Nanodiscs for use in removing lipid accumulation in the eye

Abstract

The present disclosure generally relates to compositions comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, methods of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, and uses of such compositions.

Description

PARTICLE COMPOSITIONS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefits of, and priority to 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 each of which are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (283912000240seqlist.xml; Size: 20,955 bytes; and Date of Creation: December 9, 2024) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure generally relates to compositions comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, methods of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, and uses of such a composition.BACKGROUND

[0004] Particles comprising an amphipathic polymer, for example an amphipathic polypeptide or membrane scaffold protein (MSP), and a phospholipid, provide a native-like lipid environment for the incorporation of membrane proteins, and they have become a valuable platform for the study of membrane biophysics and for therapeutical applications. However, most of these MSP / lipid compositions that have satisfying stability and processability are generated to form a phospholipid bilayer with a relatively high lipid content, namely, the MSP / lipid ratio leading to particles that are typically fully-lipidated.BRIEF SUMMARY OF THE INVENTION

[0005] In one aspect, provided herein is a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated.

[0006] In some embodiments according to the compositions described above, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than (e.g., less than about 95% of) a phospholipid-polymer saturation ratio Ns.

[0007] In some embodiments according to any of the compositions described above, the amphipathic polymer is an amphipathic polypeptide. In some embodiments, the amphipathic polypeptide comprises one or more amphipathic helix sequences of about 9 to about 30 amino acids. In some embodiments, the amphipathic polypeptide is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, and a variant thereof, such as a variant of ApoA-I. In some embodiments, the amphipathic polypeptide comprises one or more amphipathic helix sequences derived from ApoA-I. In some embodiments, the amphipathic polypeptide comprises an N-terminus truncation relative to ApoA-I. In some embodiments, the amphipathic polypeptide is selected from the group consisting of MSP ID 1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and a variant thereof. In some embodiments, the amphipathic polypeptide is a variant of any of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP2N2, wherein the variant does not comprise the N- terminal His-tag portion (his-tag removed variant) (hereinafter also referred to as MSPIDI(-), MSPIEIDI(-), MSP1E2D10, MSP1E3D10, and MSP2N20, respectively). In some embodiments, the amphipathic polypeptide is selected from the group consisting of MSPIDI(-), MSPIEIDI(-), MSP1E2D10, MSP1E3D10, MSP2N20, and a variant thereof. In some embodiments, the amphipathic polypeptide is (i) an MSP ID 1 His-tag removed variant (i.e., MSP1D10) comprising the amino acid sequence of SEQ ID NO: 11; (ii) an MSP1E3D1 His-tag removed variant (i.e., MSP1E3D10) comprising the amino acid sequence of SEQ ID NO: 12; (iii) an MSP1E1D1 His-tag removed variant (i.e., MSP1E1D1(- )) comprising the amino acid sequence of SEQ ID NO: 17; (iv) an MSP1E2D1 His-tag removed variant (i.e., MSP1E2D10) comprising the amino acid sequence of SEQ ID NO: 18; or (v) an MSP2N2 His-tag removed variant (i.e., MSP2N20) comprising the amino acid sequence of SEQ ID NO: 19.

[0008] In some embodiments according to any of the compositions described above, the phospholipid comprises one or more acyl chains and a glycol backbone. In some embodiments, the one or more acyl chains has 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 according to any of the compositions described above, the amphipathic polymer is MSP ID 1, MSPIDI(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 81. In some embodiments, the molar ratio of DMPC to the amphipathic polymer in the composition is less than about 78: 1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer in the composition is from about 65: 1 to about 75: 1, or from about 70: 1 to about 75: 1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer 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, or about 55: 1.

[0010] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP ID 1, MSPIDI(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 81. In some embodiments, the molar ratio of DPPC to the amphipathic polymer in the composition is less than about 75: 1.

[0011] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP ID 1, MSPIDI(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 62. In some embodiments, the molar ratio of POPC to the amphipathic polymer in the composition is less than about 58: 1.

[0012] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP1E1D1, MSPIEIDI(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 107. In some embodiments, the molar ratio of DMPC to the amphipathic polymer (e.g., MSP1E1D1 or His-tag removed variant thereof) in the composition is less than about 100: 1, such as from about 60:1 to about 65: 1.

[0013] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP1E1D1, MSPIEIDI(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 107. In some embodiments, the molar ratio of DPPC to the amphipathic polymer (e.g., MSP1E1D1) in the composition is less than about 100: 1.

[0014] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP1E1D1, MSPIEIDI(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 80. In some embodiments, the molar ratio of POPC to the amphipathic polymer (e.g., MSP1E1D1 or His-tag removed variant thereof) in the composition is less than about 76: 1.

[0015] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP1E2D1, MSP1E2D1(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 135. In some embodiments, the molar ratio of DMPC to theamphipathic polymer (e.g., MSP1E2D1 or His-tag removed variant thereof) in the composition is less than about 128: 1.

[0016] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP1E2D1, MSP1E2D1(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 135. In some embodiments, the molar ratio of DPPC to the amphipathic polymer (e.g., MSP1E2D1 or His-tag removed variant thereof) in the composition is less than about 128: 1.

[0017] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP1E2D1, MSP1E2D1(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 102. In some embodiments, the molar ratio of POPC to the amphipathic polymer (e.g., MSP1E2D1 or His-tag removed variant thereof) in the composition is less than about 96: 1.

[0018] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP1E3D1, MSP1E3D1(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 167. In some embodiments, the molar ratio of DMPC to the amphipathic polymer (e.g., MSP1E3D1 or His-tag removed variant thereof) in the composition is less than about 158: 1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer (e.g., MSP1E3D1 or His-tag removed variant thereof) 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, about 63: 1, or about 62: 1.

[0019] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP1E3D1, MSP1E3D1(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 167. In some embodiments, the molar ratio of DPPC to the amphipathic polymer (e.g., MSP1E3D1 or His-tag removed variant thereof) in the composition is less than about 158: 1.

[0020] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP1E3D1, MSP1E3D1(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 126. In some embodiments, the molar ratio of POPC to the amphipathic polymer (e.g., MSP1E3D1 or His-tag removed variant thereof) in the composition is less than about 118: 1.

[0021] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP2N2, MSP2N2(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 387. In some embodiments, the molar ratio of DMPC to the amphipathic polymer in the composition is less than about 367: 1.

[0022] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP2N2, MSP2N2(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 387. In some embodiments, the molar ratio of DPPC to the amphipathic polymer in the composition is less than about 367: 1.

[0023] In some embodiments according to any of the compositions described above, the amphipathic polymer is MSP2N2, MSP2N2(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 291. In some embodiments, the molar ratio of POPC to the amphipathic polymer in the composition is less than about 276: 1.

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

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

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

[0027] In another aspect, provided herein is a method of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated, the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and 2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles.

[0028] In some embodiments according to the preparation methods described above, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a polymer-phospholipid saturation ratio Ns.

[0029] In some embodiments according to any of the preparation methods described above, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than or equal to the molar ratio of the phospholipid to the amphipathic polymer in the composition. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than about 95% of the molar ratio of the phospholipid to the amphipathic polymer in the composition.

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

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

[0032] In some embodiments according to any of the preparation methods described above, the concentration of the amphipathic 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 according to any of the preparation methods described above, the method further comprises isolating the particles. In some embodiments, the particles are isolated by chromatography.

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

[0035] In some embodiments according to any of the preparation methods described above, the amphipathic polymer is an amphipathic polypeptide. In some embodiments, the amphipathic polypeptide comprises one or more amphipathic helix sequences of about 9 to about 30 amino acids. In some embodiments, the amphipathic polypeptide is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, and a variant thereof, such as a variant of ApoA-I. In some embodiments, the amphipathic polypeptide comprises one or more amphipathic helix sequences derived from ApoA-I. In some embodiments, the amphipathic polypeptide comprises an N-terminus truncation relative to ApoA-I. In some embodiments, the amphipathic polypeptide is selected from the group consisting of MSP1D1, MSPE1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and a variant thereof. In some embodiments, the amphipathic polypeptide is a variant of any of MSP ID 1, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP2N2, wherein the variant does not comprise the N-terminal His-tag portion (his-tag removed variant). In some embodiments, the amphipathic polypeptide is selected from the group consisting of MSPIDI(-), MSPIEIDI(-), MSP1E2D1(-), MSP1E3D1(-), MSP2N2(-), and a variant thereof. In some embodiments, the amphipathic polypeptide is (i) an MSP1D1 His-tag removed variant (i.e., MSP1D1(-)) comprising the amino acid sequence of SEQ ID NO: 11; (ii) an MSP1E3D1 His-tag removed variant (i.e., MSP1E3D1(-)) comprising the amino acid sequence of SEQ ID NO: 12; (iii) an MSP1E1D1 His-tag removed variant (i.e., MSPIEIDI(-)) comprising the amino acid sequence of SEQ ID NO: 17; (iv) an MSP1E2D1 His-tag removed variant (i.e., MSP1E2D1(-)) comprising the amino acid sequence of SEQ ID NO: 18; or (v) an MSP2N2 His-tag removed variant (i.e., MSP2N2(-)) comprising the amino acid sequence of SEQ ID NO: 19.

[0036] In some embodiments according to any of the preparation methods described above, the phospholipid comprises one or more acyl chains, such as with lengths 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 preparation methods described above, the amphipathic polymer is MSP1D1, MSPIDI(-), or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphipathic polymer (e.g., MSP ID 1 or His-tag 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 amphipathic polymer (e.g., MSP ID 1 or His- tag removed variant thereof) in the preparation mixture is less than about 65 : 1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer (e.g., MSP ID 1 or His- tag 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 amphipathic polymer (e.g., MSP1D1 or His-tag removed variant thereof) in the preparation mixture is about 55: 1.

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

[0039] In some embodiments according to any of the preparation methods described above, the amphipathic polymer is MSP1E3D1, MSP1E3D1(-), or a variant thereof, the phospholipid is DMPC, and molar ratio of DMPC to the amphipathic polymer (e.g., MSP1E3D1 or His-tag 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 amphipathic polymer (e.g., MSP1E3D1 or His-tag removed variant thereof) in the preparation mixture is less thanor equal to about 90: 1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer (e.g., MSP1E3D1 or His-tag 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 amphipathic polymer (e.g., MSP1E3D1 or His-tag removed variant thereof) in the preparation mixture is about 90: 1.

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

[0041] In another aspect, provided herein is a composition comprising the particles prepared according to any of the preparation methods described above.

[0042] In another aspect, provided herein is a method of treating or preventing a disease associated with lipid accumulation in an individual (e.g., human), comprising administering an effective amount of any of the compositions described above 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 nerve atrophy, loss of acuity, hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, or any combination thereof. In some embodiments, the disease is AMD, such as dry AMD. In some embodiments, the dry AMD is geographic atrophy (GA). In some embodiments, the composition reduces lipid (e.g., cholesterol) or prevent lipid accumulation in the individual by at least about 5%.BRIEF DESCRIPTION OF THE DRAWINGS

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

[0044] FIG. 1A shows one chromatogram of preparative size exclusion chromatography (SEC) of the exemplary DMPC / MSP1D1(-) particle composition prepared at 30: 1 ratio. (-) denotes the removal of the His-tag from the MSP IDE The dark-grey shaded area of the peak (upper panel) corresponding to an under-lipidated particle composition containing approximately 51 lipids per MSPIDI(-) (lower panel). FIG. IB shows one chromatogram of preparative SEC of an exemplary DMPC / MSP1E3D1(-) particle composition prepared at 50: 1 ratio. A single highlighted peak (upper panel) was collected, which corresponds to an under-lipidated particle composition with about 62 lipids per MSP1E3D1(-).

[0045] FIG. 2 shows gas chromatography (GC) result for determining phospholipid to MSP ratio of the exemplary DMPC / MSP1D1(-) particle composition prepared at 30: 1 ratio.

[0046] FIG. 3A shows the cholesterol efflux assay results using various under-lipidated particle compositions. The lipid to MSP ratios were measured from the final particle compositions (not preparation mixture). 2.5 pg of samples were used. Controls include a fully-lipidated particle composition with DMPC:MSP1D1(-)=81 : 1, a fully-lipidated particle composition with DMPC:MSP1D1=83: 1, lipid-free MSPIDI(-), lipid-free Apo- Al , lipid- free HDL, positive control (from the assay kit), and a PBS buffer blank (not shown). FIG. 3B shows cholesterol efflux dose dependence of a fully-lipidated particle composition DMPC:MSP1D1(-)=81 : 1 and an exemplary under-lipidated particle composition DMPC:MSP1D1(-)=51 : 1 at 2.5 pg and 10 pg doses. (-) denotes the removal of the N- terminal His-tag.

[0047] FIG. 4 shows the SEC chromatogram of an exemplary under-lipidated DMPC / MSP1D1(-) particle composition prepared at 30: 1 ratio (final composition ratio was about 51 : 1) after freezing and thawing without cryoprotectant, compared to before-freezing.

[0048] FIG. 5 shows the cholesterol efflux results of an exemplary under-lipidated DMPC:MSP1D1(-) particle composition prepared at 30: 1 ratio (final composition ratio was about 51 : 1) before and after freezing and thawing, in comparison with controls including a fully-lipidated DMPC:MSP1D1 particle composition prepared at 85: 1 (final composition ratio was 83: 1), HDL, and PBS solution.

[0049] FIGs. 6A-6D show the correspondence between the phospholipid / MSPlDl(-) ratio in the preparation mixture and the final particle composition for DMPC / MSP1D1(-) about 81:1 particle composition (preparation mixture 80:1; final composition 81±3 : 1) (FIG. 6A), DMPC / MSP1D1(-) particle composition about 75:1 (preparation mixture 55:1; final composition 75±1.5 :1) (FIG. 6B), DMPC / MSP1D1(-) about 51:1 particle composition (preparation mixture 40:1; final composition 51±1 :1) (FIG. 6C), and DMPC / MSP1D1(-) about 51:1 particle composition (preparation mixture 30:1; final composition 51±1 :1) (FIG.6D).

[0050] FIGs. 7A-7C show the correspondence between the phospholipid / MSPlE3Dl(-) ratio in the preparation mixture and the final particle composition for DMPC / MSP1E3D1(-) about 94:1 particle composition (preparation mixture 90:1; final composition 94±4 :1) (FIG. 7A), DMPC / MSP1E3D1(-) about 63:1 particle composition (preparation mixture 60:1; final composition 63±4 :1) (FIG. 7B), and DMPC / MSP1E3D1(-) about 62:1 particle composition (preparation mixture 50:1; final composition 62±0.5 :1) particle composition (FIG. 7C).

