA-I). In some embodiments, the amphiphilic polypeptide comprises one or more (e.g., 1, 2, 3 or more) additional amphiphilic helical sequences relative to ApoA-I. In some embodiments, each additional amphiphilic helical sequence is independently derived from any of ApoA-I, ApoA-II, ApoC-I, ApoC-II, Apo-E, apolipoprotein III (apoLp-III), myoglobin, or hemoglobin. In some embodiments, the additional amphiphilic helical sequence is not derived from ApoA-I, ApoA-II, ApoC-I, ApoC-II, Apo-E, apolipoprotein III, myoglobin, or hemoglobin. In some embodiments, the additional amphiphilic helical sequence is synthetic or non-naturally occurring. In some embodiments, the additional amphiphilic helical sequence comprises at least one (e.g., 1, 2, 3, 4, 5 or more) α-helix or a fragment thereof. In some embodiments, the amphiphilic polypeptide does not contain the GLOB domain of SEQ ID NO: 14. In some embodiments, the amphiphilic helical sequence of helix 4 (H4) can be inserted into the amphiphilic polypeptide MSP1D1 to generate MSP1E1D1, as defined by SEQ ID NO: 3. In some embodiments, two amphiphilic helices, helix 4 (H4) and helix 5 (H5), can be inserted into MSP1D1 to generate MSP1E2D1 (SEQ ID NO: 4). In some embodiments, three amphiphilic helices H4, H5 and H6 can be inserted into MSP1D1 to generate MSP1E3D1 as defined by SEQ ID NO: 5. In some embodiments, an additional nine amphiphilic helices are added to MSP1D1 (SEQ ID NO: 2) using a GT peptide linker to generate the extended amphiphilic polymer MSP2N2 (SEQ ID NO: 6).

[0103] In some embodiments, the amphiphilic polypeptide includes an additional helical sequence, such as one, two, or three first, second, or third helical sequences. In some embodiments, the additional amphiphilic helical sequence is added to the N-terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphiphilic helical sequence is added to the C-terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphiphilic helical sequence is inserted between the helices of the apolipoprotein (e.g., ApoA-I). In some embodiments, each additional amphiphilic helical sequence has about 10 to about 30 amino acids (including, for example, about 15 to about 25, about 24, about 20, about 22, or about 11). In some embodiments, the total length of the additional amphiphilic helical sequence is any one of about 20 to 25, about 40 to 46, about 60 to 68, or about 100 to 200 amino acids.

[0104] In some embodiments, the amphiphilic polypeptide is MSP1E1, MSP1E2, or MSP1E3. MSP1E1, MSP1E2, and MSP1E3 are all extended MSPs and are obtained by inserting an additional 22-mer helix H4, two additional helices H4+H5, or three additional helices H4-H5-H6 into the MSP1 protein (SEQ ID NO: 13).

[0105] In some embodiments, the amphiphilic polypeptide comprises an additional amphiphilic helical sequence and an N-terminal truncation relative to ApoA-I. In some embodiments, the amphiphilic polypeptide is MSP1E3D1. In some embodiments, MSP1E3D1 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amphiphilic polypeptide is a variant of MSP1E3D1 having at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity with SEQ ID NO: 5. In some embodiments, the amphiphilic polypeptide is a variant of MSP1E3D1 without the N-terminal His-tag portion (the his-tag removed variant) (hereinafter referred to as MSP1E3D1(-)), comprising the amino acid sequence of SEQ ID NO: 12.

[0106] In some embodiments, the amphiphilic polypeptide is MSP1E1D1. In some embodiments, MSP1E1D1 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amphiphilic polypeptide is a variant of MSP1E1D1 having at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity with SEQ ID NO: 3. In some embodiments, the amphiphilic polypeptide is a variant of MSP1E1D1 without the N-terminal His-tag portion (the his-tag removed variant) (hereinafter referred to as MSP1E1D1(-)), comprising the amino acid sequence of SEQ ID NO: 17.

[0107] In some embodiments, the amphiphilic polypeptide is MSP1E2D1. In some embodiments, MSP1E2D1 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the amphiphilic polypeptide is a variant of MSP1E2D1 having at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity with SEQ ID NO: 4. In some embodiments, the amphiphilic polypeptide is a variant of MSP1E2D1 without the N-terminal His-tag portion (the his-tag removed variant) (hereinafter referred to as MSP1E2D1(-)), comprising the amino acid sequence of SEQ ID NO: 18.

[0108] In some embodiments, the amphiphilic polypeptide is MSP2N2. In some embodiments, MSP2N2 comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the amphiphilic polypeptide is a variant of MSP2N2 having at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity with SEQ ID NO: 6. In some embodiments, the amphiphilic polypeptide is a variant of MSP2N2 without the N-terminal His-tag portion (the variant with the His-tag removed) (hereinafter referred to as MSP2N2(-)), which comprises the amino acid sequence of SEQ ID NO: 19.

[0109] Detailed descriptions of MSP1D1, MSP1E1D1, MSP1E2D1, and MSP1E3D1 are available in Denisov et al., J AmChem Soc., 24 March 2004, 126(11):3477-87, the full text of which is incorporated herein by reference.

[0110] MSP2N2 is generated by extending the His-marked MSP1D1, adding a GT spacer region and additional helices 2 to 10. The detailed composition and preparation procedure of MSP2N2 are described in Grinkova et al., Protein Engineering Design and Selection 2010 23, 843-848, the full text of which is incorporated herein by reference.

[0111] In some embodiments, the histidine tag is removed from MSP1D1, MSPE1D1, MSP1E2D1, MSPE3D1, or MSP2N2 by cleavage with a TEV protease to generate MSP1D1(-), MSPE1D1(-), MSP1E2D1(-), MSPE3D1(-), or MSP2N2(-), respectively. It should be understood that TEV protease cleavage at the boxed regions shown in Table 1 can produce residual glycine (G) amino acid residues at the N-terminus of the resulting MSP protein. These variants are referred to herein as "His-tag-removed variants".

[0112] In some embodiments, the polymer, such as the polypeptide, is a variant of Apo-C. The Apo-C family comprises three closely related proteins: ApoC-I, ApoC-II, and ApoC-III, which are primarily produced in the liver and to a lesser extent in the intestine. In some embodiments, ApoC-I, ApoC-II, and ApoC-III comprise the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. In some embodiments, the amphiphilic polypeptide has at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity with any of SEQ ID NO: 7-9.

[0113] In some embodiments, polymers such as peptides are variants of Apo-E. ApoE is responsible for the uptake and transport of cholesterol in the blood. In some embodiments, Apo-E comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, MSP has at least about 70% (e.g., at least about 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity with SEQ ID NO: 10.

[0114] b. Phospholipids

[0115] Phospholipids generally refer to a class of lipids that have a hydrophilic head of a phosphate moiety linked by an alcohol or glycerol backbone and two hydrophobic tails derived from fatty acids.

[0116] In some embodiments, the phospholipid is a sphingolipid. Sphingolipids generally refer to a class of lipids containing a backbone of sphingosine-based bases, which are a group of aliphatic amino alcohols including sphingosine. In some embodiments, the sphingolipid is sphingosine-1-phosphate.

[0117] In some embodiments, the phospholipid comprises one or more acyl chains. In some embodiments, each acyl chain is independently about 5 to about 30 carbon units in length, such as about 10 to about 22 carbon units or about 10 to about 16 carbon units. In some embodiments, the phospholipid has a glycerol backbone. In some embodiments, the phospholipid is phosphatidylcholine (PC). PC generally refers to a class of phospholipids that contain choline as a head group, a glycerophosphate unit, and various fatty acids. In some embodiments, the phospholipid comprises a PC selected from the group consisting of: 1-oleoyl-2-palmitoyl-sn-glycerol-3-phosphocholine (OPPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 2-disorcinoyl-sn-glycerol-3-phosphocholine (DEPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-dilauroyl-sn-glycerol-3-phosphocholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1,2-distearatel-sn-glycerol-3-phosphocholine (DSPC), and any combination thereof. In some embodiments, the phospholipid is selected from the group consisting of: DMPC, DMPS, POPC, DPPC, DSPC, POPS, PiP2, and any combination thereof. In some embodiments, the phospholipid is DMPC, DPPC, or POPC. In some embodiments, the phospholipid is a single type of PC. In some embodiments, the phospholipid comprises a mixture of at least two types of PC.

[0118] In some embodiments, one or more acyl chains of the phospholipid are fully saturated. In some embodiments, the phospholipid is selected from the group consisting of DMPC, DSPC, DPPC, and DMPS. In some embodiments, the phospholipid is DMPC. DMPC is a synthetic phospholipid comprising two myristoyl fatty acids attached to the first and second carbons of glycerol via ester bonds; and choline attached to the third carbon of glycerol via a phosphodiester bond. In some embodiments, the phospholipid is DPPC. DPPC is a synthetic phospholipid comprising two palmitoyl fatty acids attached to the first and second carbons of glycerol via ester bonds; and choline attached to the third carbon of glycerol via a phosphodiester bond. In some embodiments, the phospholipid is 1,2-dimyristoyl-sn-glycerol-3-phosphate-L-serine (DMPS), such as 14:0 DMPS. In some embodiments, the phospholipid is DSPC. DSPC is a synthetic phospholipid containing two stearyl fatty acids attached to the first and second carbons of glycerol via ester bonds, and choline attached to the third carbon of glycerol via phosphodiester bonds.

[0119] In some embodiments, one or more acyl chains of the phospholipid are unsaturated. In some embodiments, the phospholipid is POPC, phosphatidylinositol 4,5-bisphosphate (PiP2), or palmitoyl-oleoylphosphatidylserine (POPS). In some embodiments, the phospholipid is POPC. POPC is a synthetic phospholipid comprising two fatty acids, palmitic acid and oleic acid, which are respectively attached to the first and second carbons of glycerol via ester bonds; and choline attached to the third carbon of glycerol via a phosphodiester bond. In some embodiments, the phospholipid is PiP2, which may also be referred to as PI(4,5)P2. In some embodiments, PiP2 is 18:1 PI(4,5)P2 or 18:0-20:4 PI(4,5)P2. In some embodiments, PiP2 is 18:1 PI(4,5)P2. In some embodiments, PiP2 is 18:0-20:4 PI(4,5)P2. In some embodiments, the phospholipid is DOPC. DOPC is a synthetic phospholipid comprising two oleoyl fatty acids attached to the first and second carbons of glycerol via ester bonds, and choline attached to the third carbon of glycerol via a phosphodiester bond. In some embodiments, the phospholipid is DEPC. DEPC is a synthetic phospholipid comprising two erucic acid fatty acids attached to the first and second carbons of glycerol via ester bonds, and choline attached to the third carbon of glycerol via a phosphodiester bond.

[0120] In some implementations, the phospholipid is cationic, anionic, or zwitterionic, or any combination thereof.

[0121] c. The molar ratio between phospholipids and amphiphilic polymers in the particulate composition

[0122] In some embodiments, without being bound by any scientific theory, the amount of phospholipid required to saturate the particles can depend on the properties of the polymer and the phospholipid. In some embodiments, the phospholipid may contain a phosphatidylcholine (PC) head group and more than 12 hydrocarbon tails. In some embodiments, without being bound by any scientific theory, the phospholipid may have a tendency to condense into a bilayer structure. In some embodiments, without being bound by any scientific theory, the amphiphilic polymer may be in contact with a bilayer formed by the phospholipid, wherein the hydrophobic portion of the polymer remains close to the hydrophobic tail of the phospholipid, and the hydrophilic portion of the polymer remains close to the solvent in the composition. In some embodiments, without being bound by any scientific theory, the amphiphilic polymer is a variant of ApoA-I, and the amino acids forming the globular domain and the first 11 amino acids of helix 1 (H1, table) are not in contact with the lipid (Denisov et al., J Am Chem Soc. 24 March 2004. 126(11):3477-87).

[0123] Variants of MSPs that do not have a globular domain and / or do not have an H1 domain or a portion thereof and / or do not have any amino or carboxyl-terminal tags and protease cleavage sites, such as MSP1D1(-), MSPE1D1(-), MSPE2D1(-), MSPE3D1(-) and MSP2N2(-) provided herein, may have the same molar ratio of phospholipids to amphiphilic polymers in the composition required to saturate the particles, compared to their parent proteins (e.g., MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1 and MSP2N2, respectively).

[0124] In some embodiments, the compositions provided herein comprise a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-esterified. In some embodiments, the molar ratio of phospholipids to the amphiphilic polymer in the composition is less than the polymer-phospholipid saturation ratio N. S In some implementations, unbound by any scientific theory, the polymer-phospholipid saturation ratio N... SThe results can be calculated based on the work of Denisov et al. (J Am Chem Soc. 24 March 2004, 126(11):3477-87) and Denisov and Sligar (Chem Rev. 22 March 2017, 117(6): 4669-4713). The full text of these documents is incorporated herein by reference. Denisov et al. disclosed the number of lipids (polymer-phospholipid saturation ratio N) required to saturate particles in helical amino acid polymers containing amphiphilic helical polypeptides of length M forming a lipid-binding scaffold. S ) is a function of length M and the average area occupied by a single phospholipid type. In some implementations, N S It can be calculated using Equation 1.1:

[0125] (Equation 1.1)

[0126] Where M is the number of amino acid residues in the amphiphilic polymer helical band surrounding the phospholipid bilayer, L is the helical pitch of each amphiphilic polymer residue (e.g., about 0.15 nm), and r is the average radius of the amphiphilic polymer α-helix (e.g., about 0.55 nm). In some embodiments, A of Equation 1.1 L It depends on the constant of the phospholipid, for example, it could be the area occupied by a single phospholipid in the bilayer (in nm). 2 (Calculation). In some implementations, unbound by any scientific theory, area A L It can depend on the characteristics of the lipids used and the assembly temperature relative to the bulk bilayer phase transition temperature.

[0127] In some implementations, M can be calculated according to Equation 1.2:

[0128] (Equation 1.2)

[0129] Where r is the average radius of the amphiphilic polymer α-helix (e.g., about 0.55 nm or about 5.5 Å), L is the helical pitch of each amphiphilic polymer residue (e.g., about 0.15 nm or about 1.5 Å), and A L M is the average surface area of ​​each phospholipid, and N is the number of phospholipids in each phospholipid bilayer. In some embodiments, M can be referenced from Table 2.

[0130] In some implementations, the polymer-phospholipid saturation ratio N S It can be calculated using Equation 1.1, where A of DPPC and DMPC L It is approximately 0.52, and the A of POPC L It is approximately 0.69.

[0131] In some embodiments, the length (M) of the helical band of the contact lipid is the same for MSP variants with the H(0.5) helical sequence as for the parental MSP protein containing H1 (see Table 1). In some embodiments, this helical band length (M) may also be the same, with or without added histidine or other tags. For example, used to calculate N S The helical strip lengths M of MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1, and MSP2N2 are respectively related to the lengths used to calculate N. S The helical band length M of MSP1D1(-), MSPE1D1(-), MSPE2D1(-), MSPE3D1(-), and MSP2N2(-) is the same because the tag and protease cleavage site do not participate in stabilizing the resulting particles. In some embodiments, the amount of lipids (DMPC, DPPC, and POPC) required to saturate the MSP1D1, MSPE1D1, MSPE2D1, and MSPE3D1 particles can be calculated using Equation 1.1, and the calculation results are summarized in Table 2.

[0132] Table 2. N of exemplary particulate compositions S value

[0133]

[0134] In some embodiments, without being bound by any scientific theory, the stoichiometry of the amphiphilic polymer and phospholipids is crucial to the functionality, shape uniformity, and size distribution of the particulate composition. In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the composition is less than the phospholipid-polymer saturation ratio N. S Such as less than the phospholipid-polymer saturation ratio N S Approximately 95% (e.g., less than any one of approximately 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the exemplary combination of lipids and amphiphilic polymers in N S As shown in Table 2.

[0135] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1D1 or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 81:1. S Such as a phospholipid-polymer saturation ratio of less than about 81:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DMPC in the composition to the amphiphilic polymer MSP1D1 or a His-label-removed variant thereof (e.g., SEQ ID NO: 11) is less than or equal to about 78:1, such as less than or equal to any one of about 77:1, 76:1, 75:1, 74:1, 72:1, 70:1, 65:1, 60:1, 55:1, 51:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of DMPC to MSP1D1 or its His-label-removed variant (e.g., SEQ ID NO: 11) in the composition is from about 10:1 to about 80:1, such as from about 10:1 to about 78:1, from about 20:1 to about 78:1, from about 20:1 to about 78:1, from about 30:1 to about 78:1, from about 70:1 to about 80:1, from about 74:1 to about 77:1, from about 40:1 to about 60:1, from about 65:1 to about 75:1, from about 70:1 to about 75:1, from about 72:1 to about 78:1, from about 46:1 to about 55:1, from about 75:1, from about 54:1 to about 56:1, from about 65:1, from about 60:1, from about 55:1, from about 51:1, from about 50:1, from about 45:1, from about 40:1, from about 35:1, or from about 30:1.

[0136] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is from about 72:1 to about 78:1, such as about 75:1.

[0137] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or a His-tag-removed variant thereof (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is from about 46:1 to about 55:1, such as about 51:1. In some embodiments, the amphiphilic polymer is MSP1D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1D1 containing the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1D1 as defined by SEQ ID NO: 11.

[0138] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1D1 or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is DPPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 81:1. S Such as a phospholipid-polymer saturation ratio of less than about 81:1 N SThe content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC in the composition to the amphiphilic polymer MSP1D1 or a variant thereof with its His tag removed (e.g., SEQ ID NO: 11) is less than or equal to about 75:1, such as less than or equal to any one of about 70:1, 65:1, 60:1, 58:1, 55:1, 51:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of DPPC in the composition to MSP1D1 or its His-label-removed variant (e.g., SEQ ID NO:11) is from about 10:1 to about 80:1, such as from about 10:1 to about 78:1, from about 20:1 to about 75:1, from about 20:1 to about 60:1, from about 30:1 to about 60:1, from about 40:1 to about 60:1, from about 65:1 to about 75:1, from about 70:1 to about 75:1, from about 72:1 to about 78:1, from about 46:1 to about 55:1, from about 75:1, from about 54:1 to about 56:1, from about 65:1, from about 60:1, from about 55:1, from about 51:1, from about 50:1, from about 45:1, from about 40:1, from about 35:1, or from about 30:1. In some embodiments, the molar ratio of DPPC in the composition to the amphiphilic polymer MSP1D1 or a His-tag-removed variant thereof (e.g., SEQ ID NO: 11) is about 58:1, 50:1, about 54:1 to about 56:1, or about 55:1. In some embodiments, the amphiphilic polymer is MSP1D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1D1 as defined by SEQ ID NO: 11.

[0139] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1D1 or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 62:1. S Such as a phospholipid-polymer saturation ratio of less than about 62:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of POPC in the composition to the amphiphilic polymer MSP1D1 or a His-labeled variant thereof (e.g., SEQ ID NO: 11) is less than or equal to about 60:1, such as less than or equal to any one of about 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, or less. In some embodiments, the molar ratio of the phospholipid POPC in the composition to the amphiphilic polymer MSP1D1 or a His-tag-removed variant thereof (e.g., SEQ ID NO: 11) is from about 10:1 to about 60:1, such as from about 20:1 to about 60:1, from about 30:1 to about 55:1, from about 40:1 to about 55:1, from about 55:1, from about 50:1, from about 45:1, from about 40:1, from about 35:1, or from about 30:1. In some embodiments, the amphiphilic polymer is MSP1D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1D1 as defined by SEQ ID NO: 11.

[0140] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E1D1 (e.g., SEQ ID NO: 3) or a variant thereof with its His tag removed (MSP1E1D1(-); e.g., SEQ ID NO: 17), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E1D1 or a variant thereof with its His tag removed (MSP1E1D1(-); e.g., SEQ ID NO: 17), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 107:1. S Such as a phospholipid-polymer saturation ratio of less than about 107:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DMPC in the composition to the amphiphilic polymer MSP1E1D1 or its His-labeled variant is less than or equal to about 105:1, such as less than or equal to any one of about 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of the phospholipid DMPC in the composition to the amphiphilic polymer MSP1E1D1 or a His-tag-free variant thereof is from about 30:1 to about 100:1, such as from about 40:1 to about 90:1, from about 50:1 to about 80:1, from about 50:1 to about 70:1, from about 60:1 to about 70:1, from about 60:1 to about 65:1, from about 70:1, from about 65:1, from about 60:1, or from about 55:1. In some embodiments, the molar ratio of the DMPC in the composition to the amphiphilic polymer MSP1E1D1 or a His-tag-free variant thereof is from about 60:1, 62:1, from about 64:1 to about 66:1, or about 68:1. In some embodiments, the amphiphilic polymer is MSP1E1D1. In some embodiments, the amphiphilic polymer is a His-tag-free variant of MSP1E1D1. In some embodiments, MSP1E1D1(-) contains the amino acid sequence of SEQ ID NO: 17 (or is substantially composed of or consists of the amino acid sequence of that sequence).

[0141] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E1D1 (e.g., SEQ ID NO: 3) or a variant thereof with its His tag removed (MSP1E1D1(-); e.g., SEQ ID NO: 17), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E1D1 or a variant thereof with its His tag removed (MSP1E1D1(-); e.g., SEQ ID NO: 17), the phospholipid is DPPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 107:1. S Such as a phospholipid-polymer saturation ratio of less than about 107:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC in the composition to the amphiphilic polymer MSP1D1E1 (or MSP1E1D1(-)) is less than or equal to about 105:1, such as less than or equal to any one of about 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of the phospholipid DPPC in the composition to the amphiphilic polymer MSP1E1D1 or a His-label-removed variant thereof is from about 30:1 to about 100:1, such as from about 40:1 to about 90:1, from about 50:1 to about 80:1, from about 50:1 to about 70:1, from about 60:1 to about 70:1, from about 60:1 to about 65:1, from about 70:1, from about 65:1, from about 60:1, or from about 55:1. In some embodiments, the molar ratio of the DPPC in the composition to the amphiphilic polymer MSP1E1D1 or a His-label-removed variant thereof is from about 60:1, 62:1, from about 64:1 to about 66:1, or about 68:1. In some embodiments, the amphiphilic polymer is MSP1E1D1. In some embodiments, the amphiphilic polymer is a His-label-removed variant of MSP1E1D1. In some embodiments, MSP1E1D1(-) contains the amino acid sequence of SEQ ID NO: 17 (or is substantially composed of or consists of the amino acid sequence of that sequence).

[0142] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E1D1 (e.g., SEQ ID NO: 3) or a variant thereof with its His tag removed (MSP1E1D1(-); e.g., SEQ ID NO: 17), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E1D1 or a variant thereof with its His tag removed (MSP1E1D1(-); e.g., SEQ ID NO: 17), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 80:1. S Such as a phospholipid-polymer saturation ratio of less than about 80:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of POPC in the composition to the amphiphilic polymer MSP1E1D1 or a His-labeled variant thereof is less than or equal to about 76:1, such as less than or equal to any one of about 76:1, 70:1, 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, or less. In some embodiments, the molar ratio of the phospholipid POPC in the composition to the amphiphilic polymer MSP1E1E1 (or MSP1E1D1(-)) is from about 10:1 to about 75:1, such as from about 20:1 to about 75:1, from about 20:1 to about 60:1, from about 30:1 to about 60:1, from about 40:1 to about 60:1, from about 65:1, from about 60:1, from about 55:1, from about 50:1, from about 45:1, from about 40:1, from about 35:1, or from about 30:1. In some embodiments, the molar ratio of the POPC in the composition to the amphiphilic polymer MSP1E1D1 or a His-label-removed variant thereof is from about 50:1, from about 54:1 to about 56:1, or from about 55:1. In some embodiments, the amphiphilic polymer is MSP1E1D1. In some embodiments, the amphiphilic polymer is a His-label-removed variant of MSP1E1D1. In some embodiments, MSP1E1D1(-) contains the amino acid sequence of SEQ ID NO: 17 (or is substantially composed of or consists of the amino acid sequence of that sequence).

[0143] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E2D1 (e.g., SEQ ID NO: 4) or a variant thereof with its His tag removed (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E2D1 or a variant thereof with its His tag removed (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 135:1. S Such as a phospholipid-polymer saturation ratio of less than about 135:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DMPC in the composition to the amphiphilic polymer MSP1E2D1 or a His-labeled variant thereof is less than or equal to about 128:1, such as less than or equal to any one of about 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, or less. In some embodiments, the molar ratio of the phospholipid DMPC in the composition to the amphiphilic polymer MSP1E2D1 or a His-tag-removed variant thereof is from about 50:1 to about 130:1, such as from about 50:1 to about 120:1, from about 60:1 to about 120:1, from about 70:1 to about 110:1, from about 70:1 to about 100:1, from about 70:1, from about 75:1, from about 80:1, or from about 85:1. In some embodiments, the amphiphilic polymer is MSP1E2D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E2D1. In some embodiments, MSP1E2D1(-) comprises (or is substantially composed of or consists of) the amino acid sequence of SEQ ID NO: 18.

[0144] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E2D1 (e.g., SEQ ID NO: 4) or a variant thereof with its His tag removed (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E2D1 or a variant thereof with its His tag removed (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is DPPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 135:1. S Such as a phospholipid-polymer saturation ratio of less than about 135:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC in the composition to the amphiphilic polymer MSP1E2D1 or a His-labeled variant thereof is less than or equal to about 128:1, such as less than or equal to any one of about 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, or less. In some embodiments, the molar ratio of the phospholipid DPPC in the composition to the amphiphilic polymer MSP1E2D1 or a His-tag-removed variant thereof is from about 50:1 to about 130:1, such as from about 50:1 to about 120:1, from about 60:1 to about 120:1, from about 70:1 to about 110:1, from about 70:1 to about 100:1, from about 70:1, from about 75:1, from about 80:1, or from about 85:1. In some embodiments, the amphiphilic polymer is MSP1E2D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E2D1. In some embodiments, MSP1E2D1(-) comprises (or is substantially composed of or consists of) the amino acid sequence of SEQ ID NO: 18.

[0145] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E2D1 (e.g., SEQ ID NO: 4) or a variant thereof with its His tag removed (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E2D1 or a variant thereof with its His tag removed (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 102:1. S Such as a phospholipid-polymer saturation ratio of less than about 102:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of POPC in the composition to the amphiphilic polymer MSP1E2D1 or a His-labeled variant thereof is less than about 96:1, such as less than any one of about 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, 50:1, or less. In some embodiments, the molar ratio of the phospholipid POPC in the composition to the amphiphilic polymer MSP1E2D1 or a His-tag-removed variant thereof is from about 40:1 to about 95:1, such as from about 50:1 to about 90:1, from about 60:1 to about 90:1, from about 70:1 to about 90:1, from about 70:1 to about 80:1, from about 70:1, from about 75:1, from about 80:1, or from about 85:1. In some embodiments, the amphiphilic polymer is MSP1E2D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E2D1. In some embodiments, MSP1E2D1(-) comprises (or is substantially composed of or consists of) the amino acid sequence of SEQ ID NO: 18.

[0146] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 167:1. S Such as a phospholipid-polymer saturation ratio of less than about 167:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DMPC in the composition to the amphiphilic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (e.g., SEQ ID NO: 12) is less than or equal to about 160:1, such as less than or equal to any one of about 158:1, 155:1, 150:1, 145:1, 140:1, 135:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 98:1, 95:1, 94:1, 90:1, 85:1, 80:1, 75:1, or less. In some embodiments, the phospholipid DMPC in the composition is combined with the amphiphilic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (e.g., SEQ ID NO: 5). The molar ratio of NO:12 is from about 50:1 to about 160:1, such as from about 50:1 to about 150:1, from about 60:1 to about 140:1, from about 60:1 to about 130:1, from about 60:1 to about 120:1, from about 60:1 to about 100:1, from about 60:1 to about 70:1, from about 90:1 to about 100:1, from about 87:1 to about 98:1, from about 87:1 to about 96:1, from about 58:1 to about 68:1, from about 60:1, from about 62:1, from about 63:1, from about 65:1, from about 70:1, from about 80:1, from about 90:1, from about 93:1, from about 94:1, from about 100:1, or from about 110:1.

[0147] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 87:1 to about 98:1, or about 87:1 to about 96:1 such as about 93:1 or about 94:1, or about 58:1 to about 68:1 such as about 63:1 or about 62:1.

[0148] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a His-tagged variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 87:1 to about 98:1 or about 87:1 to about 96:1. In some embodiments, the amphiphilic polymer is MSP1E3D1. In some embodiments, the amphiphilic polymer is a His-tagged variant of MSP1E3D1 containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphiphilic polymer is a His-tagged variant (or MSP1E3D1(-)) of MSP1E3D1 as defined by SEQ ID NO: 12.

[0149] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a His-tag-removed variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is from about 58:1 to about 68:1, such as about 63:1. In some embodiments, the amphiphilic polymer is MSP1E3D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 as defined by SEQ ID NO: 12.

[0150] In some embodiments, the amphiphilic polymer is MSP1E3D1, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphiphilic polymer in the composition is from about 58:1 to about 68:1, such as about 62:1. In some embodiments, the amphiphilic polymer is MSP1E3D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 12 (MSP1E3D1(-)). In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 as defined by SEQ ID NO: 12.

[0151] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DPPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 167:1. S Such as a phospholipid-polymer saturation ratio of less than about 167:1 N SThe content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC in the composition to the amphiphilic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (e.g., SEQ ID NO: 12) is less than or equal to about 160:1, such as less than or equal to any one of about 155:1, 150:1, 145:1, 140:1, 135:1, 130:1, 125:1, 120:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, or less. In some embodiments, the phospholipid DPPC in the composition is combined with the amphiphilic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (e.g., SEQ ID NO: 5). 12) The molar ratio is from about 70:1 to about 160:1, such as from about 80:1 to about 150:1, from about 70:1 to about 140:1, from about 70:1 to about 130:1, from about 80:1 to about 120:1, from about 80:1 to about 100:1, from about 80:1, from about 90:1, from about 100:1, from about 87:1 to about 98:1, from about 87:1 to about 96:1, from about 58:1 to about 68:1, from about 60:1, from about 62:1, from about 63:1, from about 65:1, from about 70:1, from about 80:1, from about 90:1, from about 93:1, from about 94:1, from about 100:1, or from about 110:1. In some embodiments, the amphiphilic polymer is MSP1E3D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 as defined by SEQ ID NO: 12.

[0152] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 126:1. S Such as a phospholipid-polymer saturation ratio of less than about 126:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of POPC in the composition to the amphiphilic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (e.g., SEQ ID NO: 12) is less than or equal to about 120:1, such as less than or equal to any one of about 118:1, 115:1, 110:1, 105:1, 100:1, 95:1, 90:1, 85:1, 80:1, 75:1, 70:1, 65:1, or less. In some embodiments, the molar ratio of the phospholipid POPC in the composition to the amphiphilic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or its His-tag-removed variant (e.g., SEQ ID NO: 12) is about 60:1 to about 120:1, such as about 70:1 to about 120:1, about 70:1 to about 110:1, about 70:1 to about 100:1, about 80:1 to about 110:1, about 80:1 to about 100:1, about 80:1, about 90:1, about 100:1, or about 110:1. In some embodiments, the amphiphilic polymer is MSP1E3D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 as defined by SEQ ID NO: 12.

[0153] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or a variant thereof with its His tag removed (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP2N2 or a variant thereof with its His tag removed (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 387:1. S Such as a phospholipid-polymer saturation ratio of less than approximately 387:1 N S Approximately 95% (e.g., less than any of approximately 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DMPC in the composition to the amphiphilic polymer MSP2N2 or a His-labeled variant thereof is less than or equal to about 370:1, such as less than or equal to about 367:1, 360:1, 350:1, 340:1, 330:1, 320:1, 310:1, 300:1, 290:1, 280:1, 270:1, 260:1, 250:1, 240:1, 230:1, 220:1, 210:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1 or less. In some embodiments, the molar ratio of the phospholipid DMPC in the composition to the amphiphilic polymer MSP2N2 or a His-tag-removed variant thereof is from about 150:1 to about 350:1, such as from about 200:1 to about 350:1, from about 250:1 to about 350:1, from about 250:1 to about 330:1, from about 280:1 to about 320:1, from about 280:1 to about 300:1, from about 280:1, from about 290:1, from about 300:1, or from about 310:1. In some embodiments, the amphiphilic polymer is MSP2N2. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP2N2. In some embodiments, MSP2N2(-) comprises (or is substantially composed of or consists of) the amino acid sequence of SEQ ID NO: 19.

[0154] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or a variant thereof with its His tag removed (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or a variant thereof with its His tag removed (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is DPPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 387:1. S Such as a phospholipid-polymer saturation ratio of less than approximately 387:1 N S Approximately 95% (e.g., less than any of approximately 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of DPPC in the composition to the amphiphilic polymer MSP2N2 or a His-labeled variant thereof is less than or equal to about 370:1, such as less than or equal to about 367:1, 360:1, 350:1, 340:1, 330:1, 320:1, 310:1, 300:1, 290:1, 280:1, 270:1, 260:1, 250:1, 240:1, 230:1, 220:1, 210:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1 or less. In some embodiments, the molar ratio of the phospholipid DPPC in the composition to the amphiphilic polymer MSP2N2 or a His-tag-removed variant thereof is from about 150:1 to about 350:1, such as from about 200:1 to about 350:1, from about 250:1 to about 350:1, from about 250:1 to about 330:1, from about 280:1 to about 320:1, from about 280:1 to about 300:1, from about 280:1, from about 290:1, from about 300:1, or from about 310:1. In some embodiments, the amphiphilic polymer is MSP2N2. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP2N2. In some embodiments, MSP2N2(-) comprises (or is substantially composed of or consists of) the amino acid sequence of SEQ ID NO: 19.

[0155] In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or a variant thereof with its His tag removed (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles is under-esterified. In some embodiments, a composition is provided comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein the amphiphilic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or a variant thereof with its His tag removed (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 291:1. S Such as a phospholipid-polymer saturation ratio of less than 291:1 N S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of POPC in the composition to the amphiphilic polymer MSP2N2 or a His-labeled variant thereof is less than or equal to about 280:1, such as less than or equal to any one of about 276:1, 270:1, 260:1, 250:1, 240:1, 230:1, 220:1, 210:1, 200:1, 190:1, 180:1, 170:1, 160:1, 150:1, 140:1, 130:1, 120:1, 110:1, 100:1, or less. In some embodiments, the molar ratio of the phospholipid POPC in the composition to the amphiphilic polymer MSP2N2 or a His-tag-removed variant thereof is from about 100:1 to about 280:1, such as from about 100:1 to about 260:1, from about 120:1 to about 260:1, from about 140:1 to about 260:1, from about 160:1 to about 240:1, from about 180:1 to about 220:1, from about 180:1, from about 190:1, from about 200:1, or from about 210:1. In some embodiments, the amphiphilic polymer is MSP2N2. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP2N2. In some embodiments, MSP2N2(-) comprises (or is substantially composed of or consists of) the amino acid sequence of SEQ ID NO: 19.

[0156] d. Characteristics

[0157] In some embodiments, the composition comprises particles purified by chromatography, such as size exclusion chromatography (SEC). In some embodiments, at least about 85% of the plurality of particles are under-esterified. In some embodiments, each of the plurality of particles contains an amphiphilic polymer and a phospholipid.

[0158] In some embodiments, the average particle size is any one of about 2 nm to about 100 nm, about 5 nm to about 75 nm, about 5 nm to about 50 nm, about 5 nm to about 40 nm, about 5 nm to about 30 nm, about 5 nm to about 20 nm, or about 10 nm to about 20 nm. In some embodiments, the average particle size is about 2 nm to about 20 nm, such as about 10 nm to about 20 nm. In some embodiments, the average particle size can be tuned by the length of the amphiphilic polymer.

[0159] In some embodiments, the average diameter of the particles is any one of about 2 nm to about 100 nm, about 5 nm to about 75 nm, about 5 nm to about 50 nm, about 5 nm to about 40 nm, about 5 nm to about 30 nm, about 5 nm to about 20 nm, or about 10 nm to about 20 nm. In some embodiments, the average diameter of the particles is about 2 nm to about 20 nm. In some embodiments, the average diameter of the particles can be tuned by the length of the amphiphilic polymer.

[0160] In some embodiments, the polydispersity index (a measure of heterogeneity) of the particle size in the composition is less than about 0.5, such as less than any one of about 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. In some embodiments, the particle size and size distribution in the composition can be determined by combining multi-angle light scattering with size exclusion chromatography (SEC-MALS). In some embodiments, at least about 60% of the particles in the composition, such as at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99%, have a size of about 2 nm to about 20 nm. In some embodiments, after one or more freezing and thawing cycles, such as freezing at -80°C and thawing with or without a cryoprotectant, at least about 60% of the particles, such as any one of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99%, retain a size of about 2 nm to about 20 nm. In some embodiments, after one or more freezing and thawing cycles, such as freezing at -80°C and thawing with or without a cryoprotectant, at least about 60% of the particles in the particulate composition, such as any one of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99%, have a substantially unchanged size (e.g., the size variation of the particles in the particulate composition is less than any one of about 20%, 15%, 10%, 5%, 2%, or 1%).