[0051] FIG. 8 shows cholesterol: MSPIDI(-) protein ratio as a function of time, which was tested by mixing an under-lipidated DMPC:MSP1D1(-)=51:1 particle composition and LUVs containing 70% POPC: 30% cholesterol.

[0052] FIG. 9A displays a chromatogram of the formation of POPC MSP ID 1 particle composition at different POPC to MSP ID 1 ratios. The results indicate that a ratio of 65: 1 forms a fully-lipidated particle composition. FIG. 9B displays a chromatogram of the formation of DMPC MSP1D1 particle composition at different DMPC to MSP1D1 ratios. The results indicate that fully-lipidated particle composition form at 85:1 DMPC to MSP1D1 ratio.

[0053] FIG. 10 shows the percent of cholesterol efflux from macrophages in vitro using various particle compositions with different lipid to MSP ratios. Lipid A refers to DMPC, and Lipid B refers to POPC.

[0054] FIG. 11 shows that a fully-lipidated DMPC MSP ID 1 particle composition with a lipid to MSP ratio of 85:1 has the ability to remove almost two times more cholesterol from macrophages than naturally occurring ApoA-I after 4 hours of incubation.

[0055] FIGs. 12A-12C shows the percent of cholesterol efflux from human retinal pigment epithelial (RPE) cells in vitro using a fully-lipidated particle composition with POPC to MSP1D1 ratio of 65:1 (MSP1D1 Lipid B), a fully-lipidated particle composition with DMPC to MSP1D1 ratio of 85: 1 (MSP1D1 Lipid A), and a MSP1D1 protein alone after 2 hours (FIG. 12A), 4 hours (FIG. 12B), and 6 hours (FIG. 12C) of incubation.

[0056] FIGs. 13A-13B show fluorescence images following intravitreal delivery of a GFP- labeled exemplary fully-lipidated particle composition into the murine eye in vivo. From left to right, FIG. 13A shows fluorescence images at 15 minutes, 30 minutes, 60 minutes, 6 hours, and 24 hours after delivery of a GFP-labeled exemplary fully-lipidated particle composition into the murine eye in vivo. The first row in FIG. 13B, from left to right, shows fluorescence images at 7 minutes, 7 minutes, 10 minutes, 15 minutes, and 20 minutes after delivery of a GFP-labeled exemplary fully-lipidated particle composition into the murine eye. The second row in FIG. 13B, from left to right, shows fluorescence images at 30 minutes, 30 minutes, 33 minutes, and 47 minutes after delivery of a GFP-labeled exemplary fully- lipidated particle composition into the murine eye. The third row in FIG. 13B, from left to right, shows fluorescence images at 6 hours, 6 hours, and 24 hours after delivery of a GFP- labeled exemplary fully-lipidated particle composition into the murine eye.

[0057] FIG. 14A is a schematic depicting schedule for intravitreal injection of fluorescent labeled exemplary fully-lipidated particle composition and tissue harvest. FIG. 14B shows representative images of immune-stained retina sections taken from injected mice depicting retinal neurons (solid arrows) and fluorescent labeled exemplary fully-lipidated particle composition (blank arrows). FIG. 14B, from left to right, shows fluorescence images at 0 minutes, 15 minutes, 30 minutes, 1 hour, 6 hours and 24 hours after delivery of a fluorescent- labelled exemplary fully-lipidated particle composition into the murine eye. In FIG. 14B, from top to bottom, the images show the fluorescence signals at retinal pigment epithelium (RPE), outer nuclear layer (ONL), and inner nuclear layer (INL).

[0058] FIG. 15A is a schematic depicting schedule for laser injury -induced choroidal neovascularization (CNV), intravitreal injection of the exemplary fully-lipidated particle composition, intravenous injection of FITC Dextran, and tissue harvest. FIG. 15B shows representative confocal image of CNV area labeled by FITC Dextran and outlined in a circle following laser injury in the mouse. FIG. 15C shows dot plots showing CNV area in mice that received 0 (‘Cntl’), 1 (‘particle composition), or 2 (‘particle composition + d3) intravitreal injections of the exemplary fully-lipidated particle composition. Data (n=8-10 mice per group) are presented as a mean (±SEM).

[0059] FIG. 16A shows a schematic depicting schedule for high-fat diet administration, intravitreal injections, baseline (TO) and follow-up (Tl) imaging and tissue harvest for Abcal / gl-rod / -rod mice. FIGs. 16B-16C show line graphs showing full-field scotopic (FIG. 16B) and light bleach recovery (FIG. 16C) response amplitudes and ratios in vehicle (‘Vehicle’) and exemplary particle composition-treated (‘Disc’) mouse groups. Data(Vehicle, n=3; Disc, n=4) are presented as mean (±SEM). FIG. 16D shows representative images of hematoxylin and eosin-stained (H&E) sections of whole eyes (left) and retinas (right). FIG. 16E shows representative images of immune-stained retina sections showing retinal neuron (solid arrows) and complement-3 (blank arrows) expression in the central (left) and mid-peripheral (right) retina. FIG. 16F shows representative baseline (top) and follow-up (bottom) optical coherence tomography (OCT) images of living mouse retina cross section showing incidence of (Vehicle) and absence of (Disc) inner / outer segment abnormalities (arrowhead in the bottom left image) and retinal pigment epithelium detachments (arrowhead in the bottom right image) in reference to baseline (asterisk and arrowhead in the top images).

[0060] FIG. 17A shows a schematic depicting schedule for high-fat diet administration, intravitreal injections, baseline (TO) and follow-up (Tl) imaging and blood collection, and tissue harvest for Abcal / gl-rod / -rod mice. FIGs. 17B-17C show line graphs showing fullfield scotopic (FIG. 17B) and light bleach recovery (FIG. 17C) response amplitudes and ratios in vehicle (‘Vehicle’) and the exemplary particle composition-treated (‘Disc’) mouse groups. The y-axis shows Amplitude (pV). Data (Vehicle, n=3; Disc, n=4) are presented as mean (±SEM). FIG. 17D shows box plots showing cholesteryl ester (CE) proportions of retinal lipids measured in Vehicle- and particle composition- treated mice. Data (Vehicle, n=3; Disc, n=4) is presented as a median with interquartile range, minimum, and maximum values indicated.

[0061] FIG. 18 displays an exemplary chromatogram of a fully-lipidated particle composition preparation.DETAILED DESCRIPTION

[0062] The present invention provides compositions comprising a plurality of particles comprising an amphipathic polymer (e.g., membrane scaffold protein (MSP)) and a phospholipid, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated, z.e., the particle comprises less phospholipids than those required to saturate the particle. Such compositions are hereinafter also referred to as “under-lipidated particle compositions.” Preparation methods of such under-lipidated particle compositions are also provided.

[0063] Particle compositions comprising MSP and lipid are normally generated with a relatively high lipid content to satisfy stability and processability. Such particle compositions are hereinafter referred to as “fully-lipidated particle compositions,” z.e., the particle comprises the amount of phospholipids required to saturate the particle. Fully-lipidatedparticle compositions were shown to be able to remove lipids such as cholesterol and cholesterol esters. For example, see, U.S. Provisional Application No. 63 / 450,945 filed on March 8, 2023, the content of which is incorporated herein by reference in its entirety. In some embodiments, fully lipidated particles may have some capacity to adsorb cholesterol and cholesterol esters. The capacity for a particle to uptake of cholesterol and cholesterol esters is an important parameter in any therapeutic applications, both for overall efficacy and in order to minimize the xenobiotic burden on the recipient.

[0064] Inventors of the present invention surprisingly discovered that under-lipidated particle compositions were able to be successfully made and could be homogeneous. This was unknown at the time of the invention. These stable under-lipidated particles demonstrated several unexpected superior effects. The inventors discovered that for the under-lipidated particles described herein, the capacity of the particles to absorb cholesterol and / or cholesterol esters may correlate to the degree of lipid saturation of the particles. The under- lipidated particle compositions described herein have novel physical and chemical properties, and may lead to improved therapeutic solutions. First, the under-lipidated particle compositions showed greater lipid (e.g., cholesterol) efflux capacity compared to fully- lipidated particle compositions of equivalent dose, or even at a much lower dose compared to the fully-lipidated particle compositions, demonstrating promising use in the removal of lipids (e.g., cholesterol and cholesterol esters) from cells and tissues and related disease therapies. The cholesterol efflux capacity of under-lipidated particle compositions were found to be much better than high-density lipoprotein (HDLs), lipid-free MSP proteins, and lipid- free Apo-AI. Second, the under-lipidated particle compositions showed superior stability, such as after freeze and thaw cycles without or without cryoprotectants. The cholesterol efflux capacity of under-lipidated particle compositions after freeze and thaw cycles was not affected. Third, the methods provided herein for generating the under-lipidated particle compositions were not only able to yield the surprisingly stable under-lipidated particle compositions with superior cholesterol efflux activity, but also were simple and require very few purification steps, sometimes only a single purification. The obtained under-lipidated particle compositions had surprisingly narrow size distribution and narrow distribution of the molar ratio between phospholipid and the amphipathic polymer. Fourth, the under-lipidated particle compositions showed high lipid (e.g., cholesterol) binding capacity at equilibrium when mixed with lipid (e.g., cholesterol)-containing Large Unilamellar Vesicles (LUVs). For example, an exemplary under-lipidated particle composition was shown to bind 7 mols ofcholesterol per 1 mol of the particle composition at equilibrium. The maximum lipid (e.g., cholesterol) binding capacity of the under-lipidated particle compositions can be even higher.

[0065] In one aspect, provided herein is a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a polymer-phospholipid saturation ratio Ns. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a polymer-phospholipid saturation ratio Ns described herein. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 95% (e.g., less than about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower) of the polymer-phospholipid saturation ratio Ns. Inventors of the present invention further discovered that certain particular ratios of lipid to amphipathic polypeptide may give rise to a homogeneous particle with maximum uptake capacity for cholesterol and cholesterol esters. This increased ability is critical for the therapeutic applications described herein. Several exemplary under-lipidated particle compositions are provided herein with different amphipathic polymer components, as well as different phospholipid to the amphipathic polymer ratios.

[0066] In another aspect, there is provided a method of preparing a composition comprising a plurality of particles, wherein the plurality of particles each comprises an amphipathic polymer and a phospholipid, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated, the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and 2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a polymer-phospholipid saturation ratio Ns. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is equal to or less than (e.g., less than about any of 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower of) the molar ratio of the phospholipid to the amphipathic polymer in the composition. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is approximately equal to about any of the molar ratio of the phospholipid to the amphipathic polymer in the composition. For example, the molar ratio ofthe phospholipid to the amphipathic polymer in the preparation mixture deviates from the molar ratio of the phospholipid to the amphipathic polymer in the composition by less than about any of 20%, 15%, 10%, 5%, 2%, 1%, or 0.1%. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture described herein yields a homogeneous under-lipidated particle composition with no extraneous material.

[0067] The compositions and methods of treatment described herein are particularly helpful in treating a broad range of diseases characterized by lipid accumulation, such as eye diseases including macular degeneration and atrophy in the eye.I. Definitions

[0068] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise.

[0069] The term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ± 20%, ± 15%, ± 10%, ± 5%, or ± 1%. In certain embodiments, the term “about” indicates the designated value ± one standard deviation of that value.

[0070] The terms “effective amount” and “pharmaceutically effective amount” refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and / or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0071] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, in some embodiments a mammal, and in some embodiments, a human, having a complement system, including a human in need of therapy for, or susceptible to, a condition or its sequelae. The individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, rabbits, hamsters, guinea pigs, monkeys, mice, and humans. In some embodiments, the individual is a human.

[0072] The term “treatment” typically refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with disease ordisorder are mitigated or eliminated, including, but not limited to, decreasing the frequency and / or severity of a sign and / or symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and / or prolonging survival of individuals. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0073] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a polypeptide sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0074] As used herein, “amphipathic” refers a molecule (e.g., polypeptide) having both a polar water-soluble (hydrophilic) group and a water-insoluble non-polar (hydrophobic) group. Amphipathic molecules have both hydrophilic and hydrophobic properties. Examples include phospholipids, cholesterol, amphipathic peptides, glycolipids and fatty acids. In some embodiments, amphipathic peptides include, but are not limited to, membrane scaffold protein (MSP), such as the MSP1, MSP1D1, MSP1E1, MSP1E1D1, MSP1E2, MSP1E2D1, MSP1E3, and MSPE3Dl described herein.

[0075] It is understood that embodiments of the invention described herein include “consisting of’ and / or “consisting essentially of’ embodiments.II. Particle Compositions

[0076] In one aspect, provided herein is a composition (e.g., pharmaceutical composition) comprising a plurality of particles (hereinafter also referred to as “particle composition”) comprising a polymer (e.g., an amphipathic polymer) and a phospholipid, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, the molar ratio of thephospholipid to the amphipathic polymer in the composition is less than (e.g., less than about any of 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower of) a polymer-phospholipid saturation ratio Ns. In some embodiments, the amphipathic polymer is an amphipathic polypeptide, such as ApoA-I or a membrane scaffold protein (MSP), e.g., ApoAI-derived MSPs.

[0077] In some embodiments, the amphipathic polymer is ApoA-I, ApoA-II, ApoC, ApoE, or a variant thereof. In some embodiments, the amphipathic polypeptide is a variant of ApoA-I. In some embodiments, the amphipathic polypeptide comprises one or more amphipathic helix sequences derived from ApoA-I. In some embodiments, the amphipathic polypeptide comprises an N-terminus truncation relative to ApoA-I. In some embodiments, the amphipathic polypeptide is selected from the group consisting of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and a variant thereof (e.g., a variant with N-terminal His-tag portion removed). In some embodiments, the phospholipid comprises one or more acyl chains, optionally with 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 can 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.