[0161] In some embodiments, the polydispersity index (a measure of heterogeneity) of the lipid content of the particles in the composition is less than about 0.5, such as less than any one of about 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. In some embodiments, the molar ratio between at least about 60%, such as at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, of the phospholipids and polymers is less than the phospholipid-polymer saturation ratio N. S Approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, after freezing and thawing once or more, such as freezing at -80°C and thawing with or without a cryoprotectant, the molar ratio between the phospholipid and polymer of at least about 60%, such as at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, is less than the phospholipid-polymer saturation ratio N. SAbout 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, after freezing and thawing once or more, such as freezing at -80°C and thawing with or without a cryoprotectant, the molar ratio between at least about 60%, such as at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, of the phospholipid to the polymer remains substantially constant (e.g., the particle size variation in the particulate composition is less than any one of about 20%, 15%, 10%, 5%, 2%, or 1%).

[0162] In some embodiments, after storage at room temperature for at least about 1 hour, such as at least about 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, or 24 hours, or 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 15 days, 30 days, 40 days, 50 days, or 100 days, the molar ratio between phospholipids and polymers in the composition remains substantially constant (e.g., the particle size variation in the particulate composition is less than any of about 20%, 15%, 10%, 5%, 2%, or 1%). In some embodiments, after storage at -80°C for at least about 1 month, such as at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 15 months, 30 months, 40 months, 50 months, or 100 months, the molar ratio between phospholipids and MSP remains substantially constant (e.g., the particle size variation in the particulate composition is less than any of about 20%, 15%, 10%, 5%, 2%, or 1%).

[0163] In some embodiments, the molecular weight of the particles in the composition is any one of about 10,000 g / mol to about 400,000 g / mol, about 80,000 g / mol to about 350,000 g / mol, about 100,000 g / mol to about 350,000 g / mol, about 150,000 g / mol to about 300,000 g / mol, about 150,000 g / mol to about 250,000 g / mol, about 150,000 g / mol, about 200,000 g / mol, about 250,000 g / mol, about 300,000 g / mol, or about 320,000 g / mol.

[0164] In some embodiments, the particulate composition described herein increases the cholesterol efflux rate by at least about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 15 times or more) compared to the cholesterol efflux rate achieved in an untreated state (e.g., before application of the particles) or by ApoA-I or high-density lipoprotein (HDL) alone. In some embodiments, compared to a fully esterified particulate composition containing the same amphiphilic polymer and phospholipids (the molar ratio of phospholipids to amphiphilic polymers in the composition is approximately the phospholipid-polymer saturation ratio N), the particulate composition increases the cholesterol efflux rate by at least about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 15 times or more). S In contrast, the hypolipidemic particulate composition described herein increases the cholesterol efflux rate by at least about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 15 times or more).

[0165] In some embodiments, the particulate compositions described herein are compared with i) ApoA-I alone, ii) HDL alone, iii) MSP alone (e.g., MSP1D1(-)) and / or iv) fully esterified particulate compositions containing the same amphiphilic polymer and phospholipids (the molar ratio of phospholipids to amphiphilic polymers in the composition is approximately the phospholipid-polymer saturation ratio N). S The particulate composition described herein may have a high equilibrium lipid (e.g., cholesterol) binding capacity (or lipid (e.g., cholesterol) partition coefficient) of at least about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 15 times or more). In some embodiments, the particulate composition described herein may bind at least about 2 mol of lipid (e.g., cholesterol) per mol of particulate composition at equilibrium, such as binding at least about 3 mol, 4 mol, 5 mol, 6 mol, 7 mol, 8 mol, 9 mol, 10 mol, 15 mol, 20 mol or more of lipid (e.g., cholesterol) per mol of particulate composition at equilibrium. In some embodiments, the equilibrium lipid (e.g., cholesterol) binding capacity (or lipid (e.g., cholesterol) partition coefficient) is measured using a LUV assay containing lipids (e.g., cholesterol) (such as the assay described in Example 8).

[0166] In some embodiments, this document provides compositions comprising particles prepared according to any of the preparation methods detailed herein.

[0167] In some embodiments, the compositions provided herein are pharmaceutical compositions. In some embodiments, the compositions further comprise a preventative or therapeutic agent. In some embodiments, the compositions do not comprise a preventative or therapeutic agent.

[0168] III. Preparation Method

[0169] In one aspect, a method is provided for preparing any of the under-esterified particulate compositions described herein. In another aspect, a method is provided for preparing a composition comprising a plurality of particles containing a polymer (e.g., an amphiphilic polymer) and phospholipids, wherein at least about 85% (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-esterified. In some embodiments, the method comprises: 1) incubating the amphiphilic polymer and phospholipids to obtain a preparation mixture, wherein the phospholipids are dissolved with a detergent or organic compound prior to incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain particles. In some embodiments, the molar ratio of phospholipids to the amphiphilic polymer in the composition is less than the phospholipid-polymer saturation ratio N. S (For example, less than any of about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower). In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture is less than or equal to the molar ratio of phospholipids to amphiphilic polymers in the composition. In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture is less than about 95% of the molar ratio of phospholipids to amphiphilic polymers in the composition (e.g., less than any of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower). In some embodiments, the method further includes, for example, purifying the obtained particles by SEC (once or multiple times). In some embodiments, the method further includes validating the obtained particulate composition, for example, after a final purification step. Also see the exemplary methods for preparing, purifying, and validating the under-esterified particulate compositions described herein in Examples 1-3. In some embodiments, the method further includes testing one or more properties of the obtained particulate composition: lipid (e.g., cholesterol) binding capacity at equilibrium, cholesterol efflux capacity, and / or stability (e.g., after freeze-thaw cycles), such as using any of the test methods described herein (e.g., see Examples 4, 5, 8, 10, 11, and 13).

[0170] In some embodiments, the particles can be synthesized by incubating dissolved phospholipids with an amphiphilic polymer (e.g., an amphiphilic polypeptide). In some embodiments, the phospholipids are dissolved using a detergent or organic compound. In some embodiments, the dissolved phospholipids are prepared by adding a solution of a detergent or organic compound to the phospholipid film. Exemplary organic compounds include, but are not limited to, alkyl glucosides such as dodecyl-β-D-maltodextrin (DDM), octyl-β-glucoside (OG), Triton X-100, 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS), 2,2-decylpropane-1,3-bis-β-D-malopyranoside (LMNG), cholates, and any combination thereof. In some embodiments, the detergent or organic compound comprises a cholate, such as sodium cholate. In some embodiments, the amphiphilic polymer is provided in a solution containing a buffer. In some embodiments, the pH of the buffer is from about 5 to about 9, such as from about 6 to about 8, about 7, or about 7.5. In some embodiments, the amphiphilic polymer in the solution is at least about 0.1 mg / mL, such as any one of at least about 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or 2.5 mg / mL. In some embodiments, the amphiphilic polymer in the solution is from about 0.5 mg / mL to about 10 mg / mL, such as any one of about 1 mg / mL to about 8 mg / mL, about 1 mg / mL to about 5 mg / mL, about 1.5 mg / mL to about 6 mg / mL, about 2 mg / mL to about 5 mg / mL, or about 2.5 mg / mL to about 4 mg / mL.

[0171] In some embodiments, the preparation method includes removing detergents or organic compounds from the preparation mixture to obtain granules. In some embodiments, organic compounds (e.g., bile salts) are removed, such as by incubation with absorbent polymer beads. In some embodiments, the absorbent polymer beads comprise styrene-divinylbenzene (macromesh) beads (e.g., Amberlite). ® XAD-2 ® (Beads). In some embodiments, the formation and size of the particles can be determined by methods known in the art, such as SEC and electron microscopy.

[0172] In some embodiments, the preparation method further includes separating the particles. In some embodiments, the particles are separated by chromatography. In some embodiments, the preparation method further includes subjecting the particles to one or more freeze-thaw cycles with or without a cryoprotectant.

[0173] In some embodiments, the preparation method further includes purifying the prepared particles, such as separating particles with different phospholipid to MSP ratios. In some embodiments, purification includes using SEC. In some embodiments, the preparation method includes more than one purification cycle until the desired distribution of the phospholipid to MSP ratio is achieved (e.g., less than the phospholipid-polymer saturation ratio N). S ).

[0174] In some embodiments, the preparation method further includes measuring the actual phospholipid to MSP ratio in the particulate composition. In some embodiments, the actual phospholipid to MSP ratio in the particulate composition can be determined by measuring the fatty acid content using gas chromatography (GC).

[0175] In some embodiments, the polymer is any polymer described herein (e.g., an amphiphilic polymer). In some embodiments, the amphiphilic polymer is an amphiphilic polypeptide. In some embodiments, the amphiphilic polypeptide comprises one or more amphiphilic helical sequences of about 9 to about 30 amino acids. In some embodiments, the amphiphilic polypeptide is selected from the group consisting of apolipoprotein AI (ApoA-I), ApoA-II, ApoC, ApoE, and variants thereof. In some embodiments, the amphiphilic polypeptide is a variant of ApoA-I. In some embodiments, the amphiphilic polypeptide comprises one or more amphiphilic helical sequences derived from ApoA-I. In some embodiments, the amphiphilic polypeptide comprises an N-terminus truncated relative to ApoA-I. In some embodiments, the amphiphilic polypeptide is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, and variants thereof. In some embodiments, the amphiphilic polypeptide is selected from the group consisting of MSP1D1, MSPE1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and variants thereof. In some embodiments, the amphiphilic polypeptide is MSP1D1. In some embodiments, the amphiphilic polypeptide is MSP1E3D1. In some embodiments, the amphiphilic polypeptide is a variant of any of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP2N2, wherein the variant does not contain an N-terminal His tag portion (a variant with the His tag removed). In some embodiments, the amphiphilic polypeptide is (i) a His-tagged variant of MSP1D1 containing the amino acid sequence of SEQ ID NO: 11 (MSP1D1(-)); (ii) a His-tagged variant of MSP1E3D1 containing the amino acid sequence of SEQ ID NO: 12 (MSP1E3D1(-)); (iii) a His-tagged variant of MSP1E1D1 containing the amino acid sequence of SEQ ID NO: 17 (i.e., MSP1E1D1(-)); (iv) a His-tagged variant of MSP1E2D1 containing the amino acid sequence of SEQ ID NO: 18 (i.e., MSP1E2D1(-)); or (v) a His-tagged variant of MSP2N2 containing the amino acid sequence of SEQ ID NO: 19 (i.e., MSP2N2(-)).

[0176] In some embodiments, the phospholipid is any of the phospholipids described herein. In some embodiments, the phospholipid comprises a sphingolipid. In some embodiments, the sphingolipid is sphingosine-1-phosphate. In some embodiments, the phospholipid comprises one or more acyl chains, optionally having a length of about 10 to about 22 carbon units. In some embodiments, the phospholipid has a glycerol backbone. In some embodiments, the acyl chains on the phospholipid are fully saturated. In some embodiments, the phospholipid is selected from the group consisting of DMPC, DPPC, or DMPS. In some embodiments, the phospholipid is DMPC. In some embodiments, one or more acyl chains of the phospholipid are unsaturated. In some embodiments, the phospholipid is POPC, POPS, or PiP2. In some embodiments, the phospholipid is cationic, anionic, zwitterionic, or any combination thereof.

[0177] In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture (e.g., the mixture from step (1) of the preparation method described above) is the same as the molar ratio of phospholipids to amphiphilic polymers in the obtained particulate composition. In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture (e.g., the mixture from step (1) of the preparation method described above) is less than the molar ratio of phospholipids to amphiphilic polymers in the obtained particulate composition (e.g., less than any one of about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture is less than about 80% of the molar ratio of phospholipids to amphiphilic polymers in the particulate composition. In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture is determined such that at least about 60%, such as at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, of the prepared particles are under-esterified as described herein. In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the preparation mixture is determined such that at least about 60%, such as at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, of the prepared particles have a molar ratio of phospholipids to amphiphilic polymers less than the phospholipid-polymer saturation ratio N. S Approximately 95% (e.g., less than any of approximately 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%).

[0178] In some embodiments, the amphiphilic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC or DPPC, and the molar ratio of phospholipid to amphiphilic polymer in the preparation mixture (e.g., the mixture in step (1) of the preparation method described above) is less than a phospholipid-polymer saturation ratio N of about 81:1. S The content is approximately 95% (e.g., less than any one of about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%). In some embodiments, the molar ratio of phospholipid to amphiphilic polymer in the mixture is at least about 10:1, such as any one of about 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, or 45:1. In some embodiments, the molar ratio of phospholipid to amphiphilic polymer in the mixture is from about 10:1 to about 85:1, such as from about 10:1 to about 70:1, from about 20:1 to about 60:1, from about 30:1 to about 70:1, from about 40:1 to about 70:1, from about 65:1, from about 60:1, from about 50:1, or about 40:1.

[0179] In some embodiments, a method for preparing a composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% of the plurality of particles are under-esterified, wherein the amphiphilic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein the method comprises: (1) incubating the amphiphilic polymer and phospholipids to obtain a preparation mixture, wherein the phospholipids are dissolved with a detergent or organic compound prior to incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain particles. In some embodiments, the molar ratio of phospholipids to the amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 81:1. S In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the mixture is less than (e.g., less than about 95%) or equal to the molar ratio of phospholipids to amphiphilic polymers in the composition.

[0180] In some embodiments, the amphiphilic polymer is MSP1D1 or a His-tagged variant thereof (MSP1D1(-)), the phospholipid is DMPC, and the ratio of phospholipid to amphiphilic polymer in the composition is from about 72:1 to about 78:1. In some embodiments of the foregoing, the molar ratio of DMPC to MSP1D1 or a His-tagged variant thereof in the mixture is less than or equal to about 70:1, such as less than or equal to any one of about 65:1, 60:1, 55:1, 50:1, 45:1, or 40:1. In some embodiments, the molar ratio of DMPC to MSP1D1 or a His-tagged variant thereof in the composition is about 75:1, and the molar ratio of DMPC to MSP1D1 or a His-tagged variant thereof in the mixture is about 55:1. In some embodiments, the amphiphilic polymer is MSP1D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1D1 defined by the amino acid sequence of SEQ ID NO: 11.

[0181] Therefore, in some embodiments, a method for preparing a composition is provided, the composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% of the plurality of particles are under-esterified, wherein the amphiphilic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), and the phospholipid is DMPC, wherein the method comprises: (1) incubating the amphiphilic polymer and phospholipids to obtain a preparation mixture, wherein the phospholipids are dissolved with a detergent or organic compound prior to incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain particles; wherein the molar ratio of phospholipids to the amphiphilic polymer in the composition is about 72:1 to about 78:1 (e.g., about 75:1), and wherein the molar ratio of phospholipids to the amphiphilic polymer in the preparation mixture is about 45:1 to about 65:1 (e.g., about 55:1). In some embodiments, the molar ratio of DMPC to MSP1D1 or a His-tag-free variant thereof in the composition is about 75:1, and the molar ratio of DMPC to MSP1D1 or a His-tag-free variant thereof in the preparation mixture is about 55:1. In some embodiments, the amphiphilic polymer is a His-tag-free variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the method further includes purifying the obtained composition.

[0182] In some embodiments, the amphiphilic polymer is MSP1D1 or a His-tagged variant thereof (MSP1D1(-)), the phospholipid is DMPC, and the phospholipid to amphiphilic polymer ratio in the composition is from about 48:1 to about 55:1. In some embodiments of the foregoing, the molar ratio of phospholipid DMPC to amphiphilic polymer MSP1D1 or a His-tagged variant thereof in the preparation mixture is less than or equal to about 50:1, such as less than or equal to any one of about 45:1, 40:1, 35:1, 30:1, 25:1, or 20:1. In some embodiments, the molar ratio of phospholipid DMPC to amphiphilic polymer MSP1D1 or a His-tagged variant thereof in the composition is from about 46:1 to about 55:1, about 51:1, or about 55:1, and the molar ratio of phospholipid DMPC to amphiphilic polymer MSP1D1 or a His-tagged variant thereof in the preparation mixture is about 40:1 or about 30:1. In some embodiments, the amphiphilic polymer is MSP1D1. In some embodiments, the amphiphilic polymer is a variant of MSP1D1 containing the amino acid sequence of SEQ ID NO: 11 with the His tag removed.

[0183] Therefore, in some embodiments, a method for preparing a composition is provided, the composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% of the plurality of particles are under-esterified, wherein the amphiphilic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or a variant thereof with its His tag removed (MSP1D1(-); e.g., SEQ ID NO: 11), and the phospholipid is DMPC, wherein the method comprises: (1) incubating the amphiphilic polymer and phospholipids to obtain a preparation mixture, wherein the phospholipids are dissolved with a detergent or organic compound prior to incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain particles; wherein the molar ratio of phospholipids to the amphiphilic polymer in the composition is about 46:1 to about 55:1 (e.g., about 51:1), and wherein the molar ratio of phospholipids to the amphiphilic polymer in the preparation mixture is about 30:1 to about 45:1 (e.g., about 40:1 or about 30:1). In some embodiments, the molar ratio of DMPC to MSP1D1 or a His-tag-free variant thereof in the composition is about 51:1, and the molar ratio of DMPC to MSP1D1 or a His-tag-free variant thereof in the preparation mixture is about 40:1 or about 30:1. In some embodiments, the amphiphilic polymer is a His-tag-free variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the method further includes purifying the obtained composition.

[0184] In some embodiments, a method for preparing a composition comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-esterified, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, and wherein the method comprises: (1) incubating the amphiphilic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is dissolved with a detergent or organic compound prior to incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain particles. In some embodiments, the molar ratio of phospholipid to amphiphilic polymer in the composition is less than a phospholipid-polymer saturation ratio N of about 167:1. S In some embodiments, the molar ratio of phospholipids to amphiphilic polymers in the mixture is less than (e.g., less than about 95%) or equal to the molar ratio of phospholipids to amphiphilic polymers in the composition.

[0185] In some embodiments, the amphiphilic polymer is MSP1E3D1 or a variant thereof with its His tag removed (MSP1E3D1(-)), the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphiphilic polymer in the mixture is from about 30:1 to about 100:1, including, for example, any one of about 30:1 to 100:1, 30:1 to 90:1, 40:1 to 80:1, about 50:1, about 55:1, about 60:1, or about 65:1. In some embodiments, the molar ratio of the phospholipid to the amphiphilic polymer in the mixture is at least about 30:1, such as any one of about 30:1, 35:1, 45:1, 50:1, 55:1, 60:1, 65:1, or 70:1. In some embodiments, the molar ratio of phospholipid to amphiphilic polymer in the mixture is from about 40:1 to about 90:1, such as any one of about 45:1, 50:1, 55:1 or 60:1.

[0186] In some embodiments, the amphiphilic polymer is MSP1E3D1 or a His-tagged variant thereof (MSP1E3D1(-)), the phospholipid is DMPC, and the ratio of phospholipid to amphiphilic polymer in the composition is from about 90:1 to about 100:1. In some embodiments of the foregoing, the molar ratio of DMPC to MSP1E3D1 or a His-tagged variant thereof in the mixture is less than or equal to about 90:1, such as less than or equal to any one of about 85:1, 80:1, 75:1, 70:1, 65:1, 60:1, 55:1, or 50:1. In some embodiments, the molar ratio of phospholipid DMPC to a His-tagged variant of the amphiphilic polymer in the composition is from about 87:1 to about 98:1 or from about 87:1 to about 96:1, and the molar ratio of phospholipid DMPC to a His-tagged variant of the amphiphilic polymer in the mixture is about 90:1. In some embodiments, the amphiphilic polymer is MSP1E3D1 or a His-tagged variant thereof, the phospholipid is DMPC, and the ratio of phospholipid to amphiphilic polymer in the composition is from about 60:1 to about 80:1. In some embodiments of the foregoing, the molar ratio of DMPC to MSP1E3D1 or a His-tagged variant thereof in the mixture is less than or equal to about 60:1, such as less than or equal to any one of about 55:1, 50:1, 45:1, 40:1, 35:1, or 30:1. In some embodiments, the molar ratio of DMPC to MSP1E3D1 or a His-tagged variant thereof in the composition is from about 58:1 to about 68:1, about 63:1, or about 62:1, and the molar ratio of DMPC to MSP1E3D1 or a His-tagged variant thereof in the mixture is about 60:1. In some embodiments, the amphiphilic polymer is MSP1E3D1. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphiphilic polymer is a His-tag-removed variant of MSP1E3D1 as defined by SEQ ID NO: 12.

[0187] Therefore, in some embodiments, a method for preparing a composition is provided, the composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% of the plurality of particles are under-esterified, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), and the phospholipid is DMPC, wherein the method comprises: (1) incubating the amphiphilic polymer and phospholipids to obtain a preparation mixture, wherein the phospholipids are dissolved with a detergent or organic compound prior to incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain particles; wherein the molar ratio of phospholipids to the amphiphilic polymer in the composition is about 58:1 to about 68:1 (e.g., about 63:1), and wherein the molar ratio of phospholipids to the amphiphilic polymer in the preparation mixture is about 60:1. In some embodiments, the molar ratio of DMPC to MSP1E3D1 or a His-tag-free variant thereof in the composition is about 63:1, and the molar ratio of DMPC to MSP1D1 or a His-tag-free variant thereof in the preparation mixture is about 60:1. In some embodiments, the amphiphilic polymer is a His-tag-free variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the method further includes purifying the obtained composition.

[0188] In some embodiments, the molar ratio of DMPC to MSP1E3D1 or its His-tagged variant (MSP1E3D1(-)) in the composition is about 62:1, and the molar ratio of DMPC to MSP1E3D1 or its His-tagged variant in the mixture preparation is about 50:1. In some embodiments, the amphiphilic polymer is MSP1E3D1. In some embodiments, the amphiphilic polymer is a His-tagged variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphiphilic polymer is a His-tagged variant of MSP1E3D1 as defined by SEQ ID NO: 12.

[0189] Therefore, in some embodiments, a method for preparing a composition is provided, the composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% of the plurality of particles are under-esterified, wherein the amphiphilic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or a variant thereof with its His tag removed (MSP1E3D1(-); e.g., SEQ ID NO: 12), and the phospholipid is DMPC, wherein the method comprises: (1) incubating the amphiphilic polymer and phospholipids to obtain a preparation mixture, wherein the phospholipids are dissolved with a detergent or organic compound prior to incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain particles; wherein the molar ratio of phospholipids to the amphiphilic polymer in the composition is about 58:1 to about 68:1 (e.g., about 62:1), and wherein the molar ratio of phospholipids to the amphiphilic polymer in the preparation mixture is about 50:1. In some embodiments, the molar ratio of DMPC to MSP1E3D1 or a His-tag-free variant thereof in the composition is about 62:1, and the molar ratio of DMPC to MSP1D1 or a His-tag-free variant thereof in the preparation mixture is about 50:1. In some embodiments, the amphiphilic polymer is a His-tag-free variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the method further includes purifying the obtained composition.

[0190] IV. Instructions for Use

[0191] In one aspect, this document provides a method for removing lipids (e.g., cholesterol) from cells or preventing lipid accumulation in cells, the method comprising contacting cells with an effective amount of any composition described herein (e.g., a pharmaceutical composition), wherein the composition comprises a plurality of particles containing an amphiphilic polymer and phospholipids. In some embodiments, at least about 85% of the plurality of particles are under-lipinated. In some embodiments, a method for removing lipids (e.g., cholesterol) from cells or preventing lipid accumulation in cells is provided, the method comprising contacting cells with an effective amount of any under-lipinated particle composition described herein. In one aspect, this document provides a method for removing lipids (e.g., cholesterol) from an individual (e.g., a person) or preventing lipid accumulation in an individual, the method comprising administering to the individual an effective amount of any composition described herein (e.g., a pharmaceutical composition). In one aspect, this document provides a method for treating or preventing a disease in an individual associated with lipid (e.g., cholesterol) accumulation, the method comprising administering to the individual an effective amount of any under-lipinated particle composition described herein (e.g., a pharmaceutical composition). The composition (e.g., a pharmaceutical composition) may be any of the following: DMPC:MSP1D1(-)=72:1 granule composition, DMPC:MSP1D1(-)=51:1 granule composition, DMPC:MSP1D1(-)=75:1 granule composition, DMPC:MSP1E3D1(-)=62:1 granule composition, DMPC:MSP1E3D1(-)=63:1 granule composition or DMPC:MSP1E3D1(-)=94:1 granule composition.

[0192] In some embodiments, this document provides a method for removing lipids (e.g., cholesterol) or preventing lipid accumulation in the eyes of an individual (e.g., a person), the method comprising administering an effective amount of any of the compositions described herein (e.g., pharmaceutical compositions) to the individual's eye. The composition (e.g., pharmaceutical composition) may be any of the following: a DMPC:MSP1D1(-)=72:1 particle composition, a DMPC:MSP1D1(-)=51:1 particle composition, a DMPC:MSP1D1(-)=75:1 particle composition, a DMPC:MSP1E3D1(-)=62:1 particle composition, a DMPC:MSP1E3D1(-)=63:1 particle composition, or a DMPC:MSP1E3D1(-)=94:1 particle composition. In some embodiments, the lipid used to remove or prevent accumulation in the eye is cholesterol. In some embodiments, the composition is administered intravitreally. In another aspect, this document provides a method for preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual (e.g., a person), the method comprising administering an effective amount of any of the compositions described herein (e.g., pharmaceutical compositions) to the individual's eye. In some embodiments, the lipid used to remove or prevent accumulation in the eye is cholesterol. In some embodiments, the eye disease characterized by lipid accumulation is photoreceptor neurodegeneration, retinal neurovascular disease, age-related macular degeneration (AMD), or atrophy. In some embodiments, the eye disease characterized by lipid accumulation is AMD, such as dry AMD. In some embodiments, dry AMD is geographic atrophy (GA). In some embodiments, the eye disease characterized by lipid accumulation is wet AMD. In some embodiments, the eye disease is choroidal neovascularization (CNV). In some embodiments, any of the compositions described herein may be administered intravitreally.

[0193] In some embodiments, a method for preventing or treating AMD in the eye of an individual (e.g., a person) is provided, the method comprising administering an effective amount of any of the compositions described herein (e.g., pharmaceutical compositions) to the individual's eye. The composition (e.g., pharmaceutical composition) may be any of the following: a DMPC:MSP1D1(-)=72:1 particle composition, a DMPC:MSP1D1(-)=51:1 particle composition, a DMPC:MSP1D1(-)=75:1 particle composition, a DMPC:MSP1E3D1(-)=62:1 particle composition, a DMPC:MSP1E3D1(-)=63:1 particle composition, or a DMPC:MSP1E3D1(-)=94:1 particle composition. In some embodiments, the AMD is dry AMD. In some embodiments, the dry AMD is GA. In some embodiments, the AMD is wet AMD. In some embodiments, the wet AMD includes CNV.

[0194] In some embodiments, the lipids used to remove or prevent accumulation in the eye are selected from the group consisting of fats, sterols, phospholipids, and any combination thereof. In some embodiments, the lipids include cholesterol esters (ChE), wax esters (WE), diesters, triacylglycerols (TG), free cholesterol, free fatty acids (FA), (O-acyl)-ω-hydroxy fatty acids (OAHFA), or any combination thereof. In some embodiments, the lipids are fatty acids or derivatives thereof (including triglycerides, diglycerides, monoglycerides, and phospholipids). In some embodiments, the lipids used to remove or prevent accumulation in the eye comprise two or more lipids described herein.

[0195] In some implementations, eye diseases characterized by lipid accumulation are selected from the group consisting of: AMD, photoreceptor neurodegeneration, optic atrophy, vision loss, hemianopsia, visual agnosia, strabismus, retinal neurovascular disease, lipid corneal disease, corneal lipid deposition, and any combination thereof. In some implementations, exemplary eye diseases characterized by lipid accumulation include, but are not limited to, AMD (including dry AMD (atrophic AMD) and wet AMD (neovascular AMD)), choroidal neovascularization (CNV), retinal angiomyolipoma (RAP), retinal neovascularization (RNV), juvenile macular degeneration (e.g., Stargardt's disease), macular telangiectasia, macular diseases (e.g., age-related macular degeneration (ARM) and diabetic macular degeneration (DMP), including partially ischemic DMP), macular edema (e.g., diabetic macular edema (DME), including clinically significant DME, focal DME and diffuse DME, Irvine-Gass syndrome (postoperative macular edema), and post-RVO macular edema, including central RVO and branch RVO), and retinopathy (e.g., diabetes). Retinopathy (DR), including in patients with DME, proliferative vitreoretinopathy (PVR), Purtscher retinopathy and radiation retinopathy, retinal artery occlusion (RAO, such as central and branch RAO), retinal vein occlusion (RVO, such as central RVO, including central RVO with macular cystoid edema (CME), and branch RVO, including branch RVO with CME), retinitis (e.g., Kurtz's disease (exudative retinitis) and retinitis pigmentosa (RP)), choroidal retinitis, choroiditis (e.g., creeping choroiditis), uveitis (including anterior uveitis, intermediate uveitis, posterior uveitis with or without CME, panuveitis and non-infectious uveitis), retinal detachment (e.g. in von Hippel-Lindau disease), retinal pigment epithelium (RPE) detachment, rod and / or cone dystrophy, and any combination thereof.

[0196] In some implementations, eye diseases characterized by lipid accumulation are photoreceptor neurodegeneration or retinal neurovascular disorders.

[0197] In some implementations, the eye disease characterized by lipid accumulation is AMD. In some implementations, AMD is dry AMD. Dry AMD is typically characterized by the accumulation of pale yellow deposits called choroidal verrucous deposits under the retina and usually affects vision in both eyes, although vision loss usually occurs in one eye before the other. A later stage of dry AMD is called geographic atrophy (GA), in which the macular region becomes thin (atrophic), leading to severe vision loss.

[0198] Dry AMD can progress to wet AMD. Wet AMD is typically characterized by the growth of leaking vessels under the macula. This growth of leaking vessels is called choroidal neovascularization. The leakage of blood and fluid from these vessels damages the macula and causes blurred and distorted central vision. Wet macular degeneration is associated with severe vision loss that can rapidly worsen. High levels of intracellular cholesterol have been shown to significantly increase choroidal neovascularization. Therefore, in some embodiments, an eye disease characterized by lipid accumulation is wet AMD. In some embodiments, an eye disease characterized by lipid accumulation is choroidal neovascularization (CNV). In some embodiments, wet AMD includes CNV and / or RAP.

[0199] In some embodiments, the methods described herein for removing lipids or preventing lipid accumulation or for treating diseases associated with the accumulation of lipids (e.g., cholesterol) in cells, tissues, or organs can achieve one or more of the following effects: i) removing lipids (e.g., cholesterol) from cells, tissues, or organs (such as the eye) with accumulated lipids (e.g., by active removal and / or passive removal; e.g., removal of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); ii) increasing the amount of lipids (e.g., cholesterol) in cells, tissues, or organs (such as the eye) with accumulated lipids. The rate of lipid (e.g., cholesterol) efflux from cells, tissues, or organs (such as the eye) (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 2, 2.5, 5, 10, or more); iii) reducing the likelihood of lipid accumulation and / or diseases associated with lipid (e.g., cholesterol) accumulation in cells, tissues, or organs (such as the eye) (e.g., reducing the likelihood of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%). (iv) Delay the onset of lipid accumulation in cells, tissues, or organs (such as the eye) and / or diseases associated with lipid (e.g., cholesterol) accumulation (e.g., delaying the onset of diseases for at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, or more); (v) Slow the progression of lipid accumulation in cells, tissues, or organs (such as the eye) and / or diseases associated with lipid (e.g., cholesterol) accumulation. vi) increasing the cholesterol efflux rate by at least about 2 times (e.g., at least about 2.5 times, 3 times, 4 times, 5 times, 10 times, 15 times or more) compared to the cholesterol efflux rate achieved in the untreated state or by ApoA-I or HDL or MSP alone (e.g., MSP1D1); v) increasing the cholesterol efflux rate by at least about 5% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, or at least about 2 times, 2.5 times) compared to a fully esterified composition.vi) to increase the lipid (e.g., cholesterol) binding capacity (or lipid (e.g., cholesterol) partition coefficient) at equilibrium by at least about 5% compared to (a) ApoA-I alone, (b) HDL alone, (c) MSP alone (e.g., MSP1D1(-)) and / or (d) a fully esterified particulate composition containing the same amphiphilic polymer and phospholipids. (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, or at least about 2%, 2.5%, 3%, 4%, 5%, 10%, 15%, or more).

[0200] In some embodiments, the disease is an ocular disease characterized by lipid accumulation, i.e., GA, and the methods for preventing or treating GA provided herein achieve one or more of the following effects: i) reducing lipids (e.g., cholesterol) in an individual's eye by at least about 5% (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) or preventing lipid accumulation in an individual's eye; ii) reducing the total area of ​​GA lesions in an individual's eye by at least about 5% (such as at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and iii) slowing the progression of the total area of ​​GA lesions in an individual's eye by at least about 5% (such as at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The total area of ​​GA lesions can be determined by any method known in the art, such as autofluorescence imaging. In some implementations, autofluorescence is fundus autofluorescence (FAF).

[0201] Therefore, in some embodiments, a method is provided to reduce the total area of ​​GA lesions in the eye of an individual (e.g., a person) and / or slow the progression of the total area of ​​GA lesions in the eye of an individual (e.g., a person), the method comprising administering an effective amount of any of the compositions described herein (e.g., pharmaceutical compositions) to the individual's eye. In some embodiments, the method described herein inhibits neovascularization in the eye of an individual suffering from AMD (e.g., inhibits at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). In some embodiments, the method delays the development of AMD or CNV (e.g., delays at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, or more). In some embodiments, the method treats and / or reduces the risk of developing retinal neovascularization (RNV) (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). In some embodiments, the method treats and / or reduces the risk of developing retinal angiomyolipoma (RAP) (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). Therefore, in some embodiments, methods are provided for i) inhibiting neovascularization, ii) delaying the development of AMD or CNV, and iii) treating RNV and / or RAP in the eye of an individual (e.g., a human) and / or reducing their development risk, the method comprising administering an effective amount of any of the compositions described herein (e.g., pharmaceutical compositions) to the individual's eye. The composition (e.g., a pharmaceutical composition) may be any of the following: DMPC:MSP1D1(-)=72:1 granule composition, DMPC:MSP1D1(-)=51:1 granule composition, DMPC:MSP1D1(-)=75:1 granule composition, DMPC:MSP1E3D1(-)=62:1 granule composition, DMPC:MSP1E3D1(-)=63:1 granule composition or DMPC:MSP1E3D1(-)=94:1 granule composition.

[0202] In some embodiments, an individual has AMD and is at risk of or has RAP. In some embodiments, an individual has AMD and is at risk of or has CNV. In some embodiments, an individual has AMD and is at risk of or has RNV. In some embodiments, an individual has AMD and is at risk of or has RNV. In some embodiments, an individual has been identified as having one or more genetic polymorphisms that increase the risk of AMD. In some embodiments, the methods described herein for the prevention or treatment of eye diseases characterized by lipid accumulation (e.g., AMD) further include determining whether an individual has a genetic polymorphism that increases the risk of AMD.

[0203] In some embodiments, a method is provided to improve visual impairment and / or reduce the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in the eye of an individual (e.g., a person), the method comprising administering an effective amount of any of the compositions described herein (e.g., pharmaceutical compositions) to the individual's eye. The compositions (e.g., pharmaceutical compositions) may be any of the following: a DMPC:MSP1D1(-)=72:1 granule composition, a DMPC:MSP1D1(-)=51:1 granule composition, a DMPC:MSP1D1(-)=75:1 granule composition, a DMPC:MSP1E3D1(-)=62:1 granule composition, a DMPC:MSP1E3D1(-)=63:1 granule composition, or a DMPC:MSP1E3D1(-)=94:1 granule composition. Visual impairment and / or vision loss may be associated with any of the eye diseases described herein. In some implementations, visual impairment or loss is associated with atrophic AMD (including non-central and / or central GA), or visual improvement may occur in subjects with atrophic AMD or AMD.

[0204] In some embodiments, a method is provided to improve an individual's (e.g., a person's) normal luminance best-corrected visual acuity (NL-BCVA) score and / or low luminance best-corrected visual acuity (LL-BCVA) score, the method comprising administering an effective amount of any of the compositions described herein (e.g., pharmaceutical compositions) to the individual's eye. In some embodiments, the method provided herein increases an individual's NL-BCVA score and / or LL-BCVA score by at least about 5%, such as at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. The NL-BCVA score and / or LL-BCVA score can be determined by any method known in the art. The composition (e.g., a pharmaceutical composition) may be any of the following: DMPC:MSP1D1(-)=72:1 granule composition, DMPC:MSP1D1(-)=51:1 granule composition, DMPC:MSP1D1(-)=75:1 granule composition, DMPC:MSP1E3D1(-)=62:1 granule composition, DMPC:MSP1E3D1(-)=63:1 granule composition or DMPC:MSP1E3D1(-)=94:1 granule composition.