[0078] In some embodiments, all particles within the composition are under-lipidated. In some embodiments, at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or more) of the particles within the composition are under-lipidated. In some embodiments, the composition further comprises lipid-free amphipathic polymer (e.g., lipid- free MSP) that is not assembled into particles. In some embodiments, no more than about 15% (e.g., no more than about any of 10%, 5%, 2%, 1%, or lower) of the particles within the composition are fully- lipidated. In some embodiments, the under-lipidated particle composition is further purified (e.g., by SEC), e.g., by 1, 2, 3, or more times of purifications. In some embodiments, the under-lipidated particle composition is not purified. In some embodiments, the under-lipidated particle composition comprises a plurality of particles that have a narrow distribution of the molar ratio between the phospholipid and the polymer (e.g., amphipathic polypeptide), for example, wherein the poly dispersity index of the phospholipid content of the particles in the composition is less than about any of 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-lipidatedparticle composition is stable when subjected to freezing or thawing (e.g., 1, 2, 3, or more cycles), for example, the molar ratio between the phospholipid and the polymer (e.g., amphipathic polypeptide) in the particle composition changes less than about any of 20%, 10%, 5%, 1%, or lower after at least one (e.g., 1, 2, 3, 4, or 5) freeze-and-thaw cycle (compared to before freezing) with or without a cryoprotectant. In some embodiments, the lipid (e.g., cholesterol) efflux capacity of the under-lipidated particle composition subjected to freezing or thawing (e.g., 1, 2, 3, or more cycles) changes less than about any of 20%, 10%, 5%, 1%, or lower compared to before freezing with or without a cryoprotectant. In some embodiments, the particles may be in the form of a nanocomposite, wherein a lipid bilayer comprising the phospholipid is surrounded by a stabilizing belt comprising the polymer (e.g., amphipathic polypeptide). In some embodiments, the average size of the particles in the composition is about 2 nm to about 20 nm in diameter. In some embodiments, the poly dispersity index of the size of the particles in the composition is less than about 0.2, such as less than about any of 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 poly dispersity index of the lipid content of the particles in the composition is less than about any of 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01.

[0079] In some embodiments, there is provided a composition comprising a plurality of particles comprising MSP1D1 (or His-tag removed variant thereof, e.g., SEQ ID NO: 11) and DMPC, wherein the molar ratio of DMPC to MSP ID 1 (or His-tag removed variant thereof (or MSP ID 1(-)), e.g., SEQ ID NO: 11) in the composition is about 40: 1 to about 81 : 1, such as about 65:1 to about 75: 1, about 70: 1 to about 75: 1, or about 75: 1. In some embodiments, there is provided a composition comprising a plurality of particles comprising MSP ID 1 (or His-tag removed variant thereof (or MSP1D1(-)), e.g., SEQ ID NO: 11) and DMPC, wherein the molar ratio of DMPC to MSP1D1 (or His-tag removed variant thereof, e.g., SEQ ID NO: 11) in the composition is about 40: 1 to about 60: 1, such as about 46: 1 to about 55: 1, about 51 : 1, about 55: 1, or about 50: 1. In some embodiments, there is provided a composition comprising a plurality of particles comprising MSP1E3D1 (or His-tag removed variant thereof (or MSP1E3D1(-)), e.g., SEQ ID NO: 12) and DMPC, wherein the molar ratio of DMPC to MSP1E3D1 (or His-tag removed variant thereof, e.g., SEQ ID NO: 12) 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. In some embodiments, the average size of the particles in the composition is about 2 nm to about 20 nm in diameter. In some embodiments, the poly dispersity index of the size of the particles in the composition isless than about 0.2, such as less than about any of 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 poly dispersity index of the lipid content of the particles in the composition is less than about 0.2, such as less than about any of 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01. a. Polymers

[0080] In some embodiments, the particles described herein comprise an amphipathic polymer. In some embodiments, each particle comprises 1, 2, or 3 amphipathic polymers. In some embodiments each particle comprises 2 amphipathic polymers, such as any of the amphipathic polypeptides described herein. As used herein, “polymer” refers to a composition of individual building blocks of organic or inorganic compounds that result in an amphipathic material of an extended amphipathic nature. In some embodiments, the polymer is a polypeptide. In some embodiments, the number of individual building blocks in the case of amino acid-based polymers (e.g., polypeptide) may range from roughly 2 to 1000, such as roughly any of 5-75, 5-50, 5-25, 25-500, 25-250, 25-200, 25-150, 25-100, or 25-50. In some embodiments, the amphipathic polypeptide may be any of amphipathic polypeptides that can support a lipid bilayer structure, such as membrane scaffold protein (MSP) as described herein. In some embodiments, the amphipathic polypeptide may include, but not limited to MSP1D1, MSP1E1D1, MSP1E2D1, MSPE3D1, and MSP2N2 as described herein. In some embodiments, the amphipathic polypeptide is MSP1, MSP1E1, MSP1E2, MSP1E3, or a variant thereof (e.g., His-tag removed variant thereof). In some embodiments, the polypeptide is an apolipoprotein or a variant thereof. In some embodiments, the polypeptide is a membrane scaffold protein (MSP). In some embodiments, the polymer is an amphipathic polymer. In some embodiments, the amphipathic polymer is an amphipathic polypeptide. In some embodiment, about any of 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 polypeptide in the particles is an amphipathic polypeptide described herein. In some embodiments, the amphipathic polypeptide comprises one or more amphipathic helix sequences of about 9 to about 30 amino acids, such as any of about 10 to about 25 amino acids, about 11 to about 22 amino acids, about 11 amino acids, or about 22 amino acids.

[0081] The apolipoproteins may include, but are not limited to, the apolipoprotein A (Apo- A) such as Apo-AI, 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, apo ApoC-II, apo ApoC-III, and ApoC-IV; apolipoprotein D (Apo-D); apolipoprotein E (Apo-E); and apolipoprotein M(ApoM). In some embodiment, the apolipoproteins may be from any organism, including but not limited to, human, cows, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, mice, rats, cats, guinea pigs, hamsters, duck, chicken, salmon, and eel. In some embodiments, the apolipoprotein is human apolipoprotein.

[0082] In some embodiments, the polymer (e.g., amphipathic 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 derived from a natural apolipoprotein selected from the group consisting of ApoA-I, ApoA-II, Apo-C, Apo-E, and Apo-M. In some embodiments, the polymer comprises a mutation (e.g., insertion, deletion, substitution, or any combination thereof) compared to a naturally existing apolipoprotein (e.g., ApoA-I). In some embodiments, the polymer comprises one or more substitutions of the amino acid cysteine (Cys). In some embodiments, the mutation (e.g., Cys substitution) is at the N terminus, at the C terminus, and / or among the amphipathic helices of the polymer. In some embodiments, the Cys substitution is in one of the helixes 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 luminescent small molecules). In some embodiments, the polymer (e.g., amphipathic polypeptide) is fused with a fluorescent protein such as GFP, RFP, or YFP. In some embodiments, the polymer (e.g., amphipathic polypeptide) comprises a tag, such a tag commonly used in protein expression and / or purification. In some embodiments, the tag is a polyhistidine, c-Myc, FLAG, biotin, or any combination thereof. In some embodiments, the polymer (e.g., amphipathic polypeptide) comprises an enzymatic cleavage site, e.g., Tobacco Etch Protease (TEV) recognition site (boxed in Table 1).

[0083] In some embodiments, the polymer (e.g., amphipathic polymer, such as amphipathic polypeptide) is an MSP. In some embodiments, the MSP comprises a His-tag. In some embodiments, the MSP does not comprise a His-tag. The symbol “(-)” as used herein refers to an amphipathic polypeptide (e.g., MSP) with the His-tag removed. In these constructs the His-tag is followed by a TEV protease recognition site. Cleavage with TEV leaves an additional glycine (G) residue at the amino terminus of the MSPs. In some embodiments, the MSP is derived from a naturally occurring ApoA-I, e.g., human ApoA-I. TEV protease recognition site sequence is boxed in Table 1.

[0084] ApoA-I is the major protein component of high-density lipoprotein (HDL) particles in human plasma. Human ApoA-I contains an N-terminal globular domain (GLOB) made up of about 43 residues followed by 10 amphipathic helices (H1-H10) made up of 11, 22, or 24residues each. Seven of the helices are 22 amino acids in length, one helix (H10) is 24 amino acids in length, while two helices (H3, H9) are 11 amino acids in length. The helices are separated by glycine or proline Residues. The sequences of different domains are indicated for human ApoA-I in Table 1. A Full-length human ApoA-I comprises from N’ to C’ : GLOB-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10. Human ApoA-I comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the GLOB domain comprises the amino acid sequence of DEPPQSPWDRVKDLATVYVDVLKDSGRDYVSQFEGSALGKQLN (SEQ ID NO: 14).

[0085] MSPs containing alternate amphipathic helical polypeptide sequences can be generated by adding or removing one or more helical segments (e.g., Hl, H2, etc.) or partial helical segments (e.g., removing partial Hl sequence of ApoA-I to arrive at H(0.5)), or adding, removing, or substituting amino acid residues, based off an ApoA-I (e.g., human ApoA-I) protein. In some embodiments, an MSP can be derived from ApoA-I (e.g., human ApoA-I) by removing the GLOB domain. In some embodiments, the MSP 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.

[0086] Exemplary MSPs and their contained domains and sequences are indicated in Table 1. Other MSPs with variations relative to those in Table 1 can also be used herein. For example, by adding or removing one or more helical segments or portion thereof, and / or by adding, removing, and / or substituting one or more amino acid residues (e.g., adding a peptide linker in between helical segments) at N-terminus, internally, and / or at C-terminus, and / or by adding, removing, and / or substituting a tag-sequence.Table 1. Sequences of ApoA-I and Exemplary Membrane Scaffold Proteins (MSPs)

[0087] ApoA-1 can be from any organism. In some embodiments, ApoA-1 is derived from animals selected from the group consisting of human, cows, horses, sheep, monkeys, baboons, goats, rabbits, dogs, hedgehogs, badgers, mice, rats, cats, guinea pigs, hamsters, duck, chicken, salmon, eel, and any combination thereof. In some embodiments, ApoA-1 is a human ApoA-1. In some embodiments, the human ApoA-1 comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 1.

[0088] In some embodiments, the amphipathic polypeptide is a variant of ApoA-I. In some embodiments, the amphipathic polypeptide has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or higher) sequence identity to SEQ ID NO: 1. In some embodiments, the amphipathic polypeptide is selected from the group consisting of MSP ID 1 (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 amphipathic polypeptide is a variant of any of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP1N2, such as a variant with insertion(s), deletion(s), and / or substitution(s) (e.g., conservative substitution(s)). A variant of any of the amphipathic polypeptides described herein can have at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or higher) sequence identity to its corresponding reference amphipathic polypeptide.

[0089] In some embodiments, the amphipathic polypeptide comprises an N-terminus truncation relative to ApoA-I. In some embodiments, the truncation comprises deleting 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 deleting about 10-70 amino acids from the N-terminus of ApoA-I. In some embodiments, the N-terminus GLOB domain of ApoA-I is removed in the amphipathic polypeptide. In someembodiments, the first 11 amino acids of Hl of ApoA-I are removed in the amphipathic polypeptide, resulting in a H(0.5) helical portion of STFSKLREQLG (SEQ ID NO: 16) (see H(0.5) in Table 1). In some embodiments, both the N-terminus GLOB domain and the first 11 amino acids of Hl of ApoA-I is removed in the amphipathic polypeptide.

[0090] In some embodiments, the amphipathic polypeptide comprises an amino acid sequence of (SEQ ID NO: 15) at the N-terminus,hereinafter also referred to as “His-tag” or “His-tag portion.” The TEV protease recognition site is indicated by boxing. In some embodiments, the amphipathic polypeptide does not comprise the N-terminal His-tag portion (his-tag removed), herein denoted with “(-)” at the end of the name of the amphipathic polypeptide. In some embodiments, the N-terminal His- tag (SEQ ID NO: 15) is removed from the amphipathic polypeptide by cleaving with the TEV protease. It is understood that cleavage at the TEV protease recognition site may result in a residual glycine (G) residue at the N-terminus of the resulting MSP sequence. For example, MSPIDI(-) (SEQ ID NO: 11) is obtained by cleaving MSP1D1 (SEQ ID NO: 2) with TEV protease; MSPIEIDI(-) (SEQ ID NO: 17) is obtained by cleaving MSP1E1D1 (SEQ ID NO: 3) with TEV protease; MSP1E2D1(-) (SEQ ID NO: 18) is obtained by cleaving MSP1E2D1 (SEQ ID NO: 4) with TEV protease; and MSP1E3D10 (SEQ ID NO: 12) is obtained by cleaving MSP1E3D1 (SEQ ID NO: 5) with TEV protease. It is also understood for MSPs wherein the indicated His-tag (SEQ ID NO: 15) is not removed, the N-terminal methionine residue will be often removed during heterologous expression, e.g., if expressed in bacteria such as E. coli.

[0091] In some embodiments, the amphipathic 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 without the globular N-terminal domain of native ApoA-1. In some embodiments, the amphipathic polypeptide has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 13.

[0092] In some embodiments, the amphipathic polypeptide is MSP1D1 and comprises the amino acid sequence of SEQ ID NO: 2. MSP ID 1 is derived from the sequence of ApoA-1, but without the globular N-terminal domain of native ApoA-1 and removing 11 amino acids from the first helix labeled Hl in the parent ApoA-I, generating the half helix labeled H(0.5). In some embodiments, the amphipathic polypeptide is a variant of MSP1D1 having at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 2. In some embodiments, the amphipathic polypeptide is a variant ofMSP ID 1 that does not comprise the N-terminal His-tag portion (his-tag removed variant) (hereinafter referred to as MSP1D1(-)), which comprises the amino acid sequence of SEQ ID NO: 11.

[0093] In some embodiments, the amphipathic polypeptide comprises one or more amphipathic helix sequences, such as one or more amphipathic helix sequences derived from an MSP or an apolipoprotein (e.g., ApoA-I). In some embodiments, the amphipathic polypeptide comprises one or more (e.g., 1, 2, 3, or more) additional amphipathic helix sequences relative to ApoA-I. In some embodiments, each additional amphipathic helix sequence is independently derived from any of ApoA-I, ApoA-II, ApoC-I, ApoC-II, Apo-E, apolipophorin III (apoLp-III), myoglobin, or hemoglobin. In some embodiments, the additional amphipathic helix sequence is not derived from ApoA-I, ApoA-II, ApoC-I, ApoC- II, Apo-E, apolipophorin III, myoglobin, or hemoglobin. In some embodiments, the additional amphipathic helix sequence is synthetic or not naturally occurring. In some embodiments, the additional amphipathic helix sequence comprises at least one (e.g., 1, 2, 3, 4, 5, or more) a-helixes or fragments thereof. In some embodiments, the amphipathic polypeptide does not comprise the GLOB domain of SEQ ID NO: 14. In some embodiments, an amphipathic helix sequence of helix 4 (H4) can be inserted to the amphipathic polypeptide MSP1D1 to generate MSP1E1D1, as defined by SEQ ID NO: 3. In some embodiments, 2 amphiphilic helices of Helix 4 (H4) and Helix 5 (H5) can be inserted into MSP ID 1, generating MSP1E2D1 (SEQ ID NO:4). In some embodiments, three amphiphilic helices, H4, H5 and H6, can be inserted into MSP1D1, generating MSP1E3D1 defined by SEQ ID NO: 5. In some embodiments, an additional 9 amphipathic helices is added to MSP1D1 (SEQ ID NO: 2) with a GT peptide linker to generate an extended amphipathic polymer MSP2N2 (SEQ ID NO: 6).