[0205] In some implementations, any of the following methods are: i) removing lipids or preventing lipid accumulation compared to a baseline state (or untreated state) before treatment, natural progression without any treatment, treatment with placebo, or treatment with ApoA-I or MSP protein alone (e.g., MSP1D1); ii) preventing or treating diseases associated with lipid (e.g., cholesterol) accumulation in cells, tissues, or organs (such as the eye); iii) preventing or treating eye diseases characterized by lipid accumulation; v) reducing and / or slowing the progression of the total area of ​​GA lesions; v) inhibiting angiogenesis; vi) delaying the development of AMD or CNV; vii) treating and / or reducing the risk of developing RNV and / or RAP; viii) improving visual impairment and / or reducing the likelihood of vision loss associated with eye diseases characterized by lipid accumulation; and iv) improving the NL-BCVA score and / or LL-BCVA score mentioned herein.

[0206] Route of administration and dosage

[0207] The particle-containing compositions described herein may be administered via any suitable route of application. In some embodiments, any of the compositions described herein (e.g., pharmaceutical compositions) may be administered topically, for example, via topical ocular application.

[0208] In some embodiments, suitable routes of administration include, but are not limited to, intravitreal, topical, periocular injection, intraocular or periocular implants, intravitreal implants and choroidal implants or particle or polymer compositions, or any release system such as emulsions, solid non-biodegradable or degradable implants or tablets, or micropumps. In some embodiments, any composition described herein (e.g., a pharmaceutical composition) may be administered directly to the eye via ocular tissue injection (e.g., periocular, conjunctival, subfascial, anterior chamber, intravitreal, intraocular, subretinal, subconjunctival, retrobulbar, choroidal, or intracanal injection). In some embodiments, a composition (e.g., a pharmaceutical composition) may be administered directly to the eye using a catheter or other placement device (e.g., retinal pellets, intraocular inserts, suppositories, or implants comprising porous, non-porous, or gel-like materials). In some embodiments, a composition (e.g., a pharmaceutical composition) may be administered via topical ocular drops or ointments. In some embodiments, the composition (e.g., a pharmaceutical composition) is administered via a sustained-release device into the fornix, or implanted near the sclera (via the sclera), within the sclera (intrascleral), on the choroid, or inside the eye. Intra-anterior chamber injection can be administered through the cornea into the anterior chamber to allow the agent to reach the trabecular meshwork. Intratubular injection can be administered through a venous collector channel draining from a Schrem tube or into the Schrem tube itself. In some embodiments, the composition (e.g., a pharmaceutical composition) is administered intravitreally.

[0209] In some embodiments, the composition (e.g., a pharmaceutical composition) may be an isotonic sterile saline solution (sodium dihydrogen phosphate or disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or a mixture of such salts), a sterile aqueous dispersion (e.g., a water-oil emulsion), or a dry, especially lyophilized composition, which may constitute an injectable solution after the addition of sterile water or physiological saline, depending on the circumstances.

[0210] In some embodiments, the composition (e.g., a pharmaceutical composition) further comprises a pharmaceutically acceptable excipient for topical application. Suitable pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically or ophthalmologically acceptable preservatives, solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, isotonic agents, stabilizers, pH adjusters, or water, to form an aqueous sterile ophthalmic suspension or solution. In some embodiments, the retention of the pharmaceutical composition may be further improved by viscosity-building agents such as hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, hyaluronic acid, etc.

[0211] In some embodiments, the composition (e.g., a pharmaceutical composition) may be applied as an ophthalmic ointment composition. In some embodiments, a pharmaceutically acceptable excipient is contained within a suitable medium, such as mineral oil, liquid lanolin, or white petrolatum.

[0212] In some embodiments, the composition (e.g., a pharmaceutical composition) may be an anhydrous dosage form comprising multiple particles. Such an anhydrous dosage form can be prepared using anhydrous or low-moisture components and low-moisture or low-humidity conditions.

[0213] In some embodiments, the composition (e.g., a pharmaceutical composition) may also contain a buffer. In some embodiments, the buffer is an isotonic buffer. In some embodiments, the isotonic buffer is phosphate-buffered saline (PBS). In some embodiments, the isotonic buffer does not contain glycerol (tris).

[0214] In some embodiments, the composition (e.g., a pharmaceutical composition) may also contain a preservative. Suitable preservatives include, but are not limited to, sodium bisulfite, sodium bisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, methylparaben, polyvinyl alcohol, and phenylethanol.

[0215] In some implementations, the excipients may be selected by those skilled in the art. Exemplary excipients include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (eds.), 6th edition (2009), the contents of which are incorporated herein by reference in their entirety.

[0216] In some implementation schemes, doctors may determine what they consider the most appropriate dosage based on preventative or therapeutic treatment, as well as on age, weight, condition, and other factors specific to the individual being treated.

[0217] In some implementations, the frequency and dosage may vary based on individual-specific factors, depending on the specific therapy (e.g., therapeutic or prophylactic), route of administration, and the individual's age, physical condition, weight, response, and medical history. In some implementations, the effective dose may be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0218] In some embodiments, the dosage of any composition described herein (e.g., a pharmaceutical composition) may be from about 1 μg / kg to about 1 g / kg.

[0219] In some cases, it may be necessary to use dosages of any compositions described herein (e.g., pharmaceutical compositions) that are outside the scope disclosed herein, as will be apparent to those skilled in the art. Furthermore, it should be noted that clinicians or treating physicians will know and when to interrupt, adjust, or discontinue therapy in conjunction with an individual's response.

[0220] Exemplary Implementation

[0221] Implementation Scheme 1. A composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% of the plurality of particles are under-esterified.

[0222] Implementation Scheme 2. The composition according to Implementation Scheme 1, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than the phospholipid-polymer saturation ratio N. S .

[0223] Implementation Scheme 3. The composition according to Implementation Scheme 2, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than the phospholipid-polymer saturation ratio N. S Approximately 95%.

[0224] Implementation Scheme 4. The composition according to any one of Implementation Schemes 1 to 3, wherein the amphiphilic polymer is an amphiphilic polypeptide.

[0225] Implementation Scheme 5. The composition according to Implementation Scheme 4, wherein the amphiphilic polypeptide comprises one or more amphiphilic helical sequences of about 9 to about 30 amino acids.

[0226] Implementation Scheme 6. The composition according to Implementation Scheme 4 or 5, wherein the amphiphilic polypeptide is selected from the group consisting of apolipoprotein AI (ApoA-I), ApoA-II, ApoC, ApoE, and variants thereof.

[0227] Implementation Scheme 7. The composition according to Implementation Scheme 6, wherein the amphiphilic polypeptide is a variant of ApoA-I.

[0228] Implementation Scheme 8. The composition according to Implementation Scheme 6 or 7, wherein the amphiphilic polypeptide comprises one or more amphiphilic helical sequences derived from ApoA-I.

[0229] Implementation Scheme 9. The composition according to Implementation Scheme 7 or 8, wherein the amphiphilic polypeptide comprises an N-terminus truncated relative to ApoA-I.

[0230] Implementation Scheme 10. The composition according to any one of Implementation Schemes 4 to 9, wherein the amphiphilic polypeptide is selected from the group consisting of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2 and variants thereof.

[0231] Implementation Scheme 11. The composition according to Implementation Scheme 10, wherein the amphiphilic polypeptide is a variant of any one of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1 and MSP2N2, wherein the variant does not contain an N-terminal His tag portion (a variant with the His tag removed).

[0232] Implementation Scheme 12. The composition according to Implementation Scheme 11, wherein the amphiphilic polypeptide is (i) a His-tag-removed variant of MSP1D1 comprising the amino acid sequence of SEQ ID NO: 11; or (ii) a His-tag-removed variant of MSP1E3D1 comprising the amino acid sequence of SEQ ID NO: 12.

[0233] Implementation Scheme 13. The composition according to any one of Implementation Schemes 1 to 10, wherein the phospholipid comprises one or more acyl chains optionally having a length of about 10 to about 22 carbon units, and a glycerol backbone, and wherein the phospholipid is cationic, anionic, zwitterionic, or any combination thereof.

[0234] Implementation Scheme 14. The composition according to any one of Implementation Schemes 1 to 13, wherein the phospholipid comprises DMPC, DPPC, DMPS, POPC, POPS, or PiP2.

[0235] Embodiment 15. The composition according to any one of Embodiments 1 to 14, wherein the amphiphilic polymer is MSP1D1 or a variant thereof with its His tag removed, the phospholipid is DMPC, and the N... S It is approximately 81.

[0236] Implementation Scheme 16. The composition according to Implementation Scheme 15, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 78:1.

[0237] Implementation Scheme 17. The composition according to Implementation Scheme 15, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 65:1 to about 75:1.

[0238] Implementation Scheme 18. The composition according to Implementation Scheme 15, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 40:1 to about 60:1.

[0239] Implementation Scheme 19. The composition according to Implementation Scheme 15 or 18, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is from about 46:1 to about 55:1.

[0240] Implementation Scheme 20. The composition according to any one of Implementation Schemes 1 to 14, wherein the amphiphilic polymer is MSP1D1 or a variant thereof with its His tag removed, the phospholipid is DPPC, and the N... S It is approximately 81.

[0241] Implementation Scheme 21. The composition according to Implementation Scheme 20, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 75:1.

[0242] Implementation Scheme 22. The composition according to any one of Implementation Schemes 1 to 14, wherein the amphiphilic polymer is MSP1D1 or a variant thereof with its His tag removed, the phospholipid is POPC, and the N... S It is approximately 62.

[0243] Implementation Scheme 23. The composition according to Implementation Scheme 22, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 58:1.

[0244] Implementation Scheme 24. The composition according to any one of Implementation Schemes 1 to 11, 13 or 14, wherein the amphiphilic polymer is MSP1E1D1 or a variant thereof with its His tag removed, the phospholipid is DMPC, and the N S It is approximately 107.

[0245] Implementation Scheme 25. The composition according to Implementation Scheme 24, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 100:1.

[0246] Implementation Scheme 26. The composition according to Implementation Scheme 24 or 25, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 60:1 to about 65:1.

[0247] Implementation Scheme 27. The composition according to any one of Implementation Schemes 1 to 11, 13 or 14, wherein the amphiphilic polymer is MSP1E1D1 or a variant thereof with the His tag removed, the phospholipid is DPPC, and the N S It is approximately 107.

[0248] Implementation Scheme 28. The composition according to Implementation Scheme 27, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 100:1.

[0249] Implementation Scheme 29. The composition according to any one of Implementation Schemes 1 to 11, 13 or 14, wherein the amphiphilic polymer is MSP1E1D1 or a variant thereof with the His tag removed, the phospholipid is POPC, and the NS It is approximately 80.

[0250] Implementation Scheme 30. The composition according to Implementation Scheme 29, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 76:1.

[0251] Implementation Scheme 31. The composition according to any one of Implementation Schemes 1 to 11, 13 or 14, wherein the amphiphilic polymer is MSP1E2D1 or a variant thereof with its His tag removed, the phospholipid is DMPC, and the N S It is approximately 135.

[0252] Implementation Scheme 32. The composition according to Implementation Scheme 31, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 128:1.

[0253] Implementation Scheme 33. The composition according to any one of Implementation Schemes 1 to 11, 13 or 14, wherein the amphiphilic polymer is MSP1E2D1 or a variant thereof with its His tag removed, the phospholipid is DPPC, and the N S It is approximately 135.

[0254] Implementation Scheme 34. The composition according to Implementation Scheme 33, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 128:1.

[0255] Implementation Scheme 35. The composition according to any one of Implementation Schemes 1 to 11, 13 or 14, wherein the amphiphilic polymer is MSP1E2D1 or a variant thereof with its His tag removed, the phospholipid is POPC, and the N S It is approximately 102.

[0256] Implementation Scheme 36. The composition according to Implementation Scheme 35, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 96:1.

[0257] Implementation Scheme 37. The composition according to any one of Implementation Schemes 1 to 14, wherein the amphiphilic polymer is MSP1E3D1 or a variant thereof with its His tag removed, the phospholipid is DMPC, and the N... S It is approximately 167.

[0258] Implementation Scheme 38. The composition according to Implementation Scheme 37, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 158:1.

[0259] Implementation Scheme 39. The composition according to Implementation Scheme 37 or 38, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is from about 50:1 to about 100:1.

[0260] Implementation Scheme 40. The composition according to Implementation Scheme 39, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 87:1 to about 96:1, about 58:1 to about 68:1, about 63:1, or about 62:1.

[0261] Implementation Scheme 41. The composition according to any one of Implementation Schemes 1 to 14, wherein the amphiphilic polymer is MSP1E3D1 or a variant thereof with its His tag removed, the phospholipid is DPPC, and the N... S It is approximately 167.

[0262] Implementation Scheme 42. The composition according to Implementation Scheme 41, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 158:1.

[0263] Implementation Scheme 43. The composition according to any one of Implementation Schemes 1 to 14, wherein the amphiphilic polymer is MSP1E3D1 or a variant thereof with its His tag removed, the phospholipid is POPC, and the N... S It is approximately 126.

[0264] Implementation Scheme 44. The composition according to Implementation Scheme 43, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 118:1.

[0265] Implementation Scheme 45. The composition according to any one of Implementation Schemes 1 to 11, 13 or 14, wherein the amphiphilic polymer is MSP2N2 or a variant thereof with the His tag removed, the phospholipid is DMPC, and the N S It is approximately 387.

[0266] Implementation Scheme 46. The composition according to Implementation Scheme 45, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 367:1.

[0267] Implementation Scheme 47. The composition according to any one of Implementation Schemes 1 to 11, 13 or 14, wherein the amphiphilic polymer is MSP2N2 or a variant thereof with the His tag removed, the phospholipid is DPPC, and the N S It is approximately 387.

[0268] Implementation Scheme 48. The composition according to Implementation Scheme 47, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 367:1.

[0269] Implementation Scheme 49. The composition according to any one of Implementation Schemes 1 to 11, 13 or 14, wherein the amphiphilic polymer is MSP2N2 or a variant thereof with the His tag removed, the phospholipid is POPC, and the N S It is approximately 291.

[0270] Implementation Scheme 50. The composition according to Implementation Scheme 49, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than about 276:1.

[0271] Implementation Scheme 51. The composition according to any one of Implementation Schemes 1 to 50, wherein the average size of the particles in the composition is about 2 nm to about 20 nm in diameter.

[0272] Implementation Scheme 52. The composition according to Implementation Scheme 51, wherein the polydispersity index of the particle size in the composition is less than about 0.2.

[0273] Implementation Scheme 53. The composition according to any one of Implementation Schemes 1 to 52, wherein the polydispersity index of the phospholipid content of the particles in the composition is less than about 0.2.

[0274] Implementation Scheme 54. The composition according to any one of Implementation Schemes 1 to 53, wherein the composition is a pharmaceutical composition.

[0275] Implementation Scheme 55. A method for preparing a composition comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-esterified, the method comprising: (1) incubating the amphiphilic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is dissolved with a detergent or an organic compound prior to the incubation; and (2) removing the detergent or organic compound from the preparation mixture to obtain the particles.

[0276] Implementation Scheme 56. The method according to Implementation Scheme 55, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than the phospholipid-polymer saturation ratio N. S .

[0277] Implementation Scheme 57. The method according to Implementation Scheme 55 or 56, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the preparation mixture is less than or equal to the molar ratio of the phospholipid to the amphiphilic polymer in the composition.

[0278] Implementation Scheme 58. The method according to Implementation Scheme 57, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the preparation mixture is less than about 95% of the molar ratio of the phospholipid to the amphiphilic polymer in the composition.

[0279] Implementation Scheme 59. The method according to any one of Implementation Schemes 55 to 58, wherein the detergent or organic compound comprises a bile salt.

[0280] Implementation Scheme 60. The method according to any one of Implementation Schemes 55 to 59, wherein the amphiphilic polymer is provided in a solution containing a buffer.

[0281] Implementation Scheme 61. The method according to any one of Implementation Schemes 55 to 60, wherein the concentration of the amphiphilic polymer in the solution is about 1 mg / mL to about 5 mg / mL.

[0282] Implementation Scheme 62. The method according to any one of Implementation Schemes 55 to 61, wherein the concentration of the amphiphilic polymer in the solution is about 2.5 mg / mL to about 4 mg / mL.

[0283] Implementation Scheme 63. The method according to any one of Implementation Schemes 55 to 62, wherein the method further comprises separating the particles.

[0284] Implementation Scheme 64. The method according to Implementation Scheme 63, wherein the particles are separated by chromatography.

[0285] Implementation Scheme 65. The method according to any one of Implementation Schemes 55 to 64, wherein the method further comprises subjecting the particles to one or more freeze-thaw cycles with or without a cryoprotectant.

[0286] Implementation Scheme 66. The method according to any one of Implementation Schemes 55 to 65, wherein the amphiphilic polymer is an amphiphilic polypeptide.

[0287] Implementation Scheme 67. The method according to Implementation Scheme 66, wherein the amphiphilic polypeptide comprises one or more amphiphilic helical sequences of about 9 to about 30 amino acids.

[0288] Implementation Scheme 68. The method according to Implementation Scheme 66 or 67, wherein the amphiphilic polypeptide is selected from the group consisting of apolipoprotein AI (ApoA-I), ApoA-II, ApoC, ApoE, and variants thereof.

[0289] Implementation Scheme 69. The method according to Implementation Scheme 68, wherein the amphiphilic polypeptide is a variant of ApoA-I.

[0290] Implementation Scheme 70. The method according to Implementation Scheme 68 or 69, wherein the amphiphilic polypeptide comprises one or more amphiphilic helical sequences derived from ApoA-I.

[0291] Implementation Scheme 71. The method according to Implementation Scheme 69 or 70, wherein the amphiphilic polypeptide comprises an N-terminus truncated relative to ApoA-I.

[0292] Implementation Scheme 72. The method according to any one of Implementation Schemes 66 to 71, wherein the amphiphilic polypeptide is selected from the group consisting of MSP1D1, MSPE1D1, MSP1E2D1, MSP1E3D1, MSP2N2 and variants thereof.

[0293] Implementation Scheme 73. The method according to Implementation Scheme 72, wherein the amphiphilic polypeptide is a variant of any one of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1 and MSP2N2, wherein the variant does not contain an N-terminal His tag portion (a variant with the His tag removed).

[0294] Implementation Scheme 74. The method according to Implementation Scheme 73, wherein the amphiphilic polypeptide is (i) a His-tag-removed variant of MSP1D1 containing the amino acid sequence of SEQ ID NO: 11; or (ii) a His-tag-removed variant of MSP1E3D1 containing the amino acid sequence of SEQ ID NO: 12.

[0295] Implementation Scheme 75. The method according to any one of Implementation Schemes 55 to 74, wherein the phospholipid comprises one or more acyl chains, optionally having a length of about 10 to about 22 carbon units.

[0296] Implementation Scheme 76. The method according to any one of Implementation Schemes 55 to 75, wherein the phospholipid has a glycerol backbone.

[0297] Implementation Scheme 77. The method according to any one of Implementation Schemes 55 to 76, wherein the phospholipid is cationic, anionic, zwitterionic, or any combination thereof.

[0298] Implementation Scheme 78. The method according to any one of Implementation Schemes 55 to 77, wherein the phospholipid comprises DMPC, DPPC, DMPS, POPC, POPS, or PiP2.

[0299] Implementation Scheme 79. The method according to any one of Implementation Schemes 55 to 78, wherein the amphiphilic polymer is MSP1D1 or a variant thereof with the His tag removed, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 65:1 to about 75:1.

[0300] Implementation Scheme 80. The method according to Implementation Scheme 79, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the preparation mixture is less than about 65:1.

[0301] Implementation Scheme 81. The method according to Implementation Scheme 79 or 80, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 65:1 to about 75:1, and the molar ratio of the phospholipid to the amphiphilic polymer in the preparation mixture is about 55:1.

[0302] Implementation Scheme 82. The method according to any one of Implementation Schemes 55 to 78, wherein the amphiphilic polymer is MSP1D1 or a variant thereof with the His tag removed, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 46:1 to about 55:1.

[0303] Implementation Scheme 83. The method according to Implementation Scheme 82, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the mixture is less than about 45:1.

[0304] Implementation Scheme 84. The method according to Implementation Scheme 82 or 83, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 46:1 to about 55:1, and the molar ratio of the phospholipid to the amphiphilic polymer in the preparation mixture is about 40:1 or about 30:1.

[0305] Implementation Scheme 85. The method according to any one of Implementation Schemes 55 to 78, wherein the amphiphilic polymer is MSP1E3D1 or a variant thereof with the His tag removed, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 85:1 to about 100:1.

[0306] Implementation Scheme 86. The method according to Implementation Scheme 85, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the mixture is less than or equal to about 90:1.

[0307] Implementation Scheme 87. The method according to Implementation Scheme 85 or 86, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is from about 87:1 to about 96:1, and the molar ratio of the phospholipid to the amphiphilic polymer in the preparation mixture is from about 90:1.

[0308] Implementation Scheme 88. The method according to any one of Implementation Schemes 55 to 78, wherein the amphiphilic polymer is MSP1E3D1 or a variant thereof with the His tag removed, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 55:1 to about 80:1.

[0309] Implementation Scheme 89. The method according to Implementation Scheme 88, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the mixture is less than or equal to about 60:1.

[0310] Implementation Scheme 90. The method according to Implementation Scheme 88 or 89, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 58:1 to about 68:1 or about 63:1, and the molar ratio of the phospholipid to the amphiphilic polymer in the preparation mixture is about 60:1.

[0311] Implementation Scheme 91. The method according to Implementation Scheme 88 or 89, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is about 58:1 to about 68:1 or about 62:1, and the molar ratio of the phospholipid to the amphiphilic polymer in the preparation mixture is about 50:1.

[0312] Implementation Scheme 92. A composition comprising the particles prepared according to any one of Implementation Schemes 51 to 85.

[0313] Implementation Scheme 93. A method for treating or preventing a disease associated with lipid accumulation in an individual, the method comprising administering to the individual an effective amount of a composition according to any one of Implementation Schemes 1 to 54 and 92.

[0314] Implementation Scheme 94. The method according to Implementation Scheme 93, wherein the disease is associated with lipid accumulation in the eye.

[0315] Implementation Scheme 95. The method according to Implementation Scheme 93 or 94, wherein the disease is age-related macular degeneration (AMD), photoreceptor neurodegeneration, optic atrophy, vision loss, hemianopsia, visual agnosia, strabismus, retinal neurovascular disease, lipid corneal disease, corneal lipid deposition, or any combination thereof.

[0316] Implementation Scheme 96. The method according to any one of Implementation Schemes 93 to 95, wherein the disease is AMD.

[0317] Implementation Scheme 97. The method according to Implementation Scheme 96, wherein the AMD is a dry AMD.

[0318] Implementation Scheme 98. The method according to Implementation Scheme 97, wherein the dry AMD is map-like atrophy (GA).

[0319] Implementation Scheme 99. The method according to any one of Implementation Schemes 93 to 98, wherein the composition reduces lipids in the individual by at least about 5% or prevents lipid accumulation in the individual.

[0320] Example

[0321] Example 1: Preparation of an exemplary particulate composition with insufficient esterification

[0322] A composition comprising multiple particles is prepared from phospholipids, amphiphilic polymers, and organic compounds according to the scheme provided below.

[0323] Material

[0324] MSP1D1(-) refers to MSP1D1 with the N-terminal histidine affinity tag removed, which was used in the preparation described herein. MSP1D1(-) contains the sequence of SEQ ID NO: 11. MSP1D1 refers to the form without the N-terminal histidine affinity tag removed, containing SEQ ID NO: 2. Concentration was measured by UV-Vis spectroscopy, which showed an extinction coefficient of 18450 M at 280 nm. -1 (0.84 mg / ml).

[0325] Phospholipids (e.g., DMPC, DPPC) are dissolved in chloroform and their concentration is measured by determining total phosphorus (see, for example, Chen, Toribara and Warner (1956) Anal. Chem. 28:1756-1758, the contents of which are incorporated herein by reference in their entirety).

[0326] Other reagents used in the preparation included phosphate-buffered saline (PBS), 200 mM sodium cholate detergent, Superdex S-200 size exclusion column, methanol, ethyl acetate, ethanol, hexane, concentrated hydrochloric acid, and water.

[0327] Preparation of DMPC / MSP1D1(-) particulate composition

[0328] All operations were performed at room temperature (20°C to 22°C).

[0329] Phospholipid-polymer saturation ratio N of particles containing DMPC and MSP1D1 (or MSP1D1(-)) SThe concentration was approximately 81 (see Table 2). A DMPC / chloroform stock solution was added to a glass tube to prepare a final ratio of DMPC to MSP1D1(-) of 30:1. The DMPC was dried under a nitrogen stream while the glass tube was rotated to form a film on the side of the tube. The tube containing the DMPC was then placed in a desiccator and a vacuum was applied for at least 4 hours, e.g., overnight.

[0330] The DMPC was then hydrated in water. To determine the required amount of water, the desired final sodium cholate concentration was first determined. The final cholate to lipid ratio was determined to be greater than 2. An exemplary concentration was 17 mM cholate. The amounts of water and 200 mM sodium cholate were determined such that the final sodium cholate concentration was 17 mM. The amount of protein was then determined such that the final protein concentration was approximately 3.5 mg / mL. At this point, only water was added to the dried DMPC.

[0331] The water / DMPC mixture was vortexed until the solution turned milky white. Then, 200 mM sodium cholate was added to the hydrated DMPC to obtain a final cholate concentration of 17 mM in the assembly mixture. The sodium cholate / DMPC mixture was completely dissolved by placing the tube in an ultrasonic water bath for 5 min, followed by gentle heating at approximately 40 °C, repeated twice. MSP1D1(-) was then added to the sodium cholate / DMPC mixture and thoroughly mixed to obtain a preparative mixture with a protein concentration of approximately 3.5 mg / mL. The preparative mixture was incubated at room temperature for 10 to 20 minutes.

[0332] Assembly of the particle composition is initiated by adding Amberlite XAD-2 resin, in which Amberlite absorbs the bile salt detergent. The amount of Amberlite XAD-2 resin filled is at least 50% of the volume of the assembly mixture; for example, for a 5 ml preparation mixture, Amberlite resin is added to the preparation mixture until the volume is 7.5 ml. The assembly mixture containing Amberlite resin is gently agitated by placing it on a platform shaker or tube rotator at room temperature for at least four hours. The supernatant containing the assembled particles is recovered from the Amberlite XAD resin using a pipette and placed in a new tube. The Amberlite XAD resin is washed twice with PBS, and the wash solution containing the remaining assembled particles is added to the sample tube. Large particles are removed from the sample by passing them through a 0.2-micron filter. Depending on the particle sample volume, this can also be done using a centrifuge filter (Amicon Ultra Free) or a syringe equipped with a 0.2-micron filter.

[0333] Similarly, another under-esterified particulate composition comprising DMPC and MSP1D1(-) was prepared, wherein the molar ratio of DMPC to MSP1D1(-) in the mixture was about 55:1. Similarly, a third under-esterified particulate composition comprising DMPC and MSP1D1(-) was prepared, wherein the molar ratio of DMPC to MSP1D1(-) in the mixture was about 40:1.

[0334] In a similar manner, under-esterified particulate compositions comprising DMPC and MSP1E3D1(-) were prepared using different lipid-to-MSP molar ratios in the preparation mixture. The lipid-to-MSP molar ratios used in the various DMPC / MSP1E3D1(-) compositions are also detailed in Example 6. MSP1E3D1(-) refers to MSP1E3D1 with the N-terminal histidine affinity tag removed, which was used in the preparations described herein. MSP1E3D1(-) contains the sequence of SEQ ID NO: 12. MSP1E3D1 also refers to the form without the N-terminal histidine affinity tag removed, containing SEQ ID NO: 5. The phospholipid-polymer saturation ratio of the particles containing DMPC and MSP1E3D1 (or MSP1E3D1(-)) is N... S The value was 167 (see Table 2). To prepare the under-esterified composition, the final ratio of DMPC to MSP1E3D1(-) in the mixture was approximately 50:1.

[0335] Similarly, another under-esterified particulate composition containing DMPC and MSP1E3D1(-) was prepared, wherein the molar ratio of DMPC to MSP1E3D1(-) in the mixture was about 90:1.

[0336] Particulate compositions containing other MSPs (or other phospholipid / MSP ratios) are prepared according to a similar scheme detailed herein.

[0337] As a control, fully esterified particulate compositions were also prepared. One fully esterified particulate composition was prepared starting from a preparation mixture of DMPC and MSP1D1(-) in a molar ratio of approximately 80:1. Another fully esterified particulate composition was prepared starting from a preparation mixture of DMPC and MSP1D1 in a molar ratio of approximately 85:1. The method for preparing the control particulate compositions was similar to the procedure detailed above, except that different lipid to MSP molar ratios were used in the preparation mixture. See, for example, Example 9.

[0338] Example 2: Purification of an exemplary under-esterified particulate composition

[0339] The filtered sample from Example 1 containing DMPC / MSP1D1(-) particles or DMPC / MSP1E3D1(-) particles was further purified by size exclusion chromatography (SEC). The control particle composition was purified similarly.

[0340] For the column: Use Superdex S-200 increase 10 / 300 (approximately 25 ml bed volume). The buffer is PBS. The flow rate is 0.75 ml / min. The injection volume is 0.5 mL.

[0341] The chromatogram of the preparative SEC of the DMPC / MSP1D1(-) composition (preparative mixture ratio of about 30:1) is shown in Figure 1A In the middle. For example Figure 1A As shown, two peaks were separated. The region on the right (dark gray) corresponds to a composition in which the less lipid-rich (lower lipid-to-protein ratio) particle composition contains approximately 51 lipids per MSP1D1(-). The phospholipid-to-MSP protein ratio in the final particle composition was measured using the method described in Example 3 below. The peak located at 20 minutes (see...) Figure 1A The small image at the top shows the remaining lipid-free protein.

[0342] Chromatogram of the preparative SEC of the DMPC / MSP1E3D1(-) composition (preparative mixture ratio of approximately 50:1) is shown. Figure 1B In the middle. For example Figure 1B As shown, a single highlighted peak was collected, which contains an under-esterified particulate composition of DMPC to MSP1E3D1(-) in a molar ratio of about 62:1.

[0343] Example 3: Measurement of the phospholipid to MSP ratio

[0344] This assay measures the phospholipid-to-MSP ratio by first extracting the phospholipids, then saponifying and methylating the fatty acids for rapid gas chromatography (GC) analysis. The amount of fatty acid / MSP measured is twice the amount of the starting phospholipids. Since the phospholipid DPPC is used as a standard, two fatty acids per phospholipid have been taken into account.

[0345] The ratio of phospholipids to MSP in the particulate composition was determined according to the procedure detailed below.

[0346] The MSP concentration of the particulate compositions described herein was measured by UV-Vis spectroscopy. Particulate composition samples were added to 2 mL GC vials to present approximately 1 µg of phospholipids. Each particulate composition sample was run in triplicate. The particulate composition samples were diluted to 100 µL with PBS. 10 µL of a known concentration of standard (DPPC) was added to each particulate composition sample. Total phospholipids were extracted using the modified Bligh and Dyer techniques described in conditions E and E' of the phase diagram in Figure 7 of Int. J. Mol. Sci. 2017, 18(4), 708.

[0347] Phospholipids were extracted and dried under a nitrogen flow. Fatty acids were saponified and methylated simultaneously by dissolving the particulate composition sample in 100 µL of toluene. 0.75 mL of methanol and 0.15 mL of 8% hydrochloric acid / methanol solution were added to the mixture (see J Lipid Res. 2010; 51(3):635-40). The mixture was incubated at 100 °C for 1 hour. In other batches, the mixture was incubated overnight at 45 °C.

[0348] Then, 0.5 mL of water and 0.5 mL of hexane were added to the mixture. The mixture was vortexed, and the top hexane layer was recovered for GC analysis. A standard curve of DMPC in chloroform at a known concentration was prepared by serial dilution, with 10 μL of DMPC standard added to each dilution, and saponification was performed as described in Section 176, J Lipid Res. 2010; 51(3):635-40.

[0349] The DMPC content was determined by measuring the area ratio of C14 fatty acid methyl ester (DMPC) to C16 fatty acid methyl ester (DPPC) and comparing it with a standard curve.

[0350] The lipid to MSP ratio of the particulate composition was determined from the protein concentration and DMPC concentration measured by UV-Vis spectroscopy.

[0351] Exemplary results obtained by GC method using the DMPC / MSP1D1(-) particle composition from Example 2 (preparation mixture ratio of about 30:1) are shown in Figure 2 In. Figure 2 In this context, DMPCfame is methyl myristate, and DPPCfame is methyl palmitate, which are methylated fatty acids derived from DMPC and DPPC, respectively.

[0352] Based on the measurement results, such as Figure 1AAs detailed herein, starting with a preparation mixture of DMPC and MSP1D1(-) at a molar ratio of approximately 30:1, under-esterified particulate compositions of DMPC:MSP1D1(-) at a molar ratio of approximately 72:1 (hereinafter referred to as "particulate compositions of DMPC:MSP1D1(-)=72:1") and under-esterified particulate compositions of DMPC:MSP1D1(-) at a molar ratio of approximately 51:1 (hereinafter referred to as "particulate compositions of DMPC:MSP1D1(-)=51:1") were obtained, wherein the yield of the particulate composition of DMPC:MSP1D1(-)=51:1 was optimized. Therefore, this condition was further used to prepare particulate compositions of DMPC:MSP1D1(-)=51:1.

[0353] Starting with a preparation mixture of DMPC and MSP1D1(-) at a molar ratio of approximately 55:1, an under-esterified particulate composition of DMPC and MSP1D1(-) at a molar ratio of approximately 75:1 is obtained, hereinafter referred to as "particulate composition of DMPC:MSP1D1(-)=75:1". This condition is further used to prepare particulate compositions of DMPC:MSP1D1(-)=75:1.

[0354] Starting with a preparation mixture of DMPC and MSP1D1(-) at a molar ratio of approximately 40:1, an under-esterified particulate composition with a molar ratio of DMPC to MSP1D1(-) of approximately 51:1 is obtained, hereinafter referred to as "particulate composition of DMPC:MSP1D1(-)=51:1". This condition is further used to prepare particulate compositions of DMPC:MSP1D1(-)=51:1.

[0355] Starting with a preparation mixture of DMPC and MSP1E3D1(-) in a molar ratio of about 50:1, an under-esterified particulate composition of DMPC and MSP1E3D1(-) in a molar ratio of about 62:1 is obtained, hereinafter referred to as "particulate composition of DMPC:MSP1E3D1(-)=62:1".

[0356] Starting with a preparation mixture of DMPC and MSP1E3D1(-) in a molar ratio of about 60:1, an under-esterified particulate composition of DMPC and MSP1E3D1(-) in a molar ratio of about 63:1 is obtained, hereinafter referred to as "particulate composition of DMPC:MSP1E3D1(-)=63:1".

[0357] Starting with a preparation mixture of DMPC and MSP1E3D1(-) in a molar ratio of about 90:1, an under-esterified particulate composition of DMPC and MSP1E3D1(-) in a molar ratio of about 94:1 is obtained, hereinafter referred to as "particulate composition of DMPC:MSP1E3D1(-)=94:1".

[0358] For a control, a preparative mixture with a DMPC:MSP1D1 molar ratio of about 85:1 was prepared to obtain a particulate composition (fully esterified) with a DMPC:MSP1D1 molar ratio of about 83:1, hereinafter referred to as "particulate composition of DMPC:MSP1D1=83:1".

[0359] For another control, starting with a preparation mixture of DMPC and MSP1D1(-) in a molar ratio of about 80:1, a particulate composition (fully esterified) with a molar ratio of DMPC to MSP1D1(-) of about 81:1 was obtained, hereinafter referred to as "particulate composition of DMPC:MSP1D1(-)=81:1".

[0360] Example 4: Cholesterol Efflux Measurement

[0361] Perform this assay as described in the product information for the Cholesterol Effusion Assay Kit (Cell-Based) (ab196985).

[0362] In summary, J774 mouse macrophages were cultured and placed in 96-well tissue culture plates. Cells were labeled with fluorescent cholesterol, washed, and incubated overnight. The cells were then treated for 4 hours with a culture medium containing the test particles generated as described in Examples 1 to 3. The culture medium containing the test particles was recovered, and cholesterol fluorescence (F) was measured in the medium. 培养基 Cells were lysed, and the residual fluorescence (F) in the cells was measured. 细胞 Cholesterol efflux is defined as:

[0363] % outflow = F 培养基 / (F 培养基 +F 细胞 )

[0364] Figure 3A and Figure 3B Effluent assays performed on purified exemplary particle compositions as described in Examples 1 through 3 are shown. The lipid to MSP ratio is the ratio measured from the final particle composition. In addition to the fully lipoproteinized particle composition control, MSP1D1(-) (protein only, no lipids), Apo-AI (protein only, no lipids), and high-density lipoprotein (HDL) were also used as controls. A positive control from a cholesterol efflux assay kit (ab196985) was included. PBS was used as a negative control (not shown).