[0094] In some embodiments, the amphipathic polypeptide comprises an additional helix sequence, such as one, two, or three of any of first, second, or third helix sequences. In some embodiments, the additional amphipathic helix sequence is added to the N-terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequence is added to the C-terminus of an apolipoprotein (e.g., ApoA-I). In some embodiments, the additional amphipathic helix sequence is inserted between the helixes of an apolipoprotein (e.g., ApoA-I). In some embodiments, each additional amphipathic helix sequence has about 10 to about 30 (including, for example, any of about 15 to about 25, about 24, about 20, about 22, about 11) amino acids. In some embodiments, the total additionalamphipathic helix sequence is about any of about 20-25, about 40-46, about 60-68, or about 100-200 amino acids long.

[0095] In some embodiments, the amphipathic polypeptide is MSP1E1, MSP1E2, or MSP1E3. MSP1E1, MSP1E2, and MSP1E3 are all extended MSPs, and are obtained via insertion of one 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).

[0096] In some embodiments, the amphipathic polypeptide comprises both an additional amphipathic helix sequence and an N-terminus truncation relative to ApoA-I. In some embodiments, the amphipathic polypeptide is MSP1E3D1. In some embodiments, MSP1E3D1 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amphipathic polypeptide is a variant of MSP1E3D1 having at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 5. In some embodiments, the amphipathic polypeptide is a variant of MSP1E3D1 that does not comprise the N-terminal His-tag portion (his-tag removed variant) (hereinafter referred to as MSP1E3D1(-)), which comprises the amino acid sequence of SEQ ID NO: 12.

[0097] In some embodiments, the amphipathic polypeptide is MSP1E1D1. In some embodiments, MSP1E1D1 comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amphipathic polypeptide is a variant of MSP1E1D1 having at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 3. In some embodiments, the amphipathic polypeptide is a variant of MSP1E1D1 that does not comprise the N-terminal His-tag portion (his-tag removed variant) (hereinafter referred to as MSPIEIDI(-)), which comprises the amino acid sequence of SEQ ID NO: 17.

[0098] In some embodiments, the amphipathic polypeptide is MSP1E2D1. In some embodiments, MSP1E2D1 comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the amphipathic polypeptide is a variant of MSP1E2D1 having at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 4. In some embodiments, the amphipathic polypeptide is a variant of MSP1E2D1 that does not comprise the N-terminal His-tag portion (his-tag removed variant) (hereinafter referred to as MSP1E2D1(-)), which comprises the amino acid sequence of SEQ ID NO: 18.

[0099] In some embodiments, the amphipathic polypeptide is MSP2N2. In some embodiments, MSP2N2 comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the amphipathic polypeptide is a variant of MSP2N2 having at least about 70%(e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 6. In some embodiments, the amphipathic polypeptide is a variant of MSP2N2 that does not comprise the N-terminal His-tag portion (his-tag removed variant) (hereinafter referred to as MSP2N2(-)), which comprises the amino acid sequence of SEQ ID NO: 19.

[0100] Detailed descriptions ofMSPIDl, MSP1E1D1, MSP1E2D1, and MSPlE3Dl, are disclosed in Denisov et al J Am Chem Soc. 2004 Mar 24. 126(11):3477-87, the content of which is incorporated herein by reference in its entirety.

[0101] MSP2N2 is generated by extending the His-tagged MSP ID 1, adding a GT spacer and an additional Helix2 through HelixlO. Detailed composition and preparation procedures for MSP2N2 are described in Grinkova et al. Protein Engineering Design and Selection 2010 23, 843-848, the content of which is incorporated herein by reference in its entirety.

[0102] In some embodiments, the histidine tag is removed from MSP ID 1, MSPE1D1, MSP1E2D1, MSPE3D1 or MSP2N2 to generate MSPIDI(-), MSPEIDI(-), MSP1E2D10, MSPE3D1(-) or MSP2N2(-), respectively, by cleaving with TEV protease. It is understood that TEV protease cleavage at the boxed area shown in Table 1 can result in a residual glycine (G) amino acid residue on the N-terminal of the resulting MSP protein. These variants are herein referred to as “his-tag removed variants.”

[0103] 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, that are mostly made by the liver and, to a lesser degree, in the intestine. In some embodiments, ApoC-I, ApoC-II, and ApoC-III comprises the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. In some embodiments, the amphipathic polypeptide has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to any of SEQ ID NOs: 7-9.

[0104] In some embodiments, the polymer such as the polypeptide is a variant 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, the MSP has at least about 70% (e.g., at least about any of 80%, 85%, 90%, 95%, 99%, or 100%) sequence identity to SEQ ID NO: 10. b. Phospholipids

[0105] Phospholipids generally refer to a class of lipid with a hydrophilic head of phosphate moiety and two hydrophobic tails derived from fatty acids, joined by an alcohol or glycerol backbone.

[0106] In some embodiments, the phospholipid is a sphingolipid. Sphingolipids typically refers to a class of lipids containing a backbone of sphingoid bases, which are a set of aliphatic amino alcohols that includes sphingosine. In some embodiments, the sphingolipid is sphingosine- 1 -phosphate.

[0107] In some embodiments, the phospholipid comprises one or more acyl chains. In some embodiments, each acyl chain independently has a length of about 5 to about 30 carbon units, 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 the class of phospholipids that comprise choline as a head group, glycerophosphoric unit, and a variety of fatty acids. In some embodiments, the phospholipid comprises a PC selected from the group consisting of 1- oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 2-dierucoyl-sn-glycero-3- phosphocholine (DEPC), l-palmitoyl-2- oleoyl-sn-glycero-3-phosphorylcholine (POPC), l,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), l,2-dilauroyl-sn-glycero-3 -phosphocholine (DLPC), dimyristoylphosphatidylcholine (DMPC), l ,2-distearoyl-.s / / -glycero-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.

[0108] In some embodiments, the 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 that comprises two myristoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. In some embodiments, the phospholipid is DPPC. DPPC is a synthetic phospholipid that comprises two palmitoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. In some embodiments, the phospholipid is l,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), such as a 14:0 DMPS. In some embodiments, the phospholipid is DSPC. DSPC is a synthetic phospholipid that comprises two stearoyl fatty acids attached in ester linkage to the first and second carbonof glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol.

[0109] In some embodiments, the one or more acyl chains of the phospholipid are unsaturated. In some embodiments, the phospholipid is POPC, phosphatidylinositol 4,5- bisphosphate (PiP2), or palmitoyl-oleoyl phosphatidylserine (POPS). In some embodiments, the phospholipid is POPC. POPC is a synthetic phospholipid that comprises two fatty acids, palmitic acid and oleic acid, attached in ester linkage to the first and second carbon of glycerol, respectively, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. In some embodiments, the phospholipid is PiP2, which could also be referred to as PI(4,5)P2. In some embodiments, the PiP2 is 18:1 PI(4,5)P2 or 18:0-20:4 PI(4,5)P2. In some embodiments, the PiP2 is 18:1 PI(4,5)P2. In some embodiments, the PiP2 is 18:0-20:4 PI(4,5)P2. In some embodiments, the phospholipid is DOPC. DOPC is a synthetic phospholipid that comprises two oleoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol. In some embodiments, the phospholipid is DEPC. DEPC is a synthetic phospholipid that comprises two erucoyl fatty acids attached in ester linkage to the first and second carbon of glycerol, and choline attached through a phosphodiester linkage to the third carbon of the glycerol.

[0110] In some embodiments, the phospholipid is cationic, anionic, or zwitterionic, or any combination thereof. c. Molar Ratio between Phospholipid and Amphipathic Polymer in the Particle Composition

[0111] In some embodiments, without being bound by any scientific theory, the amount of phospholipid needed to saturate a particle may depend on the identity of the polymer and the phospholipid. In some embodiments, phospholipids may comprise a phosphatidyl choline (PC) head group and more than 12 hydrocarbon tails. In some embodiments, without being bound by any scientific theory, phospholipids may have the tendency to condense into a bilayer structure. In some embodiments, without being bound by any scientific theory, the amphipathic polymer may contact with a bilayer formed by the phospholipids, wherein the hydrophobic part of the polymer stays in proximity to the hydrophobic tails of the phospholipids and the hydrophilic part of the polymer stays in proximity to a solvent in the composition. In some embodiments, without being bound by any scientific theory, the amphipathic polymer is a variant of ApoA-I, and the amino acids forming the globulardomain and those of the first 11 amino acids of Helix 1 (Hl, Table) are not in contact with the lipids (Denisov et. Al, J Am Chem Soc. 2004 Mar 24. 126(11):3477-87).

[0112] Variants of the MSPs without the globular domain, and / or without the Hl domain or portion thereof, and / or without any amino or carboxy terminal tag and protease cleavage sites, such as the MSPIDI(-), MSPEIDI(-), MSPE2D10, MSPE3D1(-) and MSP2N2(-) provided herein, may have the same molar ratio of the phospholipid to the amphipathic polymer in the composition needed to saturate the particle, compared to their parental protein, e.g., MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1, and MSP2N2, respectively.

[0113] In some embodiments, the composition provided herein comprises a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a polymer-phospholipid saturation ratio Ns. In some embodiments, without being bound by any scientific theory, the polymer- phospholipid saturation ratio Ns may be calculated according to Denisov et al. (J Am Chem Soc. 2004 Mar 24. 126(11):3477-87) and Denisov and Sligar (Chem Rev. 2017 Mar 22;117(6): 4669-4713), the contents of each of which are incorporated herein by reference in their entirety. Denisov et al. discloses that in a helical amino acid polymer comprising an amphipathic helical polypeptide with a length of M amino acid residues that form a lipid binding scaffold, the number of lipids needed to saturate the particle (polymer-phospholipid saturation ratio Ns) is a function of the length M as well as the mean area occupied by a single phospholipid type. In some embodiments, Ns may be calculated by Equation 1.1:(Equation 1.1) wherein M is the number of amino acid residues in the helical belt of the amphipathic polymer around a bilayer of the phospholipids, L is the helical pitch per amphipathic polymer residue (e.g., about 0.15 nm), and r is the mean radius of the amphipathic polymer a-helix (e.g., about 0.55 nm). In some embodiments, AL of Equation 1.1 is a constant depending on the phospholipid, e.g., may be the area occupied by a single phospholipid in a bilayer (in nm2). In some embodiments, without being bound by any scientific theory, the area AL may depend on the identity of the lipid used as well as the temperature of assembly relative to the bulk bilayer phase transition temperature.

[0114] In some embodiments, M can be calculated according to Equation 1.2:M = 2 nr + nNA / (Equation 1.2)wherein r is the mean radius of the amphipathic polymer a-helix (e.g., about 0.55 nm or about5.5 A), L is the helical pitch per amphipathic polymer residue (e.g., about 0.15 nm or about1.5 A), AL is the mean surface area per phospholipid, N is the number of phospholipid per one bilayer of the phospholipids. In some embodiments, M can be referred to from Table 2.

[0115] In some embodiments, the polymer-phospholipid saturation ratio Ns may be calculated by Equation 1.1, wherein AL is about 0.52 for DPPC and DMPC, and AL is about 0.69 for POPC.

[0116] In some embodiments, the length of the helical belt that contacts the lipids (M) is the same for the MSP variants of with the H(0.5) helical sequence, compared to their parental MSP protein containing Hl (see Table 1). In some embodiments, this helical belt length (M) may also be the same with or without an added histidine or other tag. For example, the helical belt length M of MSP1D1, MSPE1D1, MSPE2D1, MSPE3D1, and MSP2N2 for calculating Ns is the same as the helical belt length M of MSPIDI(-), MSPEIDI(-), MSPE2D1(-), MSPE3D1(-) and MSP2N2(-) for calculating Ns, respectively, as the tag and protease cleavage site is not involved in stabilizing the resulting particle. In some embodiments, the number of lipids (DMPC, DPPC and POPC) needed to saturate the MSP ID 1, MSPE1D1, MSPE2D1, MSPE3D1 particles may be calculated by Equation 1.1 and the calculation results are summarized in Table 2.Table 2. Ns values for exemplary particle compositions

[0117] In some embodiments, without being bound by any scientific theory, the stoichiometry of amphipathic polymer and phospholipid is crucial for the functions, shape homogeneity and size distribution of the particle compositions. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than the phospholipid-polymer saturation ratio Ns, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns. In some embodiments, the Ns for exemplary lipid and amphipathic polymer combinations are shown in Table 2.

[0118] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1D1 or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 81:1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 81:1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer MSP1D1 or His-tag removed variant thereof (e.g., SEQ ID NO: 11) in the composition is less than or equal to about 78:1, such as less than or equal to about any of 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 His-tag removed variant thereof (e.g., SEQ ID NO: 11) in the composition is about 10:1 to about 80:1, such as any of about 10:1 to about 78:1, about 20:1 to about 78:1, about 20:1 to about 78:1, about 30:1 to about 78:1, about 70:1 to about 80:1, about 74:1 to about 77:1, about 40:1 to about 60:1, about 65:1 to about 75:1, about 70:1 to about 75:1, about 72:1 to about 78:1, about 46:1 to about 55:1, about 75:1, about 54:1 to about 56:1, about 65:1, about 60:1, about 55:1, about 51:1, about50:1, about 45:1, about 40:1, about 35:1, or about 30:1.

[0119] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is about 72:1 to about 78:1, such as about 75:1.

[0120] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is about 46:1 to about 55:1, suchas about 51:1. In some embodiments, the amphipathic polymer is MSP ID 1. In some embodiments, the amphipathic polymer is an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amphipathic polymer is an MSP1D1 His-tag removed variant defined by SEQ ID NO: 11.

[0121] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP ID 1 or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DPPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 81: Isuch as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 81:1. In some embodiments, the molar ratio of DPPC to the amphipathic polymer MSP1D1 or His-tag removed variant thereof (e.g., SEQ ID NO: 11) in the composition is less than or equal to about 75:1, such as less than or equal to about any of 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 to the MSP1D1 or His-tag removed variant thereof (e.g., SEQ ID NO: 11) in the composition is about 10:1 to about 80:1, such as any of about 10:1 to about 78:1, about 20:1 to about 75:1, about 20:1 to about 60:1, about 30:1 to about 60:1, about 40:1 to about 60:1, about 65:1 to about 75:1, about 70:1 to about 75:1, about 72:1 to about 78:1, about 46:1 to about 55:1, about 75 : 1 , about 54:1 to about 56:1, about 65 : 1 , about 60: 1, about 55:1, about 51:1, about 50:1, about 45:1, about 40:1, about 35:1, or about 30:1. In some embodiments, the molar ratio of DPPC to the amphipathic polymer MSP1D1 or 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 amphipathic polymer is MSP ID 1. In some embodiments, the amphipathic polymer is an MSP ID 1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amphipathic polymer is an MSP1D1 His-tag removed variant defined by SEQ ID NO: 11.

[0122] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathicpolymer is MSP1D1 (e.g., SEQ ID NO: 2) or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP ID 1 or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 62: Isuch as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 62:1. In some embodiments, the molar ratio of POPC to the amphipathic polymer MSP1D1 or His-tag removed variant thereof (e.g., SEQ ID NO: 11) in the composition is less than or equal to about 60:1, such as less than or equal to about any of 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, or less. In some embodiments, the molar ratio of phospholipid POPC to the amphipathic polymer MSP1D1 or His-tag removed variant thereof (e.g., SEQ ID NO: 11) in the composition is about 10:1 to about 60:1, such as any of about 20:1 to about 60:1, about 30:1 to about 55:1, about 40: 1 to about 55:1, about 55:1, about 50:1, about 45 : 1 , about 40: 1 , about 35:1, or about 30:1. In some embodiments, the amphipathic polymer is MSP1D1. In some embodiments, the amphipathic polymer is an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amphipathic polymer is an MSP1D1 His-tag removed variant defined by SEQ ID NO: 11.

[0123] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (MSPIEIDI(-); e.g., SEQ ID NO: 17), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E1D1 or His-tag removed variant thereof (MSPIEIDI(-); e.g., SEQ ID NO: 17), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 107: Isuch as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 107:1. In some embodiments, themolar ratio of DMPC to the amphipathic polymer MSP1E1D1 or His-tag removed variant thereof in the composition is less than or equal to about 105:1, such as less than or equal to about any of 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 phospholipid DMPC to the amphipathic polymer MSP1E1D1 or His-tag removed variant thereof in the composition is about 30:1 to about 100:1, such as any of about 40:1 to about 90:1, about 50:1 to about 80:1, about 50:1 to about 70:1, about 60:1 to about 70:1, about 60:1 to about 65:1, about 70:1, about 65:1, about 60:1, or about 55:1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer MSP1E1D1 or His-tag removed variant thereof in the composition is about 60:1, 62:1, about 64:1 to about 66:1, or about 68:1. In some embodiments, the amphipathic polymer is MSP IE ID 1. In some embodiments, the amphipathic polymer is a His-tag removed variant of MSP IE ID 1. In some embodiments, MSPIEIDI(-) comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 17.

[0124] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (MSPIEIDI(-); e.g., SEQ ID NO: 17), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E1D1 or His-tag removed variant thereof (MSPIEIDI(-); e.g., SEQ ID NO: 17), the phospholipid is DPPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 107: Isuch as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 107:1. In some embodiments, the molar ratio of DPPC to the amphipathic polymer MSP1D1E1 (or MSPIEIDI(-)) in the composition is less than or equal to about 105:1, such as less than or equal to about any of 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 phospholipid DPPC to the amphipathic polymer MSP1E1D1 or His-tag removed variant thereof in the composition is about 30:1 to about 100:1, such as any of about 40:1 to about 90:1, about 50:1 to about 80:1, about 50:1 to about 70:1, about 60:1 to about 70:1, about 60:1 to about 65:1, about 70:1, about 65:1, about 60:1, or about 55:1. In some embodiments, the molar ratio of DPPC to the amphipathic polymerMSP1E1D1 or His-tag removed variant thereof in the composition is about 60:1, 62:1, about 64:1 to about 66:1, or about 68:1. In some embodiments, the amphipathic polymer is MSP1E1D1. In some embodiments, the amphipathic polymer is a His-tag removed variant of MSP1E1D1. In some embodiments, MSPIEIDI(-) comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 17.

[0125] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E1D1 (e.g., SEQ ID NO: 3) or His-tag removed variant thereof (MSPIEIDI(-); e.g., SEQ ID NO: 17), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E1D1 or His-tag removed variant thereof (MSPIEIDI(-); e.g., SEQ ID NO: 17), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 80: Isuch as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 80:1. In some embodiments, the molar ratio of POPC to the amphipathic polymer MSP1E1D1 or His-tag removed variant thereof in the composition is less than or equal to about 76:1, such as less than or equal to about any of 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 phospholipid POPC to the amphipathic polymer MSP1D1E1 (or MSPIEIDI(-)) in the composition is about 10:1 to about 75:1, such as any of about 20:1 to about 75:1, about 20:1 to about 60:1, about 30:1 to about 60:1, about 40:1 to about 60:1, about 65:1, about 60:1, about 55:1, about 50:1, about 45 : 1 , about 40: 1 , about 35 : 1 , or about 30: 1. In some embodiments, the molar ratio of POPC to the amphipathic polymer MSP IE ID 1 or His-tag removed variant thereof in the composition is about 50:1, about 54:1 to about 56:1, or about 55:1. In some embodiments, the amphipathic polymer is MSP1E1D1. In some embodiments, the amphipathic polymer is a His-tag removed variant of MSP1E1D1. In some embodiments, MSPIEIDI(-) comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 17.

[0126] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E2D1 (e.g., SEQ ID NO: 4) or His-tag removed variant thereof(MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E2D1 or His-tag removed variant thereof (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 135: Isuch as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 135:1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer MSP1E2D1 or His-tag removed variant thereof in the composition is less than or equal to about 128:1, such as less than or equal to about any of 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 phospholipid DMPC to the amphipathic polymer MSP1E2D1 or His-tag removed variant thereof in the composition is about 50:1 to about 130:1, such as any of about 50:1 to about 120:1, about 60:1 to about 120:1, about 70:1 to about 110:1, about 70:1 to about 100:1, about 70:1, about 75:1, about 80:1, or about 85:1. In some embodiments, the amphipathic polymer is MSP1E2D1. In some embodiments, the amphipathic polymer is a His-tag removed variant of MSP1E2D1. In some embodiments, MSP1E2D1(-) comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 18.

[0127] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E2D1 (e.g., SEQ ID NO: 4) or His-tag removed variant thereof (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E2D1 or His-tag removed variant thereof (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is DPPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 135: Isuch as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 135:1. In some embodiments, themolar ratio of DPPC to the amphipathic polymer MSP1E2D1 or His-tag removed variant thereof in the composition is less than or equal to about 128:1, such as less than or equal to about any of 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 phospholipid DPPC to the amphipathic polymer MSP1E2D1 or His-tag removed variant thereof in the composition is about 50:1 to about 130:1, such as any of about 50:1 to about 120:1, about 60:1 to about 120:1, about 70:1 to about 110:1, about 70:1 to about 100:1, about 70:1, about 75:1, about 80:1, or about 85:1. In some embodiments, the amphipathic polymer is MSP1E2D1. In some embodiments, the amphipathic polymer is a His-tag removed variant of MSP1E2D1. In some embodiments, MSP1E2D1(-) comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 18.

[0128] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E2D1 (e.g., SEQ ID NO: 4) or His-tag removed variant thereof (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E2D1 or His-tag removed variant thereof (MSP1E2D1(-); e.g., SEQ ID NO: 18), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 102: Isuch as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of aboutl02:l. In some embodiments, the molar ratio of POPC to the amphipathic polymer MSP1E2D1 or His-tag removed variant thereof in the composition is less than about 96:1, such as less than about any of 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 phospholipid POPC to the amphipathic polymer MSP1E2D1 or His-tag removed variant thereof in the composition is about 40:1 to about 95:1, such as any of about 50:1 to about 90:1, about 60:1 to about 90:1, about 70:1 to about 90:1, about 70:1 to about 80:1, about 70:1, about 75:1, about 80:1, or about 85:1. In some embodiments, the amphipathic polymer is MSP1E2D1. In some embodiments, the amphipathic polymer is a His-tag removed variant of MSP1E2D1. In some embodiments, MSP1E2D1(-) comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 18.

[0129] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or 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 amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 167: 1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 167: 1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (e.g., SEQ ID NO: 12) in the composition is less than or equal to about 160: 1, such as less than or equal to about any of 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 molar ratio of phospholipid DMPC to the amphipathic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (e.g., SEQ ID NO: 12) in the composition is about 50: 1 to about 160: 1, such as any of about 50: 1 to about 150: 1, about 60: 1 to about 140: 1, about 60: 1 to about 130: 1, about 60: 1 to about 120: 1, about 60: 1 to about 100: 1, about 60: 1 to about 70: 1, about 90: 1 to about 100: 1, about 87: 1 to about 98: 1, about 87: 1 to about 96: 1, about 58: 1 to about 68: 1, about 60: 1, about 62: 1, about 63: 1, about 65: 1, about 70: 1, about 80: 1, about 90: 1, about 93: 1, about 94: 1, about 100: 1, or about 110: 1.

[0130] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or 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 amphipathic 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.

[0131] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathicpolymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or 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 amphipathic polymer in the composition is about 87: 1 to about 98:1 or about 87:1 to about 96:1. In some embodiments, the amphipathic polymer is MSP1E3D1. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant (or MSP1E3D10) defined by SEQ ID NO: 12.

[0132] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or 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 amphipathic polymer in the composition is about 58: 1 to about 68:1, such as about 63:1. In some embodiments, the amphipathic polymer is MSP1E3D1. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant defined by SEQ ID NO: 12.

[0133] In some embodiments, the amphipathic polymer is MSP1E3D1, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphipathic polymer in the composition is about 58:1 to about 68:1, such as about 62:1. In some embodiments, the amphipathic polymer is MSP1E3D1. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant (MSP1E3D1(-)) comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant defined by SEQ ID NO: 12.

[0134] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DPPC, andwherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 167: 1 such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of aboutl67: l. In some embodiments, the molar ratio of DPPC to the amphipathic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (e.g., SEQ ID NO: 12) in the composition is less than or equal to about 160: 1, such as less than or equal to about any of 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 molar ratio of phospholipid DPPC to the amphipathic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (e.g., SEQ ID NO: 12) in the composition is about 70: 1 to about 160: 1, such as any of about 80: 1 to about 150: 1, about 70: 1 to about 140: 1, about 70: 1 to about 130: 1, about 80: 1 to about 120: 1, about 80: 1 to about 100: 1, about 80: 1, about 90: 1, about 100: 1, about 87: 1 to about 98: 1, about 87: 1 to about 96: 1, about 58 : 1 to about 68: 1, about 60: 1, about 62 : 1 , about 63: 1, about 65: 1, about 70: 1, about 80: 1, about 90: 1, about 93: 1, about 94: 1, about 100: 1, or about 110: 1. In some embodiments, the amphipathic polymer is MSP1E3D1. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant defined by SEQ ID NO: 12.

[0135] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 126: 1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 126: 1. In some embodiments, the molar ratio of POPC to the amphipathic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (e.g., SEQ ID NO: 12) in the composition is less than orequal to about 120:1, such as less than or equal to about any of 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 phospholipid POPC to the amphipathic polymer MSP1E3D1 (e.g., SEQ ID NO: 5) or His- tag removed variant thereof (e.g., SEQ ID NO: 12) in the composition is about 60: 1 to about 120:1, such as any of 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 amphipathic polymer is MSP1E3D1. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant defined by SEQ ID NO: 12.

[0136] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or His-tag removed variant thereof (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is DMPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP2N2 or His-tag removed variant thereof (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is DMPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 387: 1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 387:1. In some embodiments, the molar ratio of DMPC to the amphipathic polymer MSP2N2 or His-tag removed variant thereof in the composition is less than or equal to about 370: 1, such as less than or equal to about any of 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 phospholipid DMPC to the amphipathic polymer MSP2N2 or His-tag removed variant thereof in the composition is about 150: 1 to about 350:1, such as any of about 200:1 to about 350:1, about 250:1 to about 350:1, about 250:1 to about 330:1, about 280:1 to about 320:1, about 280:1 to about 300:1, about 280:1, about 290:1, about 300:1, or about 310:1. In some embodiments, the amphipathic polymer is MSP2N2. In some embodiments, the amphipathic polymer is a His-tag removed variant ofMSP2N2. In some embodiments, MSP2N2(-) comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 19.

[0137] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or His-tag removed variant thereof (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is DPPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or His-tag removed variant thereof (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is DPPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 387: 1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 387: 1. In some embodiments, the molar ratio of DPPC to the amphipathic polymer MSP2N2 or His-tag removed variant thereof in the composition is less than or equal to about 370:1, such as less than or equal to about any of 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 phospholipid DPPC to the amphipathic polymer MSP2N2 or His-tag removed variant thereof in the composition is about 150: 1 to about 350: 1, such as any of about 200: 1 to about 350: 1, about 250: 1 to about 350: 1, about 250: 1 to about 330: 1, about 280: 1 to about 320: 1, about 280: 1 to about 300: 1, about 280: 1, about 290:1, about 300: 1, or about 310: 1. In some embodiments, the amphipathic polymer is MSP2N2. In some embodiments, the amphipathic polymer is a His-tag removed variant of MSP2N2. In some embodiments, MSP2N2(-) comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 19.

[0138] In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein the amphipathic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or His-tag removed variant thereof (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is POPC, and wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, there is provided a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein theamphipathic polymer is MSP2N2 (e.g., SEQ ID NO: 6) or His-tag removed variant thereof (MSP2N2(-); e.g., SEQ ID NO: 19), the phospholipid is POPC, and wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 291 : 1, such as less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns, which is 291 : 1. In some embodiments, the molar ratio of POPC to the amphipathic polymer MSP2N2 or His-tag removed variant thereof in the composition is less than or equal to about 280:1, such as less than or equal to about any of 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 phospholipid POPC to the amphipathic polymer MSP2N2 or His-tag removed variant thereof in the composition is about 100: 1 to about 280: 1, such as any of about 100: 1 to about 260: 1, about 120: 1 to about 260: 1, about 140: 1 to about 260: 1, about 160: 1 to about 240: 1, about 180: 1 to about 220: 1, about 180: 1, about 190: 1, about 200: 1, or about 210: 1. In some embodiments, the amphipathic polymer is MSP2N2. In some embodiments, the amphipathic polymer is a His-tag removed variant of MSP2N2. In some embodiments, MSP2N2(-) comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 19. d. Characteristics

[0139] 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-lipidated. In some embodiments, the plurality of particles each comprises an amphipathic polymer and a phospholipid.