[0365] like Figure 3AAs shown, when 2.5 μg of sample is incubated with cells labeled with fluorescent cholesterol, the results show that the sample is incubated with a particle composition having a higher lipid content (e.g., a particle composition with insufficient lipidation of DMPC:MSP1D1(-) = 75:1, for example, ... Figure 6B The particulate composition shown) and the particulate composition of fully esterified DMPC:MSP1D1(-)=81:1 (e.g., Figure 6A Compared to the particulate composition shown, the particulate composition with insufficient esterification of DMPC:MSP1D1(-)=51:1 (e.g., Figure 6D The particulate composition shown exhibits a much higher cholesterol efflux rate. Figure 3A Further, it was shown that removing the N-terminal his-tag portion of the MSP protein did not affect cholesterol efflux (comparing a particle composition with fully esterified DMPC:MSP1D1(-)=81:1 and a particle composition with fully esterified DMPC:MSP1D1=83:1). The phospholipid-polymer saturation ratio of particles containing DMPC and MSP1E3D1 (or MSP1E3D1(-)) was N... S The value is 167 (see Table 2). Therefore, the particulate compositions with DMPC:MSP1E3D1(-)=62:1 and DMPC:MSP1E3D1(-)=94:1 are significantly under-esterified. Figure 3A As shown, the particle composition with DMPC:MSP1E3D1(-)=62:1 also exhibited a slightly better cholesterol efflux rate compared to the particle composition with DMPC:MSP1E3D1(-)=94:1 (which has a higher lipid content). Compared to the lipid-free MSP1D1(-) protein control, Apo-AI control, and HDL control, the under-lipidized particle compositions with DMPC:MSP1D1(-)=51:1, DMPC:MSP1E3D1(-)=62:1, and DMPC:MSP1E3D1(-)=94:1 all showed much larger cholesterol efflux rates. Figure 3B The results showed that cholesterol efflux capacity increased significantly with increasing particulate composition dosage (10 μg vs. 2.5 μg). Surprisingly, compared with 10 μg of fully esterified DMPC:MSP1D1(-)=81:1 particulate composition, 2.5 μg of under-esterified DMPC:MSP1D1(-)=51:1 particulate composition even showed stronger cholesterol efflux capacity, further demonstrating the technical superiority of the under-esterified particulate composition.

[0366] These results demonstrate that the under-esterified particulate composition prepared in this paper has a surprisingly excellent cholesterol efflux capacity.

[0367] Example 5: Stability of under-esterified particulate compositions to freeze-thaw cycles

[0368] The stability of the particulate compositions prepared in Examples 1 to 3 was tested. One exemplary DMPC / MSP1D1(-) particulate composition tested is... Figure 1A The right-hand region of the chromatographic peak corresponds to the under-esterified DMPC:MSP1D1(-)=51:1 particulate composition. The sample was frozen at -80°C with or without 10% glycerol as a cryoprotectant. The frozen sample was then removed from -80°C storage and rapidly thawed. The sample was subjected to SEC and compared with the chromatogram obtained before freezing. The results are shown in... Figure 4 In the study, the chromatograms of samples indicating freeze-thaw cycles (without glycerol) were nearly identical to those of pre-frozen samples. The chromatograms of samples frozen with or without cryoprotectant after freeze-thaw cycles were also nearly identical (data not shown). These results indicate that the prepared under-esterified particulate compositions exhibit surprisingly high stability when subjected to freeze-thaw cycles.

[0369] Freeze-thaw cycles on under-esterified particulate compositions had no effect on cholesterol efflux efficiency. Figure 5 The image shows a comparison of cholesterol efflux from a particulate composition of under-esterified DMPC:MSP1D1(-)=51:1 before and after freeze-thaw cycles. Figure 5 As shown, the cholesterol efflux efficiency of the under-lipidated particle composition remained high after freeze-thaw cycles (second column) and remained superior to the fully lipidated particle composition control with high DMPC content (particle composition with DMPC:MSP1D1=83:1), as well as higher than lipid-free HDL and blank control PBS solutions.

[0370] Example 6: Correspondence between the phospholipid / MSP1D1(-) ratio in the prepared mixture and the final particulate composition

[0371] The correspondence between the phospholipid / MSP1D1(-) ratio in the preparation mixture and the final particle composition was examined. Exemplary particle compositions containing multiple DMPC / MSP1D1(-) particles were synthesized and purified according to the procedures described in Examples 1 and 2, and the ratios were measured as in Example 3. For compositions starting with DMPC / MSP1D1(-) preparation mixture ratios of 80:1, 55:1, and 40:1, the fractions collected from the first SEC were repurified using a second SEC (repurification). For compositions starting with a DMPC / MSP1D1(-) preparation mixture ratio of 40:1, a third SEC purification was performed (repurification 2). For compositions starting with a DMPC / MSP1D1(-) preparation mixture ratio of 30:1, only one SEC step was performed. After the final purification step, the lipid-to-protein ratio was measured in the final particle composition. The results were shown in... Figures 6A to 6D In the middle. For example Figure 6A As shown, an initial DMPC / MSP1D1(-) ratio of 80:1 in the preparation mixture yields a composition containing multiple particles, wherein the DMPC / MSP1D1(-) ratio is approximately 81:1, such as 81 ± 3:1. Figure 6B As shown, an initial DMPC / MSP1D1(-) ratio of 55:1 in the preparation mixture yields a composition containing multiple particles, wherein the DMPC / MSP1D1(-) ratio is approximately 75:1, such as 75 ± 1.5:1. Figure 6C As shown, an initial DMPC / MSP1D1(-) ratio of 40:1 in the preparation mixture yields a composition containing multiple particles, wherein the DMPC / MSP1D1(-) ratio is approximately 51:1, such as 51 ± 1:1. Figure 6D As shown, an initial DMPC / MSP1D1(-) ratio of 30:1 in the preparation mixture yields a composition containing multiple particles, wherein the DMPC / MSP1D1(-) ratio is approximately 51:1, such as 51 ± 1:1. Furthermore, DMPC and MSP1D1(-) at a 30:1 (preparation ratio) are adequately separated in a single purification step. These results collectively demonstrate that low phospholipid / MSP ratios (e.g., an initial DMPC / MSP1D1(-) ratio of less than 81:1 in the preparation mixture (the phospholipid-polymer saturation ratio N of DMPC and MSP1D1) are suitable for low phospholipid / MSP ratios. S This can produce surprisingly stable, under-esterified compositions with excellent cholesterol efflux activity (see Example 4).

[0372] Example 7: Correspondence between the phospholipid / MSP1E3D1(-) ratio in the preparation mixture and the final particulate composition sex

[0373] The correspondence between the phospholipid / MSP1E3D1(-) ratio in the prepared mixture and the final particle composition was examined. Exemplary particle compositions containing multiple DMPC / MSP1E3D1(-) particles were synthesized and purified according to the procedures described in Examples 1 and 2, and the ratio was measured as in Example 3. Only one SEC step was performed. The lipid-to-protein ratio was measured in the final particle composition after the final purification step. The results were shown... Figures 7A to 7C In the middle. For example Figure 7A As shown, an initial DMPC / MSP1E3D1(-) ratio of 90:1 in the preparation mixture yields a composition containing multiple particles, wherein the DMPC / MSP1D1(-) ratio is approximately 94:1, such as 94 ± 4:1. Figure 7B As shown, an initial DMPC / MSP1E3D1(-) ratio of 60:1 in the preparation mixture yields a composition containing multiple particles, wherein the DMPC / MSP1D1(-) ratio is approximately 63:1, such as 63 ± 4:1. Figure 7C As shown, an initial DMPC / MSP1D1(-) ratio of 50:1 in the preparation mixture yields a composition containing multiple particles, wherein the DMPC / MSP1D1(-) ratio is about 62:1, such as 62 ± 0.5:1. These results collectively demonstrate that a low phospholipid / MSP ratio (e.g., an initial DMPC / MSP1E3D1(-) ratio of less than 167:1 in the preparation mixture (the phospholipid-polymer saturation ratio N of DMPC and MSP1E3D1) is suitable. S This can produce surprisingly stable, under-esterified compositions with excellent cholesterol efflux activity (see Example 4).

[0374] Example 8: Determination of Cholesterol Binding Capacity

[0375] Large monolayer vesicles (LUVs) possess a single phospholipid bilayer and can be loaded with a large amount of hydrophilic material. Cholesterol-containing LUVs can be used to measure the equilibrium binding (or partition coefficient) of cholesterol with the hypoesterified particulate compositions described herein. This type of assay represents the ability of the hypoesterified particulate composition to passively remove cholesterol from the cholesterol-containing LUV. The cholesterol-binding “capacity” measured by this assay depends on the ability of the hypoesterified particulate composition to bind cholesterol and the conditions used in the assay, and it does not reflect the maximum cholesterol-binding capacity of the hypoesterified particulate composition. The cholesterol-binding “capacity” value is an equilibrium value where the passive diffusion rate of cholesterol from the LUV to the hypoesterified particulate composition is equal to the diffusion rate of cholesterol from the hypoesterified particulate composition back to the LUV. Assay conditions, such as LUV concentration, mol% cholesterol in the LUV, and MSP concentration, can be readily adjusted to evaluate the cholesterol-binding capacity of various hypoesterified particulate compositions in vitro.

[0376] Using an under-esterified DMPC:MSP1D1(-)=51:1 particulate composition prepared from Examples 1 to 3 (starting from a preparation mixture with a DMPC to MSP1D1(-) molar ratio of about 30:1) and an exemplary LUV containing 70% POPC and 30% cholesterol, cholesterol-binding capacity at equilibrium was demonstrated using LUV-based assays.

[0377] Preparation of an exemplary LUV

[0378] POPC and cholesterol in the chloroform stock solution were combined to achieve a final POPC:cholesterol ratio of 7:3. Solvent removal was achieved by drying the composition under a nitrogen stream followed by drying in a vacuum desiccator for 4 hours. The lipids (POPC and cholesterol) were then hydrated and vortexed in phosphate-buffered saline (PBS) until a milky white suspension was obtained. LUV was formed by extruding the suspension through a 100 nm filter (Whatman Nuclepore hydrophilic membrane, 0.1 µm pore size) nine times. Prior to experiments, the clarified LUV sample was subjected to gas chromatography-mass spectrometry (GC-MS) to measure the total cholesterol concentration.

[0379] Cholesterol binding capacity measurement

[0380] At 37°C, a 51:1 ratio of under-lipidized DMPC:MSP1D1(-) particles in PBS was combined with the obtained LUV (70% POPC:30% cholesterol) to achieve a final concentration of 1 mM cholesterol and 0.02 mM MSP1D1(-) protein (given the 51:1 ratio in the particle composition, the final mixture contained approximately 1.02 mM DMPC). At different time points after mixing, 50 μl of the reactant was collected, and LUV was separated from the 51:1 DMPC:MSP1D1(-) particles by size exclusion chromatography (SEC), detected by monitoring absorbance at 280 nm. Fractions containing the 51:1 DMPC:MSP1D1(-) particles were collected, and the MSP1D1(-) protein concentration was determined by the integrated area of ​​the SEC chromatogram. The cholesterol content in the fractions containing the 51:1 DMPC:MSP1D1(-) particles was measured by GC-MS.

[0381] Figure 8 The cholesterol:MSP1D1(-) ratio as a function of time is shown in a fraction of a particulate composition containing DMPC:MSP1D1(-) = 51:1. Figure 8 The results show that equilibrium was achieved under the assay conditions approximately 8 to 10 hours after mixing at 37°C, indicating that approximately 3.5 mol of cholesterol was bound per mol of MSP1D1(-). Since each particle of the under-lipidized DMPC:MSP1D1(-)=51:1 particle composition contains two MSP1D1(-) proteins, this result indicates that approximately 7 mol of cholesterol was bound per mol of the under-lipidized DMPC:MSP1D1(-)=51:1 particle composition at equilibrium.

[0382] Example 9: Preparation of an exemplary fully esterified particulate composition

[0383] A fully esterified particulate composition assembled from phospholipids, MSP, and detergent was used as a control. See Sligar Lab, Nanodisc Technology: Protocols for Preparation of Nanodiscs (2008), University of Illinois at Urbana-Champaign, as an exemplary method, the entire contents of which are incorporated herein by reference.

[0384] In short, a frozen solution containing MSP in 20 mM glycerol (Tris) (pH 7.4), 0.1 M NaCl, 0.5 mM EDTA, and 0.01% NaN3 was thawed and filtered through a 0.22 micron filter. The concentration of MSP was determined spectrophotometrically using a molar extinction coefficient ε280 = 21,000 M. -1 cm -1 Measurement.

[0385] Phospholipid stock solutions were prepared in chloroform at 50 mM to 100 mM for long-term storage in 4 ml glass vials with Teflon-lined screw caps at -20 °C. The concentration of the stock solution was determined by phosphate analysis. The required amount of chloroform-lipid stock solution was aliquoted into disposable glass culture tubes, and the solvent was dried using a mild nitrogen stream in a fume hood. The tubes were rotated at an angle to obtain a thin film on the lower wall. Residual solvent was removed by placing the tubes in a vacuum desiccator under high vacuum for at least 4 hours.

[0386] Add a buffer containing detergent and sodium cholate to the tube to achieve a cholate to lipid ratio of 2:1. Vortex the tube, heat it in hot tap water, and sonicate it in an ultrasonic bath until the solution is clear and no lipid remains on the tube walls. Add MSP to the solution and incubate for at least 15 minutes.

[0387] To remove bile salts and initiate the self-assembly of the particulate composition, 0.5 g to 0.8 g of Amberlite XAD 2 beads (Sigma) were added per ml of the reconstitution mixture. The suspension was placed on a fixed-orbit shaker and incubated overnight. The optimal lipid-to-MSP ratios and incubation temperatures for various lipids, as well as the optimized incubation temperatures and minimum incubation times, are shown in Table 3.

[0388] Table 3

[0389]

[0390] Samples were extracted from Amberlite XAD 2 beads, filtered through a 0.22-micron filter, and fractionated in MSP standard buffer on a Superdex 200 increase 10 / 300 GL column (GE Healthcare) at a flow rate of 0.75 ml / min. Samples were filtered prior to injection, and fractions were collected every minute. Figure 18 An exemplary chromatogram of the reaction (MSP1D1+DMPC) is shown.

[0391] To construct exemplary particulate compositions, MSP1D1 was added to a sodium cholate lipid mixture at lipid-to-MSP ratios of 10:1, 32.5:1, 50:1, and 65:1 for POPC and 20:1, 30:1, 40:1, 50:1, 60:1, and 85:1 for DMPC. No lipids were added as a control (lipid-free). Figure 9A As shown, a 65:1 ratio of POPC to MSP1D1 produces a fully esterified (10 nm diameter) particulate composition. (As...) Figure 9B As shown, a fully esterified particulate composition is formed when the molar ratio of DMPC to MSP1D1 is 85:1.

[0392] Example 10: Exemplary particulate composition alters the ability of cholesterol to flow out of macrophages in vitro.

[0393] Using the method of Example 9, particle compositions containing different lipid concentrations of DMPC (lipid A) or POPC (lipid B) were formed with MSP1D1. J774 cells derived from the mouse macrophage cell line were plated and treated with 74 kBq per millimeter. 3 H-cholesterol was radiolabeled. ABCA1 was upregulated by incubation with 0.3 mM 8-(4-chlorophenylthio)-cAMP for 6 hours. Subsequently, the particulate composition and effluent medium were added and incubated for 4 hours.

[0394] All steps were performed in the presence of the acyl-CoA cholesterol acyltransferase inhibitor CP113,818 (2 μg / mL). Liquid scintillation counting was used to quantify the efflux of radioactive cholesterol from cells. The amount of radioactive cholesterol incorporated into cellular lipids was calculated by isopropanol extraction from control wells not exposed to the particle composition / culture medium. The percentage of cholesterol efflux was calculated using the following formula:

[0395]

[0396] All experiments were performed in duplicate and in accordance with publicly available literature (see Mehta, Nehal N et al. "Abnormal lipoprotein particles and cholesterol efflux capacity in patients with psoriasis." Atherosclerosis, Vol. 224, 1 (2012): 218-21. doi:10.1016 / j.atherosclerosis.2012.06.068, the full text of which is incorporated herein by reference).

[0397] like Figure 10 As shown, POPC (lipid B)-containing particle compositions with lipid to MSP1D1 ratios of 50:1 and 32.5:1 (both under-lipidized) exhibited a higher percentage of cholesterol efflux than fully lipidized POPC (lipid B) particle compositions with a lipid to MSP1D1 ratio of 65:1. Compared to fully lipidized DMPC-containing particle compositions with a lipid to MSP1D1 ratio of 85:1, POPC-containing particle compositions with lipid to MSP1D1 ratios from 32.5:1 to 65:1 all showed better cholesterol efflux properties. Furthermore, all tested particle compositions demonstrated at least approximately twice the cholesterol efflux achieved through the MSP1D1 protein itself.

[0398] like Figure 11 As shown, the cholesterol efflux percentage of an exemplary fully esterified particulate composition with a DMPC to MSP1D1 ratio of 85:1 is more than twice that of naturally occurring ApoA-I.

[0399] Example 11: Exemplary fully liposomed particulate composition desorbed from human retinal pigment epithelial cells in vitro Cholesterol

[0400] Fully esterified particulate compositions (MSP1D1 lipid B) with a POPC to MSP1D1 ratio of 65:1 and (MSP1D1 lipid A) with a DMPC to MSP1D1 ratio of 85:1 were formed using the method of Example 9. Cholesterol efflux was measured in human retinal pigment epithelial (RPE) cells using the method described in Example 10 to measure cholesterol efflux at 2, 4, and 6 hours of incubation. Passive efflux (without the addition of protein or particulate composition) and MSP1D1 protein alone were used for comparison.

[0401] Figures 12A to 12C It shows that after 2 hours of incubation ( Figure 12A ), 4 hours ( Figure 12B ) and 6 hours ( Figure 12CThe percentage of cholesterol efflux from RPE cells was then measured. These results collectively demonstrate that both fully lipoproteinized particle compositions with a POPC / MSP1D1 ratio of 65:1 and DMPC / MSP1D1 ratio of 85:1 continuously promoted cholesterol efflux from RPE cells for at least 6 hours, significantly greater than cholesterol efflux achieved by MSP1D1 protein alone. Furthermore, during the 2-4 hour incubation window, the fully lipoproteinized particle composition with a DMPC / MSP1D1 ratio of 85:1 appeared to efflux cholesterol faster than the fully lipoproteinized particle composition with a POPC / MSP1D1 ratio of 65:1, but the fully lipoproteinized particle composition with a POPC / MSP1D1 ratio of 65:1 caught up by 6 hours.

[0402] The under-lipidated particle compositions containing DMPC and MSP1D1 and the under-lipidated particle compositions containing POPC and MSP1D1 may be able to promote cholesterol efflux from RPE cells better than their fully lipidated forms.