[0140] In some embodiments, the average size of the particles is any 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 size of the particles is about 2 nm to about 20 nm, such as about 10 nm to about 20 nm. In some embodiments, the average size of the particles can be tuned by the length of the amphipathic polymer.

[0141] In some embodiments, the average diameter of the particles is any 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 20nm. In some embodiments, the average diameter of the particles is about 10 nm to about 20 nm. In some embodiments, the average diameter of the particles can be tuned by the length of the amphipathic polymer.

[0142] In some embodiments, the poly dispersity index (a measure of the heterogeneity) of the size of the particles in the composition is less than about 0.5, such as less than about any of 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 size and size distribution of the particles in the composition may be determined by combines multi-angle light scattering with size-exclusion chromatography (SEC-MALS). In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the particles in the composition have a size of about 2 nm to about 20 nm. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the particles remain to have sizes of about 2 nm to about 20 nm after being frozen and thawed for one or more times, such as being frozen at - 80°C and thawed with or without a cryoprotectant. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the particles in the particle composition have sizes substantially unchanged (e.g., a size change of the particles in the particle composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being frozen and thawed for one or more times, such as being frozen at -80 °C and thawed with or without a cryoprotectant.

[0143] In some embodiments, the poly dispersity index (a measure of the heterogeneity) of the lipid content of the particles in the composition is less than about 0.5, such as less than about any of 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, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the particles have molar ratio between phospholipid and polymer of less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the particles have molar ratio between phospholipid and polymer of less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns after being frozen and thawed for one or more times, such as being frozen at -80°C and thawed, with or without a cryoprotectant. In some embodiments, at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the particles have molar ratio between phospholipid and polymer remains substantially unchanged (e.g., a size change of theparticles in the particle composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being frozen and thawed for one or more times, such as being frozen at -80°C and thawed with or without a cryoprotectant.

[0144] In some embodiments, the molar ratio between phospholipid and polymer in the composition remains substantially unchanged (e.g., a size change of the particles in the particle composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being storied under room temperature for at least about 1 hour, such as at least about any of 2, 5, 10, 12, 15, 20, or 24 hours, or 2, 3, 4, 5, 6, 7, 8, 15, 30, 40, 50, 100 days. In some embodiments, the molar ratio between phospholipid and polymer remains substantially unchanged (e.g., a size change of the particles in the particle composition of less than about any of 20%, 15%, 10%, 5%, 2%, or 1%) after being storied under -80°C for at least about 1 month, such as at least about any of 2, 3, 4, 5, 6, 7, 8, 15, 30, 40, 50, 100 months.

[0145] In some embodiments, the molecular weight of the particles in the composition is any 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.

[0146] In some embodiments, the particle compositions described herein increase the cholesterol efflux rate by at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or more) compared to an untreated state (e.g., before administering the particles) or the cholesterol efflux rate by ApoA-I or a high density lipoprotein (HDL) alone. In some embodiments, the under-lipidated particle composition described herein increases the cholesterol efflux rate by at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or more) compared to a fully- lipidated particle composition containing the same amphipathic polymer and phospholipid (the molar ratio of the phospholipid to the amphipathic polymer in the composition is about the phospholipid-polymer saturation ratio Ns).

[0147] In some embodiments, the particle compositions described herein have at least about 20% (e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 2.5- fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, or more) higher lipid (e.g., cholesterol) binding capacity at equilibrium (or lipid (e.g., cholesterol) partition coefficient) compared to that of i) an ApoA-I alone, ii) an HDL alone, iii) an MSP (e.g., MSP1D1(-)) alone, and / or iv) a fully-lipidated particle composition containing the same amphipathic polymer and phospholipid (the molar ratio of the phospholipid to the amphipathic polymer in the composition is about the phospholipid-polymer saturation ratio Ns). In some embodiments, the particle compositions described herein can bind at least about 2 mols of lipid (e.g., cholesterol) per 1 mol of the particle composition at equilibrium, such as at least about any of 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more, mols of lipid (e.g., cholesterol) per 1 mol of the particle composition at equilibrium. In some embodiments, the lipid (e.g., cholesterol) binding capacity at equilibrium (or lipid (e.g., cholesterol) partition coefficient) is measured using a lipid (e.g., cholesterol)-containing LUV assay, such as that described in Example 8.

[0148] In some embodiments, provided herein are compositions comprising particles prepared according to any of the methods of preparation detailed herein.

[0149] In some embodiments, the composition provided herein is a pharmaceutical composition. In some embodiments, the composition further comprises a prophylactic or therapeutic agent. In some embodiments, the composition does not comprise a prophylactic or therapeutic agent.III. Methods of preparation

[0150] In one aspect, there is provided a method of preparing any of the under-lipidated particle compositions described herein. In one aspect, there is provided a method of preparing a composition comprising a plurality of particles, comprising a polymer (e.g., amphipathic polymer) and a phospholipid, wherein at least about 85% (e.g., at least about any of 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the plurality of particles are under-lipidated. In some embodiments, the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and 2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than (e.g., less than about any of 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower of) a phospholipid-polymer saturation ratio Ns. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than or equal to the molar ratio of the phospholipid to the amphipathic polymer in the composition. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than about 95% (e.g., less than about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, or lower) of the molar ratio of the phospholipid to theamphipathic polymer in the composition. In some embodiments, the method further comprises purifying the obtained particles, e.g., by SEC (one or more times). In some embodiments, the method further comprises verifying the obtained particle composition, e.g., after the final purification step. Also see Examples 1-3 for exemplary methods of preparing, purifying, as well as verification of the under-lipidated particle compositions described herein. In some embodiments, the method further comprises testing one or more properties of the obtained particle compositions: lipid (e.g., cholesterol) binding capacity at equilibrium, cholesterol efflux capacity, and / or stability (e.g., after freeze thaw cycle(s)), such as using any of the testing methods described herein (e.g., see Examples 4, 5, 8, 10, 11, and 13).

[0151] In some embodiments, the particles can be synthesized by incubating solubilized phospholipid with an amphipathic polymer (e.g., amphipathic polypeptide). In some embodiments, the phospholipid is solubilized by a detergent or an organic compound. In some embodiments, the solubilized phospholipid is prepared by adding solution of detergent or an organic compound to a thin film of phospholipid. Exemplary organic compounds include, but are not limited to, alkyglucosides such as n-dodecyl-P-D-maltoside (DDM), octyl-P-glucoside (OG), Triton X-100, 3-[(3-cholamidopropyl) dimethylammonio]-l- propanesulfonate (CHAPS), 2,2-didecylpropane-l,3-bis-P-D-maltopyranoside (LMNG), cholate, and any combination thereof. In some embodiments, the detergent or organic compound comprises a cholate, such as sodium cholate. In some embodiments, the amphipathic polymer is provided in a solution comprising a buffer. In some embodiments, the buffer has a pH of about 5 to about 9, such as about 6 to about 8, about 7, or about 7.5. In some embodiments, the amphipathic polymer in the solution is at least about 0.1 mg / mL, such as at least about any of 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 amphipathic polymer in the solution is about 0.5 mg / mL to about 10 mg / mL, such as any 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.

[0152] In some embodiments, the preparation method comprises removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles. In some embodiments, the organic compound (e.g., cholate) is removed, such as by incubation with absorbent polymer beads. In some embodiments, the absorbent polymer beads comprise styrene-divinylbenzene (macroreticular) beads (e.g., Amberlite® XAD-2® Beads). In someembodiments, the formation and size of particles can be determined by methods known in the art, such as SEC and electron microscopy.

[0153] In some embodiments, the preparation method further comprises isolating the particles. In some embodiments, the particles are isolated by chromatography. In some embodiments, the preparation method further comprises subjecting the particles to one or more freeze thaw cycles with or without cryoprotectant.

[0154] In some embodiments, the preparation method further comprises purifying the prepared particles, such as separating particles with different phospholipid to MSP ratios. In some embodiments, the purification comprises the use of SEC. In some embodiments, the preparation method comprises more than one purification cycles until a desired distribution of phospholipid to MSP ratio (e.g., less than phospholipid-polymer saturation ratio Ns) is reached.

[0155] In some embodiments, the preparation method further comprises measuring the actual phospholipid to MSP ratio in the particle compositions. In some embodiments, the actual phospholipid to MSP ratio in the particle compositions can be determined by measuring the fatty acid content using gas chromatography (GC) analysis.

[0156] In some embodiments, the polymer is any of the polymers (e.g., amphipathic polymer) described herein. In some embodiments, the amphipathic polymer is an amphipathic polypeptide. In some embodiments, the amphipathic polypeptide comprises one or more amphipathic helix sequences of about 9 to about 30 amino acids. In some embodiments, the amphipathic polypeptide is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, and a variant thereof. In some embodiments, the amphipathic polypeptide is a variant of ApoA-I. In some embodiments, the amphipathic polypeptide comprises one or more amphipathic helix sequences derived from ApoA-I. In some embodiments, the amphipathic polypeptide comprises an N-terminus truncation relative to ApoA-I. In some embodiments, the amphipathic polypeptide is selected from the group consisting of MSP1E1, MSP1E2, MSP1E3, and a variant thereof. In some embodiments, the amphipathic polypeptide is selected from the group consisting of MSP ID 1, MSPE1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and a variant thereof. In some embodiments, the amphipathic polypeptide is MSP1D1. In some embodiments, the amphipathic polypeptide is MSP1E3D1. In some embodiments, the amphipathic polypeptide is a variant of any of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP2N2, wherein the variant does not comprise the N-terminal His-tag portion (his-tag removed variant). In some embodiments, the amphipathic polypeptide is (i) an MSP1D1 His-tag removed variant (MSP1D1(-)) comprisingthe amino acid sequence of SEQ ID NO: 11; (ii) an MSP1E3D1 His-tag removed variant (MSP1E3D1(-)) comprising the amino acid sequence of SEQ ID NO: 12; (iii) an MSP1E1D1 His-tag removed variant (i.e., MSPIEIDI(-)) comprising the amino acid sequence of SEQ ID NO: 17; (iv) an MSP1E2D1 His-tag removed variant (i.e., MSP1E2D1(-)) comprising the amino acid sequence of SEQ ID NO: 18; or (v) an MSP2N2 His-tag removed variant (i.e., MSP2N2(-)) comprising the amino acid sequence of SEQ ID NO: 19.

[0157] 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 with lengths 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, the 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.

[0158] In some embodiments, the molar ratio of phospholipid to the amphipathic polymer in the preparation mixture (e.g., mixture of method of preparation step (1) described above) is the same as that in the obtained particle composition. In some embodiments, the molar ratio of phospholipid to the amphipathic polymer in the preparation mixture (e.g., mixture of method of preparation step (1) described above) is less than (e.g., less than about any of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40% of) that in the obtained particle composition. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than about 80% of the molar ratio of the phospholipid to the amphipathic polymer in the particle composition. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is determined such that at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the as-prepared particles are under- lipidated as defined herein. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is determined such that at least about 60%, such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the as- prepared particles have a molar ratio of the phospholipid to the amphipathic polymer lessthan about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns.

[0159] In some embodiments, the amphipathic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC or DPPC, and the molar ratio of phospholipid to the amphipathic polymer in the preparation mixture (e.g., mixture of method of preparation step (1) described above) is less than about 95% (e.g., less than about any of 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40%) of the phospholipid-polymer saturation ratio Ns of about 81 : 1. In some embodiments, the molar ratio of phospholipid to the amphipathic polymer in the preparation mixture is at least about 10: 1, such as about any of 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1 or 45: 1. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is about 10: 1 to about 85: 1, such as any of about 10:1 to about 70: 1, about 20: 1 to about 60: 1, about 30: 1 to about 70: 1, about 40: 1 to about 70: 1, about 65: 1, about 60: 1, about 50: 1, or about 40:1.

[0160] In some embodiments, there is provided a method of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated, wherein the amphipathic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, and wherein the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and 2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 81 : 1. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than (e.g., less than about 95% of) or equal to the molar ratio of the phospholipid to the amphipathic polymer in the composition.

[0161] In some embodiments, the amphipathic polymer is MSP ID 1 or His-tag removed variant thereof (MSP1D1(-)), the phospholipid is DMPC, and the phospholipid to the amphipathic 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 His-tag removed variant thereof in the preparation mixture is less than or equal to about 70: 1, such as less than or equal to about any of 65: 1, 60: 1, 55: 1, 50: 1, 45: 1, or 40: 1. In some embodiments, themolar ratio of DMPC to MSP ID 1 or His-tag removed variant thereof in the composition is about 75: 1, and the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the preparation mixture is about 55: 1. In some embodiments, the amphipathic polymer is MSP1D1. In some embodiments, the amphipathic polymer is an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amphipathic polymer is an MSP1D1 His-tag removed variant defined by the amino acid sequence of SEQ ID NO: 11.

[0162] Hence in some embodiments, there is provided a method of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated, wherein the amphipathic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, wherein the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and 2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles; wherein the phospholipid to the amphipathic polymer in the composition is from about 72: 1 to about 78: 1 (e.g., about 75: 1), and wherein the molar ratio of the phospholipid to the amphipathic 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 MSP ID 1 or His-tag removed variant thereof in the composition is about 75: 1, and the molar ratio of DMPC to MSP1D1 or His-tag removed variant thereof in the preparation mixture is about 55: 1. In some embodiments, the amphipathic polymer is an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the method further comprises purifying the obtained composition.

[0163] In some embodiments, the amphipathic polymer is MSP ID 1 or His-tag removed variant thereof (MSP1D1(-)), the phospholipid is DMPC, and the phospholipid to the amphipathic polymer in the composition is from about 48: 1 to about 55: 1. In some embodiments of the foregoing, the molar ratio of the phospholipid DMPC to the amphipathic polymer MSP ID 1 or His-tag removed variant thereof in the preparation mixture is less than or equal to about 50: 1, such as less than or equal to about any of 45: 1, 40: 1, 35: 1, 30:1, 25: 1, or 20: 1. In some embodiments, the molar ratio of the phospholipid DMPC to the amphipathic polymer MSP ID 1 or a His-tag removed variant thereof in the composition is about 46: 1 to about 55: 1, about 51 : 1, or about 55:1, and the molar ratio of the phospholipid DMPC to the amphipathic polymer MSP ID 1 or a His-tag removed variant thereof in the preparationmixture is about 40:1 or about 30:1. In some embodiments, the amphipathic polymer is MSP1D1. In some embodiments, the amphipathic polymer is an MSP1D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 11.

[0164] Hence in some embodiments, there is provided a method of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated, wherein the amphipathic polymer is MSP1D1 (e.g., SEQ ID NO: 2) or His-tag removed variant thereof (MSPIDI(-); e.g., SEQ ID NO: 11), the phospholipid is DMPC, wherein the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and 2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles; wherein the phospholipid to the amphipathic polymer in the composition is from about 46:1 to about 55:1 (e.g., about 51:1), and wherein the molar ratio of the phospholipid to the amphipathic 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 MSP ID 1 or His-tag removed variant thereof in the composition is about 51:1, and the molar ratio of DMPC to MSP ID 1 or His-tag removed variant thereof in the preparation mixture is about 40: 1 or about 30: 1. In some embodiments, the amphipathic polymer is an MSP ID 1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the method further comprises purifying the obtained composition.

[0165] In some embodiments, there is provided a method of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, and wherein the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and 2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns of about 167:1. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than(e.g., less than about 95% of) or equal to the molar ratio of the phospholipid to the amphipathic polymer in the composition.

[0166] In some embodiments, the amphipathic polymer is MSP1E3D1 or His-tag removed variant thereof (MSP1E3D1(-)), the phospholipid is DMPC, and the molar ratio of phospholipid to amphipathic polymer in the preparation mixture is about 30: 1 to 100: 1, including for example any 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 phospholipid to the amphipathic polymer in the preparation mixture is at least about 30: 1, such as about any of 30: 1, 35: 1, 45: 1, 50:1, 55: 1, 60: 1, 65: 1, or 70: 1. In some embodiments, the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is about 40: 1 to about 90: 1, such as any of about 45: 1, 50: 1, 55: 1, or 60: 1.

[0167] In some embodiments, the amphipathic polymer is MSP1E3D1 or His-tag removed variant thereof (MSP1E3D1(-)), the phospholipid is DMPC, and the phospholipid to the amphipathic 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 His-tag removed variant thereof in the preparation mixture is less than or equal to about 90: 1, such as less than or equal to about any of 85: 1, 80: 1, 75: 1, 70: 1, 65: 1, 60: 1, 55: 1, or 50: 1. In some embodiments, the molar ratio of the phospholipid DMPC to the amphipathic polymer His-tag removed variant thereof in the composition is about 87: 1 to about 98: 1 or about 87: 1 to about 96: 1, and the molar ratio of the phospholipid DMPC to the amphipathic polymer His-tag removed variant thereof in the preparation mixture is about 90: 1. In some embodiments, the amphipathic polymer is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the phospholipid to the amphipathic 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 His-tag removed variant thereof in the preparation mixture is less than or equal to about 60: 1, such as less than or equal to about any of 55: 1, 50: 1, 45: 1, 40: 1, 35: 1, or 30: 1. In some embodiments, the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof in the composition is about 58: 1 to about 68: 1, about 63 : 1, or about 62: 1, and the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof in the preparation mixture is about 60: 1. In some embodiments, the amphipathic polymer is MSP1E3D1. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant defined by SEQ ID NO: 12.

[0168] Hence in some embodiments, there is provided a method of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, wherein the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and 2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles; wherein the phospholipid to the amphipathic polymer in the composition is from about 58:1 to about 68:1 (e.g., about 63:1), and wherein the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is about 60:1. In some embodiments, the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof in the composition is about 63 : 1, and the molar ratio of DMPC to MSP ID 1 or His-tag removed variant thereof in the preparation mixture is about 60:1. In some embodiments, the amphipathic polymer is an MSP1E3D1 His- tag removed variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the method further comprises purifying the obtained composition.

[0169] In some embodiments, the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof (MSP1E3D1(-)) in the composition is about 62 : 1, and the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof in the preparation mixture is about 50:1. In some embodiments, the amphipathic polymer is MSP1E3D1. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the amphipathic polymer is an MSP1E3D1 His-tag removed variant defined by SEQ ID NO: 12.

[0170] Hence in some embodiments, there is provided a method of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated, wherein the amphipathic polymer is MSP1E3D1 (e.g., SEQ ID NO: 5) or His-tag removed variant thereof (MSP1E3D1(-); e.g., SEQ ID NO: 12), the phospholipid is DMPC, wherein the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and 2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles; wherein the phospholipid to the amphipathic polymer in the composition is from about 58: 1 to about 68: 1 (e.g., about 62: 1),and wherein the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is about 50: 1. In some embodiments, the molar ratio of DMPC to MSP1E3D1 or His-tag removed variant thereof in the composition is about 62: 1, and the molar ratio of DMPC to MSP ID 1 or His-tag removed variant thereof in the preparation mixture is about 50: 1. In some embodiments, the amphipathic polymer is an MSP1E3D1 His- tag removed variant comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the method further comprises purifying the obtained composition.IV. Methods of Use

[0171] In one aspect, provided herein is a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in a cell, comprising contacting the cell with an effective amount of any of the compositions (e.g., a pharmaceutical composition) described herein, wherein the composition comprises a plurality of particles comprising an amphipathic polymer and a phospholipid. In some embodiments, at least about 85% of the plurality of particles are under-lipidated. In some embodiments, there is provided a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in a cell, comprising contacting the cell with an effective amount of any of the under-lipidated particle compositions described herein. In one aspect, provided herein is a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in an individual (e.g., human), comprising administering to the individual an effective amount of any of the compositions (e.g., a pharmaceutical composition) described herein. In one aspect, provided herein is a method of treating or preventing a disease associated with lipid (e.g., cholesterol) accumulation in an individual, comprising administering to the individual an effective amount of any of the under-lipidated particle compositions (e.g., a pharmaceutical composition) described herein. The composition (e.g., pharmaceutical composition) can be any of: DMPC:MSP1D1(-)=72: 1 particle composition, DMPC:MSP1D1(-)=51 : 1 particle composition, DMPC:MSP1D1(-)=75: 1 particle composition, DMPC:MSP1E3D1(-)=62: 1 particle composition, DMPC:MSP1E3D1(- )=63 : 1 particle composition, or DMPC:MSP1E3D1(-)=94: 1 particle composition.

[0172] In some embodiments, provided herein is a method of removing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of any of the compositions (e.g., a pharmaceutical composition) described herein. The composition (e.g., pharmaceutical composition) can be any of: DMPC:MSP1D1(-)=72: 1 particle composition, DMPC:MSP1D1(-)=51 : 1 particle composition, DMPC:MSP1D1(-)=75: 1 particlecomposition, DMPC:MSP1E3D1(-)=62: 1 particle composition, DMPC:MSP1E3D1(-)=63: 1 particle composition, or DMPC:MSP1E3D1(-)=94: 1 particle composition. In some embodiments, the lipid for removal or prevention from accumulation in the eye is cholesterol. In some embodiments, the composition is administered intravitreally. In another aspect, provided herein is a method of preventing or treating an eye disease characterized by lipid (e.g., cholesterol) accumulation in an individual (e.g., human), comprising administering to the eye of the individual an effective amount of any of the compositions (e.g., a pharmaceutical composition) described herein. In some embodiments, the lipid for removal or prevention from accumulation in the eye is cholesterol. In some embodiments, the eye disease characterized by lipid accumulation is photoreceptor neurodegeneration, a retinal neurovascular disorder, 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, the dry AMD is geographical 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 can be administered intravitreally.

[0173] In some embodiments, there is provided a method of preventing or treating an AMD in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of any of the compositions (e.g., a pharmaceutical composition) described herein. The composition (e.g., pharmaceutical composition) can be any of: DMPC:MSP1D1(-)=72: 1 particle composition, DMPC:MSP1D1(-)=51 : 1 particle composition, DMPC:MSP1D1(-)=75: 1 particle composition, DMPC:MSP1E3D1(-)=62: 1 particle composition, DMPC:MSP1E3D1(-)=63: 1 particle composition, or 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 comprises CNV.

[0174] In some embodiments, the lipid for removal or prevention from accumulation in the eye is selected from the group consisting of fats, sterols, phospholipids and any combination thereof. In some embodiments, the lipid comprises cholesterol esters (ChEs), wax esters (WEs), diesters, triacylglycerol (TG), free cholesterol, free fatty acids (FAs), (O-acyl)-co- hydroxy fatty acids (OAHFAs), or any combination thereof. In some embodiments, the lipid is a fatty acid or its derivative (including tri-, di-, mono-glycerides, and phospholipids). In some embodiments, the lipid for removal or prevention from accumulation in the eye comprises two or more of the lipids described herein.

[0175] In some embodiments, the eye diseases characterized by lipid accumulation is selected from the group consisting of AMD, photoreceptor neurodegeneration, optic nerve atrophy, loss of acuity, hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, and any combination thereof. In some embodiments, 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 angiomatous proliferation (RAP), retinal neovascularization (RNV), juvenile macular degeneration (e.g., Stargardt disease), macular telangiectasia, maculopathy (e.g., age-related maculopathy (ARM) and diabetic maculopathy (DMP), including partial 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 macular edema following RVO, including central RVO and branch RVO), retinopathy (e.g., diabetic retinopathy (DR), including in patients with DME, proliferative vitreoretinopathy (PVR), Purtscher’s retinopathy, and radiation retinopathy), retinal artery occlusion (RAO, e.g., central and branch RAO), retinal vein occlusion (RVO, e.g., central RVO, including central RVO with cystoid macular edema (CME), and branch RVO, including branch RVO with CME), retinitis (e.g., Coats' disease (exudative retinitis) and retinitis pigmentosa (RP)), chorioretinitis, choroiditis (e.g., serpiginous choroiditis), uveitis (including anterior uveitis, intermediate uveitis, posterior uveitis with or without CME, pan-uveitis and non-infectious uveitis), retinal detachment (e.g., in von Hippel-Lindau disease), retinal pigment epithelium (RPE) detachment, dystrophies of rods or / and cones, and any combination thereof.

[0176] In some embodiments, the eye disease characterized by lipid accumulation is photoreceptor neurodegeneration or retinal neurovascular disorder.

[0177] In some embodiments, the eye disease characterized by lipid accumulation is AMD. In some embodiments, the AMD is dry AMD. Dry AMD is generally characterized by a buildup of yellowish deposits called drusen beneath the retina and typically affects vision in both eyes, although vision loss often occurs in one eye before the other. A more advanced stage of dry AMD is known as geographic atrophy (GA), in which areas of the macula waste away (atrophy), resulting in severe vision loss.

[0178] Dry AMD can progress into wet AMD. Wet AMD is generally characterized by the growth of leaky blood vessels underneath the macula. This growth of leaky blood vessels is called choroidal neovascularization. The vessels leak blood and fluid, which damages the macula and makes central vision appear blurry and distorted. Wet macular degeneration isassociated with severe vision loss that can worsen rapidly. It has been shown that high levels of intracellular cholesterol significantly increase choroidal neovascularization. Thus, in some embodiments, the eye disease characterized by lipid accumulation is wet AMD. In some embodiments, the eye disease characterized by lipid accumulation is choroidal neovascularization (CNV). In some embodiments, the wet AMD comprises CNV and / or RAP.

[0179] In some embodiments, the methods for removing lipid or preventing lipid accumulation, or for treating a disease associated with lipid (e.g., cholesterol) accumulation in a cell, tissue, or organ described here can achieve one or more following effects: i) removing (such as by actively removing and / or passively removing; e.g., removing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) lipid (e.g., cholesterol) in the cell, tissue, or organ with accumulated lipid, such as the eye; ii) increasing (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1- fold, 2-fold, 2.5-fold, 5-fold, 10-fold, or more) the efflux rate of the lipid (e.g., cholesterol) in the cell, tissue, or organ with accumulated lipid, such as the eye; iii) reducing (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the likelihood of the occurrence of lipid accumulation and / or disease associated with lipid (e.g., cholesterol) accumulation in a cell, tissue, or organ, such as the eye; iv) delaying (e.g., delaying at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, or more months) the onset of lipid accumulation and / or disease associated with lipid (e.g., cholesterol) accumulation in a cell, tissue, or organ, such as the eye; v) slowing the progression of lipid accumulation and / or disease associated with lipid (e.g., cholesterol) accumulation in a cell, tissue, or organ, such as the eye; vi) increasing cholesterol efflux rate by at least about 2-fold (e.g., at least about any of 2.5, 3, 4, 5, 10, 15-fold, or more) compared to an untreated state, or the cholesterol efflux rate by ApoA-I or HDL or the MSP (e.g., MSP1D1) alone; v) increasing cholesterol efflux rate by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, or at least about any of 2, 2.5, 3, 4, 5, 10, 15-fold, or more) compared to a fully lipidated composition; vi) increasing lipid (e.g., cholesterol) binding capacity at equilibrium (or lipid (e.g., cholesterol) partition coefficient) by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, or at least about any of 2, 2.5, 3, 4, 5, 10, 15-fold, or more) compared to (a) ApoA-I alone, (b) HDL alone, (c) the MSP (e.g., MSP1D1(-)) alone, and / or (d) a fully- lipidated particle composition containing the same amphipathic polymer and phospholipid.

[0180] In some embodiments, the disease is an eye disease characterized by lipid accumulation is GA, and the method of preventing or treating GA provided herein achieves one or more following effects: i) reducing lipid (e.g., cholesterol) or preventing lipid accumulation in the eye of the individual by at least about 5% (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); ii) reducing the total area of GA lesions in the eye of the individual by at least about 5% (such as by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and iii) slows the progression of the total area of GA lesions in the eye of the individual by at least about 5% (such as at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The total area of GA lesions can be determined by any methods known in the art, such as autofluorescence imaging. In some embodiments, the autofluorescence is fundus autofluorescence (FAF).

[0181] Hence in some embodiments, there is provided a method of reducing and / or slowing the progress of total area of GA lesions in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of any of the compositions (e.g., a pharmaceutical composition) described herein. In some embodiments, the methods described herein inhibits (e.g., inhibiting at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) neovascularization in the eye of an individual suffering from AMD. In some embodiments, the method delays (e.g., delaying at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, or more months) the development of AMD or CNV. In some embodiment, the method treats and / or reduces (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the risk of developing retinal neovascularization (RNV). In some embodiment, the method treats and / or reduces (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the risk of developing retinal angiomatous proliferation (RAP). Hence in some embodiments, there is provided a method of i) inhibiting neovascularization, ii) delaying the development of AMD or CNV, iii) treating and / or reducing the risk of developing RNV and / or RAP in the eye of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of any of the compositions (e.g., a pharmaceutical composition) described herein. The composition (e.g., pharmaceutical composition) can be any of: DMPC:MSP1D1(-)=72: 1 particle composition, DMPC:MSP1D1(-)=51 : 1 particle composition, DMPC:MSP1D1(-)=75: 1 particle composition, DMPC:MSP1E3D1(-)=62: 1 particle composition, DMPC:MSP1E3D1(-)=63: 1 particle composition, or DMPC:MSP1E3D1(-)=94: 1 particle composition.