[0403] Example 12: In vivo delivery of an exemplary fully liposomed particulate composition in mouse eyes

[0404] To determine the in vivo penetration and persistence of an exemplary fully lipotropic particulate composition in the eye, a fully lipotropic particulate composition comprising MSP1D1 and DMPC in a molar ratio of 1:85 was prepared, wherein MSP1D1 was linked to a green fluorescent label via cysteine ​​residues engineered into the MSP1D1 protein. The fully lipotropic particulate composition was prepared one day prior to the procedure and stored at 4°C.

[0405] Six-week-old C57Bl / 6J mice were anesthetized via intraperitoneal injection of a mixture of 87.6 mg / kg ketamine and 10 mg / kg toluenethiazide, and pain was managed with 0.5% promecaine hydrochloride eye drops. The prepared fully liposomed particulate composition was administered intravitreally at a dose of 7 μg per eye in 1.14 μl volume. A subset of mice were enlarged in the left eye with 1% tropicamide ophthalmic solution, followed by application of 2.5% Gonak hydroxypropyl methylcellulose palliative solution, and non-invasive fundus imaging was performed at various time points using a PhoenixMicron III imaging system. Figures 13A to 13BAt the time points shown, mice were euthanized and their eyes removed. The eyes were fixed in 10% neutral buffered formalin at room temperature (RT) for 2 hours, then transferred to graded sucrose (10%, then 30%) overnight. The fixed, cryopreserved eyes were mounted at the optimal cutting temperature (OCT) and slides were prepared by cutting sections (20 μM) on a Lieca 1850 cryostat. Cover slips were applied using a Vectasheild anti-fading mounting agent with Dapi to counterstain cell nuclei and avoid additional processing steps associated with other counterstaining methods. The prepared slides were imaged on an Olympus FV1000 confocal microscope using a system-optimized z-step for each corresponding objective. This allowed direct visualization of the fluorescently labeled, fully lipidized particle composition after successful cholesterol removal from the retina.

[0406] like Figures 13A to 13B As shown, the fully liposomed particulate composition was successfully delivered into the mouse eye via intravitreal injection, and the fully liposomed particulate composition was able to penetrate from the vitreous humor, reach the retinal space, enter the RPE, and enter the ocular muscles by 6 hours. By 24 hours, the fully liposomed particulate composition appeared to have left the eye. These results indicate that the exemplary fully liposomed particulate compositions have excellent permeability in vivo and do not remain in the eye for extended periods (>24 hours).

[0407] Insufficiently esterified particulate compositions can achieve similar effects to their corresponding fully esterified particulate compositions.

[0408] Example 13: In vivo safety and efficacy of an exemplary fully liposomed particulate composition in mouse eyes.

[0409] Further testing was conducted on the fully esterified DMPC:MSP1D1 = 85:1 (molar ratio) particle composition from the above embodiments to illustrate its in vivo safety and efficacy in the eye. MSP1D1 is linked to a green fluorescent label via cysteine ​​residues engineered into the MSP1D1 protein.

[0410] An exemplary, fully lipid-modified particulate composition labeled with fluorescence was delivered intravitreally into the eyes of anesthetized mice, and the diffusion path and rate of the particulate composition within the eye were visualized by imaging post-mortem retinal tissue over a 24-hour timeframe following injection (Figure 14A). As shown in Figure 14B, the fluorescence signal was detectable within 15 minutes of injection and diffused rapidly across the retina via a vitreous-to-RPE (retinal pigment epithelium) gradient, indicating that the particulate composition could be delivered into the eye and efficiently transported to the target tissue via the retina. These results collectively demonstrate that the exemplary particulate composition can be distributed to the RPE.

[0411] Next, a “wet” AMD mouse model with laser-induced Bruch’s membrane injury to induce choroidal neovascularization (CNV) was used. The exemplary fully liposomed particulate composition was injected intravitreally in conjunction with laser injury or 3 days after laser injury (“+d3”) to examine the resulting CNV area size (Fig. 15A). Importantly, the mean CNV area was similar between control and mice treated with the exemplary particulate composition, indicating that the exemplary particulate composition did not exacerbate the prominent features of AMD in mice (Figs. 15B–15C). These studies are crucial because they demonstrate that the exemplary fully liposomed particulate composition can be efficiently delivered to the retina without exacerbating the features of neovascular AMD in mice, unlike currently approved complement inhibitors for the treatment of geographic atrophy (GA).

[0412] To determine whether the exemplary fully lipotropic particulate composition could reduce lipid load in the eye, transgenic mouse species were used, allowing for the specific loss of the Abca1 and Abcg1 cholesterol efflux transporters in rod photoreceptors (Abca1 / g1-rod / -rod), resulting in lipid accumulation in the RPE and Bruch's membrane, as well as retinal neurodegeneration similar to that seen in patients with early to mid-stage dry AMD. To accelerate lipid accumulation, mice were fed a high-fat diet during the study period. As shown in Figure 16A, for the first mouse cohort (Study 1), baseline (T0) optical coherence tomography (OCT) fundus images of the mouse retina were collected prior to high-fat diet induction and intravitreal delivery or blank injection of the exemplary fully lipotropic particulate composition (“discs” in Figures 16B–16C) (“mediums” in Figures 16B–16C). A second intravitreal injection was administered 3 weeks later. Six weeks after exposure to a high-fat diet, follow-up OCT fundus imaging (T1) and blood sample collection were performed, followed by electroretinal spectroscopy (ERG) visual function testing and tissue harvesting. First, full-field scotopic vision and mild bleaching recovery ERG diagnostics, measuring photoreceptor and RPE function separately, showed no difference between mice injected with the mediator and those injected with the exemplary particulate composition (“disc”), demonstrating the safety of the exemplary fully lipotropic particulate composition for retinal electrical signal transduction (Figures 16B–16C). Further confirmation of safety was provided by examination of retinal histology, where differences in retinal inflammation or retinal integrity were identical between mouse groups. Figure 16D Additionally, immunostaining of retinal sections revealed that complement 3 (C3) staining was detectable in the mouse retina, but similar expression was observed in mice treated with the medium and mice treated with the exemplary particulate composition (“disc”), indicating that the exemplary fully lipidized particulate composition did not exacerbate the expected complement signaling in this dry AMD model. Figure 16ERPE rupture and hyperreflectivity, ellipsoidal region abnormalities, inner retinal deposition, and hyperreflective lesions are the main anatomical changes associated with AMD progression, identified using non-invasive imaging. Therefore, we examined follow-up (T1) optical coherence tomography (OCT) images for potential structural changes induced by genetic mutations and a high-fat diet. As expected, after 6 weeks of a high-fat diet absent from baseline T0 images, a high incidence of various inner segment / outer segment (IS / OS) junction abnormalities and RPE disruption (including pigment epithelial detachment (PED)) was found in images of mice injected with the vector. Figure 16F However, these abnormalities were rarely detected or absent in mouse eyes treated with the exemplary particulate composition (“disc”), indicating improved characterization of AMD during OCT imaging.

[0413] To replicate this study and to further emphasize the high-fat diet-induced phenotype, a second cohort of mice on a 6-week high-fat diet (Study 2) was tested, but the exemplary fully liposomed particulate composition (“disc”) or mediator was not injected until 2 and 4 weeks after diet induction (Fig. 17A). At 6 weeks, visual function was reassessed, and no differences were found in full-field dark vision and bright bleach recovery responses between the mediator-treated group and the group treated with the exemplary particulate composition (“disc”), confirming the safety of the fully liposomed particulate composition (Figs. 17B–17C). Analysis of follow-up OCT imaging revealed more lesions in the IS / OS junction and RPE in the mediator-treated mice, but far fewer lesions were observed in the mice injected with the exemplary fully liposomed particulate composition. Furthermore, a greater number of lesions were observed in both groups compared to the first study, indicating that exposing Abca1 / g1-rod / -rod mice to a high-fat diet for 2 weeks prior to the initial intravitreal injection worsened the disease (Table 4 below summarizes the cumulative incidence of retinopathy in mice treated with the medium and mice treated with the exemplary particulate composition via OCT imaging across the two studies). Nevertheless, treatment of mice with the exemplary fully lipidated particulate composition resulted in a nearly four-fold reduction in retinopathy in both studies compared to the medium.

[0414] Table 4 Cumulative incidence of retinal diseases

[0415]

[0416] Finally, lipidomics analysis was performed on retinal samples to examine potential changes in individual lipid species in mouse eyes after treatment with the exemplary fully lipidated particulate composition. A reduction in cholesterol ester (CE) composition (a major component of AMD causing drusen) was found in the retinas of mice treated with the particulate composition (“disc”) (Figure 17D), indicating that this treatment reduces cholesterol load in diseased mice characteristic of AMD. Furthermore, this reduction in cholesterol esters (a major lipid species found in drusen), along with the improvement in OCT lesions, provides strong support for the mechanism of action and scientific prospects of the particulate composition presented herein.

[0417] The under-esterified particulate compositions presented in this article can achieve similar or even better results than their corresponding fully esterified particulate compositions.

Claims

1. A composition comprising a plurality of particles containing an amphiphilic polymer and phospholipids, wherein at least about 85% of the plurality of particles are under-esterified.

2. The composition according to claim 1, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than the phospholipid-polymer saturation ratio N. S .

3. The composition according to claim 2, wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than the phospholipid-polymer saturation ratio N. S Approximately 95%.

4. The composition according to any one of claims 1 to 3, (i) wherein the amphiphilic polymer is an amphiphilic polypeptide selected from the group consisting of apolipoprotein AI (ApoA-I), ApoA-II, ApoC, ApoE, and their variants; and / or (ii) wherein the phospholipid comprises DMPC, DPPC, DMPS, POPC, POPS or PiP2.

5. The composition according to claim 4, wherein the amphiphilic polypeptide is selected from the group consisting of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, MSP1D1(-), MSP1E1D1(-), MSP1E2D1(-), MSP1E3D1(-), MSP2N2(-), and variants thereof.

6. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1D1, MSP1D1(-) or a variant thereof, the phospholipid is DMPC, and the N... S It is approximately 81.

7. The composition according to claim 6, wherein the molar ratio of DMPC to the amphiphilic polypeptide in the composition is (i) less than about 78:1; (ii) about 65:1 to about 75:1; (iii) about 40:1 to about 60:1; or (iv) about 46:1 to about 55:

1.

8. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1D1, MSP1D1(-) or a variant thereof, the phospholipid is DPPC, and the N... S It is approximately 81.

9. The composition according to claim 8, wherein the molar ratio of DPPC to the amphiphilic polypeptide in the composition is less than about 75:

1.

10. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1D1, MSP1D1(-) or a variant thereof, the phospholipid is POPC, and the N... S It is approximately 62.

11. The composition of claim 10, wherein the molar ratio of POPC to the amphiphilic polypeptide in the composition is less than about 58:

1.

12. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1E1D1, MSP1E1D1(-) or a variant thereof, the phospholipid is DMPC, and the N... S It is approximately 107.

13. The composition according to claim 12, wherein the molar ratio of DMPC to the amphiphilic polypeptide in the composition is (i) less than about 100:1; and / or (ii) about 60:1 to about 65:

1.

14. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1E1D1, MSP1E1D1(-) or a variant thereof, the phospholipid is DPPC, and the N... S It is approximately 107.

15. The composition of claim 14, wherein the molar ratio of DPPC to the amphiphilic polypeptide in the composition is less than about 100:

1.

16. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1E1D1, MSP1E1D1(-) or a variant thereof, the phospholipid is POPC, and the N... S It is approximately 80.

17. The composition of claim 16, wherein the molar ratio of POPC to the amphiphilic polypeptide in the composition is less than about 76:

1.

18. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1E2D1, MSP1E2D1(-) or a variant thereof, the phospholipid is DMPC, and the N... S It is approximately 135.

19. The composition of claim 18, wherein the molar ratio of DMPC to the amphiphilic polypeptide in the composition is less than about 128:

1.

20. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1E2D1, MSP1E2D1(-) or a variant thereof, the phospholipid is DPPC, and the N... S It is approximately 135.

21. The composition of claim 20, wherein the molar ratio of DPPC to the amphiphilic polypeptide in the composition is less than about 128:

1.

22. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1E2D1, MSP1E2D1(-) or a variant thereof, the phospholipid is POPC, and the N... S It is approximately 102.

23. The composition of claim 22, wherein the molar ratio of POPC to the amphiphilic polypeptide in the composition is less than about 96:

1.

24. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1E3D1, MSP1E3D1(-) or a variant thereof, the phospholipid is DMPC, and the N... S It is approximately 167.

25. The composition of claim 24, wherein the molar ratio of DMPC to the amphiphilic polypeptide in the composition is (i) less than about 158:1; (ii) about 50:1 to about 100:1; (iii) about 87:1 to about 98:1; (iv) about 87:1 to about 96:1; (v) about 58:1 to about 68:1; (vi) about 63:1; or (vii) about 62:

1.

26. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1E3D1, MSP1E3D1(-) or a variant thereof, the phospholipid is DPPC, and the N... S It is approximately 167.

27. The composition of claim 26, wherein the molar ratio of DPPC to the amphiphilic polypeptide in the composition is less than about 158:

1.

28. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP1E3D1, MSP1E3D1(-) or a variant thereof, the phospholipid is POPC, and the N... S It is approximately 126.

29. The composition of claim 28, wherein the molar ratio of POPC to the amphiphilic polypeptide in the composition is less than about 118:

1.

30. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP2N2, MSP2N2(-) or a variant thereof, the phospholipid is DMPC, and the N... S It is approximately 387.

31. The composition of claim 30, wherein the molar ratio of DMPC to the amphiphilic polypeptide in the composition is less than about 367:

1.

32. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP2N2, MSP2N2(-) or a variant thereof, the phospholipid is DPPC, and the N... S It is approximately 387.

33. The composition of claim 32, wherein the molar ratio of DPPC to the amphiphilic polypeptide in the composition is less than about 367:

1.

34. The composition according to claim 5, wherein the amphiphilic polypeptide is MSP2N2, MSP2N2(-) or a variant thereof, the phospholipid is POPC, and the N... S It is approximately 291.

35. The composition of claim 34, wherein the molar ratio of POPC to the amphiphilic polypeptide in the composition is less than about 276:

1.

36. The composition according to any one of claims 1 to 35, (i) wherein the average size of the particles in the composition is about 2 nm to about 20 nm in diameter; (ii) wherein the polydispersity index of the particle size in the composition is less than about 0.2; (iii) wherein the polydispersity index of the phospholipid content of the particles in the composition is less than about 0.2; and / or (iv) The composition thereon is a pharmaceutical composition.

37. A method for preparing a composition comprising a plurality of particles containing an amphiphilic polymer and a phospholipid, wherein at least about 85% of said plurality of particles are under-esterified, the method comprising: 1) Incubate the amphiphilic polymer and the phospholipid to obtain a mixture, wherein the phospholipid is dissolved in a detergent or organic compound prior to the incubation; as well as (2) Remove the detergent or organic compound from the prepared mixture to obtain the particles.

38. The method according to claim 37, (i) wherein the molar ratio of the phospholipid to the amphiphilic polymer in the composition is less than the phospholipid-polymer saturation ratio N. S ; (ii) wherein the molar ratio of the phospholipid to the amphiphilic polymer in the preparation mixture is less than or equal to the molar ratio of the phospholipid to the amphiphilic polymer in the composition; (iii) The method further includes separating the particles; and / or (iv) The detergent or organic compound contained in the form of bile salts.

39. The method according to claim 37 or 38, (i) wherein the amphiphilic polymer is an amphiphilic polypeptide selected from the group consisting of apolipoprotein AI (ApoA-I), ApoA-II, ApoC, ApoE, and their variants; and / or (ii) wherein the phospholipid comprises DMPC, DPPC, DMPS, POPC, POPS or PiP2.

40. The method of claim 39, wherein the amphiphilic polypeptide is selected from the group consisting of MSP1D1, MSPE1D1, MSP1E2D1, MSP1E3D1, MSP2N2, MSP1D1(-), MSP1E1D1(-), MSP1E2D1(-), MSP1E3D1(-), MSP2N2(-), and variants thereof.

41. The method of claim 40, wherein the amphiphilic polypeptide is MSP1D1, MSP1D1(-) or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphiphilic polypeptide in the composition is about 65:1 to about 75:1; and wherein the molar ratio of DMPC to the amphiphilic polypeptide in the preparation mixture is (i) less than about 65:1; and / or (ii) about 55:

1.

42. The method of claim 40, wherein the amphiphilic polypeptide is MSP1D1, MSP1D1(-) or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphiphilic polypeptide in the composition is from about 46:1 to about 55:1; and wherein the molar ratio of DMPC to the amphiphilic polypeptide in the preparation mixture is (i) less than about 45:1; (ii) about 40:1; or (iii) about 30:

1.

43. The method of claim 40, wherein the amphiphilic polypeptide is MSP1E3D1, MSP1E3D1(-) or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphiphilic polypeptide in the composition is from about 85:1 to about 100:1; and wherein the molar ratio of DMPC to the amphiphilic polypeptide in the preparation mixture is less than or equal to about 90:

1.

44. The method of claim 43, wherein the molar ratio of DMPC to the amphiphilic polypeptide in the composition is from about 87:1 to about 98:1, and the molar ratio of DMPC to the amphiphilic polypeptide in the preparation mixture is from about 90:

1.

45. The method of claim 40, wherein the amphiphilic polypeptide is MSP1E3D1, MSP1E3D1(-) or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphiphilic polypeptide in the composition is from about 55:1 to about 80:1; and wherein the molar ratio of DMPC to the amphiphilic polypeptide in the preparation mixture is less than or equal to about 60:

1.

46. ​​The method according to claim 45, (i) wherein the molar ratio of DMPC to the amphiphilic peptide in the composition is about 58:1 to about 68:1, or about 63:1, and the molar ratio of DMPC to the amphiphilic peptide in the preparation mixture is about 60:1; or (ii) wherein the molar ratio of DMPC to the amphiphilic peptide in the composition is about 58:1 to about 68:1, or about 62:1, and the molar ratio of DMPC to the amphiphilic peptide in the preparation mixture is about 50:

1.

47. A composition comprising the particles prepared by the method according to any one of claims 37 to 46.

48. A method for treating or preventing a disease associated with lipid accumulation in an individual, the method comprising administering to the individual an effective amount of the composition according to any one of claims 1 to 36 and 47.

49. The method of claim 48, wherein the disease is associated with lipid accumulation in the eye.

50. The method of claim 48 or 49, wherein the disease is age-related macular degeneration (AMD), photoreceptor neurodegeneration, optic atrophy, vision loss, hemianopsia, visual agnosia, strabismus, retinal neurovascular disease, lipid corneal disease, corneal lipid deposition, or any combination thereof.

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