[0182] In some embodiments, the individual is suffering from AMD and is at risk of or suffering from RAP. In some embodiments, the individual is suffering from AMD and is at risk of or suffering from CNV. In some embodiments, the individual is suffering from AMD and is at risk of or suffering from RNV. In some embodiments, the individual has been identified as having one or more genetic polymorphisms that increases the risk of AMD. In some embodiments, the method of preventing or treating an eye disease characterized by lipid accumulation (e.g., AMD) described herein further comprises determining whether the individual has a genetic polymorphism that increases the risk of AMD.

[0183] In some embodiments, there is provided a method of improving vision impairment and / or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation in the eye in an individual (e.g., human), comprising administering to the eye of the individual an effective amount of any of the compositions (e.g., a pharmaceutical composition) described herein. The composition (e.g., pharmaceutical composition) can be any of: DMPC:MSP1D1(-)=72: 1 particle composition, DMPC:MSP1D1(-)=51 : 1 particle composition, DMPC:MSP1D1(-)=75: 1 particle composition, DMPC:MSP1E3D1(-)=62: 1 particle composition, DMPC:MSP1E3D1(-)=63: 1 particle composition, or DMPC:MSP1E3D1(-)=94: 1 particle composition. The vision impairment and / or vision loss can be associated with any of the eye diseases described herein. In some embodiments, the vision impairment or loss is associated with atrophic AMD including non-central or / and central GA, or the vision improvement can occur in a subject with atrophic AMD or AMD.

[0184] In some embodiments, there is provided a method of improving the normal luminance best-corrected visual acuity (NL-BCVA) score and / or the low luminance best-corrected visual acuity (LL-BCVA) score of an individual (e.g., human), comprising administering to the eye of the individual an effective amount of any of the compositions (e.g., a pharmaceutical composition) described herein. In some embodiments, the method provided herein improves the NL-BCVA score and / or the LL-BCVA score of the individual by at least about 5%, such as by at least about any of 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 known methods in the art. The composition (e.g., pharmaceutical composition) can be any of: DMPC:MSP1D1(-)=72: 1 particle composition, DMPC:MSP1D1(-)=51 : 1 particle composition, DMPC:MSP1D1(-)=75: 1 particle composition, DMPC:MSP1E3D1(-)=62: 1 particle composition, DMPC:MSP1E3D1(-)=63: 1 particle composition, or DMPC:MSP1E3D1(-)=94: 1 particle composition.

[0185] In some embodiments, any of the methods of i) removing lipid or preventing lipid accumulation, ii) preventing or treating a disease associated with lipid (e.g., cholesterol) accumulation in a cell, tissue, or organ, such as the eye, iii) preventing or treating an eye disease characterized by lipid accumulation, v) reducing and / or slowing the progress of total area of GA lesions, v) inhibiting neovascularization, vi) delaying the development of AMD or CNV, vii) treating and / or reducing the risk of developing RNV and / or RAP, viii) improving vision impairment and / or reducing the likelihood of vision loss associated with an eye disease characterized by lipid accumulation, and iv) improving the NL-BCVA score and / or the LL-BCVA score mentioned herein is compared to a baseline state before treatment (or untreated state), a natural progression without any treatment, a treatment with placebo, or a treatment with ApoA-I or the MSP protein (e.g., MSP1D1) alone.Routes of administration and Dose

[0186] Compositions comprising particles described herein can be administered by any suitable administration routes. In some embodiments, any of the compositions (e.g., pharmaceutical composition) described herein can be administered locally, for example, via local ocular administration.

[0187] In some embodiments, suitable administration routes include, but are not limited to, intravitreal, topical, periocular injections, intra- or periocular implants, intravitreal implants, and suprachoroidal implants or particles or polymeric composition, or any releasing systems such as emulsions, solid non-biodegradable or degradable implants or tablets, or mini pumps. In some embodiments, any of the compositions (e.g., pharmaceutical composition) described herein can be directly administered to the eye by ocular tissue injection such as periocular, conjunctival, subtenon, intracameral, intravitreal, intraocular, subretinal, subconjunctival, retrobulbar, suprachoroidal, intracoronary, intradermal, or intracanalicular injections. In some embodiments, the composition (e.g., pharmaceutical composition) is administered by direct application to the eye using a catheter or other placement device such as a retinal pellet, intraocular insert, suppository or an implant comprising a porous, non-porous, or gelatinous material. In some embodiments, the composition (e.g., pharmaceutical composition) is administered by topical ocular drops or ointments. In some embodiments, the composition (e.g., pharmaceutical composition) is administered by a slow-release device in the cul-de-sac or implanted adjacent to the sclera (transscleral) or in the sclera (intrascleral) or suprachoroidal or within the eye. Intracameral injection may be through the cornea into the anterior chamber to allow the agent to reach the trabecular meshwork. Intracanalicularinjection may be into the venous collector channels draining Schlemm's canal or into Schlemm's canal. In some embodiments, the composition (e.g., pharmaceutical composition) is administered intravitreally.

[0188] In some embodiments, the composition (e.g., pharmaceutical composition) may be in the form of isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), sterile aqueous dispersions (e.g., water-oil emulsion), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

[0189] In some embodiments, the composition (e.g., pharmaceutical composition) further comprises a pharmaceutically acceptable excipient is administered topically. Suitable pharmaceutically acceptable excipient includes, but are not limited to, pharmaceutically or ophthalmologically acceptable preservatives, solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, isotonic agents, stabilizer, pH regulators, or water to form an aqueous, sterile ophthalmic suspension or solution. In some embodiments, the retention of the pharmaceutical composition can be further improved by viscosity building agents, such as hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, hyaluronic acid, or the like.

[0190] In some embodiments, the composition (e.g., pharmaceutical composition) can be administered as an ophthalmic ointment composition. In some embodiments, the pharmaceutically acceptable excipient comprises a preservative in an appropriate vehicle, such as mineral oil, liquid lanolin, or white petrolatum.

[0191] In some embodiments, the composition (e.g., pharmaceutical composition) can be an anhydrous dosage form comprising a plurality of particles. Such an anhydrous dosage form can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.

[0192] In some embodiments, the composition (e.g., pharmaceutical composition) can further comprise a buffer. In some embodiments, the buffer is an isotonic buffer. In some embodiments, the isotonic buffer is phosphate-buffer saline (PBS). In some embodiments, the isotonic buffer does not comprise tromethamine (tris).

[0193] In some embodiments, the composition (e.g., pharmaceutical composition) can further comprise 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 phenylethyl alcohol.

[0194] In some embodiments, the excipient can be selected by one of ordinary skill in the art. Exemplary excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), the content of which is incorporated by reference herein in its entirety.

[0195] In some embodiments, a doctor can determine the dosage which they consider as most appropriate according to a preventive or curative treatment and according to the age, weight, condition, and other factors specific to the individual to be treated.

[0196] In some embodiments, the frequency and dosage may also vary according to factors specific for each individual depending on the specific therapy (e.g., therapeutic or prophylactic), the route of administration, as well as age, body, weight, response, and the past medical history of the individual. In some embodiments, effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0197] In some embodiments, dose of any of the compositions (e.g., pharmaceutical composition) described herein can be about 1 pg / kg to about 1 g / kg.

[0198] It may be necessary to use dosages of any of the compositions (e.g., pharmaceutical composition) described herein outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with the individual’s response.EXEMPLARY EMBODIMENTS

[0199] Embodiment 1. A composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated.

[0200] Embodiment 2. The composition of embodiment 1, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipidpolymer saturation ratio Ns.

[0201] Embodiment 3. The composition of embodiment 2, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 95% of the phospholipid-polymer saturation ratio Ns.

[0202] Embodiment 4. The composition of any one of embodiments 1-3, wherein the amphipathic polymer is an amphipathic polypeptide.

[0203] Embodiment 5. The composition of embodiment 4, wherein the amphipathic polypeptide comprises one or more amphipathic helix sequences of about 9 to about 30 amino acids.

[0204] Embodiment 6. The composition of embodiment 4 or 5, wherein the amphipathic polypeptide is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, and a variant thereof.

[0205] Embodiment 7. The composition of embodiment 6, wherein the amphipathic polypeptide is a variant of ApoA-I.

[0206] Embodiment 8. The composition of embodiment 6 or 7, wherein the amphipathic polypeptide comprises one or more amphipathic helix sequences derived from ApoA-I.

[0207] Embodiment 9. The composition of embodiment 7 or 8, wherein the amphipathic polypeptide comprises an N-terminus truncation relative to ApoA-I.

[0208] Embodiment 10. The composition of any one of embodiments 4-9, wherein the amphipathic polypeptide is selected from the group consisting of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and a variant thereof.

[0209] Embodiment 11. The composition of embodiment 10, wherein the amphipathic polypeptide is a variant of any of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP2N2, wherein the variant does not comprise the N-terminal His-tag portion (his-tag removed variant).

[0210] Embodiment 12. The composition of embodiment 11, wherein the amphipathic polypeptide is (i) an MSP ID 1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 11; or (ii) an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 12.

[0211] Embodiment 13. The composition of any one of embodiments 1-10, wherein the phospholipid comprises one or more acyl chains, optionally with a length of about 10 to about 22 carbon units, and a glycol backbone, and wherein the phospholipid is cationic, anionic, zwitterionic, or any combination thereof.

[0212] Embodiment 14. The composition of any one of embodiments 1-13, wherein the phospholipid comprises DMPC, DPPC, DMPS, POPC, POPS, or PiP2.

[0213] Embodiment 15. The composition of any one of embodiments 1-14, wherein the amphipathic polymer is MSP ID 1 or His-tag removed variant thereof, the phospholipid is DMPC, and the Ns is about 81.

[0214] Embodiment 16. The composition of embodiment 15, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 78:1.

[0215] Embodiment 17. The composition of embodiment 15, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 65:1 to about 75:1.

[0216] Embodiment 18. The composition of embodiment 15, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 40: 1 to about 60: 1.

[0217] Embodiment 19. The composition of embodiment 15 or 18, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 46: 1 to about 55:1.

[0218] Embodiment 20. The composition of any one of embodiments 1-14, wherein the amphipathic polymer is MSP ID 1 or His-tag removed variant thereof, the phospholipid is DPPC, and the Ns is about 81.

[0219] Embodiment 21. The composition of embodiment 20, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 75:1.

[0220] Embodiment 22. The composition of any one of embodiments 1-14, wherein the amphipathic polymer is MSP ID 1 or His-tag removed variant thereof, the phospholipid is POPC, and the Ns is about 62.

[0221] Embodiment 23. The composition of embodiment 22, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 58:1.

[0222] Embodiment 24. The composition of any one of embodiments 1-11, 13, or 14, wherein the amphipathic polymer is MSP1E1D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the Ns is about 107.

[0223] Embodiment 25. The composition of embodiment 24, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 100:1.

[0224] Embodiment 26. The composition of embodiment 24 or 25, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 60: 1 to about 65:1.

[0225] Embodiment 27. The composition of any one of embodiments 1-11, 13, or 14, wherein the amphipathic polymer is MSP1E1D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the Ns is about 107.

[0226] Embodiment 28. The composition of embodiment 27, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 100:1.

[0227] Embodiment 29. The composition of any one of embodiments 1-11, 13, or 14, wherein the amphipathic polymer is MSP1E1D1 or His-tag removed variant thereof, the phospholipid is POPC, and the Ns is about 80.

[0228] Embodiment 30. The composition of embodiment 29, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 76: 1.

[0229] Embodiment 31. The composition of any one of embodiments 1-11, 13, or 14, wherein the amphipathic polymer is MSP1E2D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the Ns is about 135.

[0230] Embodiment 32. The composition of embodiment 31, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 128: 1.

[0231] Embodiment 33. The composition of any one of embodiments 1-11, 13, or 14, wherein the amphipathic polymer is MSP1E2D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the Ns is about 135.

[0232] Embodiment 34. The composition of embodiment 33, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 128: 1.

[0233] Embodiment 35. The composition of any one of embodiments 1-11, 13, or 14, wherein the amphipathic polymer is MSP1E2D1 or His-tag removed variant thereof, the phospholipid is POPC, and the Ns is about 102.

[0234] Embodiment 36. The composition of embodiment 35, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 96: 1.

[0235] Embodiment 37. The composition of any one of embodiments 1-14, wherein the amphipathic polymer is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the Ns is about 167.

[0236] Embodiment 38. The composition of embodiment 37, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 158: 1.

[0237] Embodiment 39. The composition of embodiment 37 or 38, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 50: 1 to about 100: 1.

[0238] Embodiment 40. The composition of embodiment 39, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 87: 1 to about 96: 1, from about 58: 1 to about 68: 1, about 63 : 1, or about 62: 1.

[0239] Embodiment 41. The composition of any one of embodiments 1-14, wherein the amphipathic polymer is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DPPC, and the Ns is about 167.

[0240] Embodiment 42. The composition of embodiment 41, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 158: 1.

[0241] Embodiment 43. The composition of any one of embodiments 1-14, wherein the amphipathic polymer is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is POPC, and the Ns is about 126.

[0242] Embodiment 44. The composition of embodiment 43, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 118: 1.

[0243] Embodiment 45. The composition of any one of embodiments 1-11, 13, or 14, wherein the amphipathic polymer is MSP2N2 or His-tag removed variant thereof, the phospholipid is DMPC, and the Ns is about 387.

[0244] Embodiment 46. The composition of embodiment 45, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 367: 1.

[0245] Embodiment 47. The composition of any one of embodiments 1-11, 13, or 14, wherein the amphipathic polymer is MSP2N2 or His-tag removed variant thereof, the phospholipid is DPPC, and the Ns is about 387.

[0246] Embodiment 48. The composition of embodiment 47, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 367: 1.

[0247] Embodiment 49. The composition of any one of embodiments 1-11, 13, or 14, wherein the amphipathic polymer is MSP2N2 or His-tag removed variant thereof, the phospholipid is POPC, and the Ns is about 291.

[0248] Embodiment 50. The composition of embodiment 49, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 276: 1.

[0249] Embodiment 51. The composition of any one of embodiments 1-50, wherein the average size of the particles in the composition is about 2 nm to about 20 nm in diameter.

[0250] Embodiment 52. The composition of embodiment 51, wherein the poly dispersity index of the size of the particles in the composition is less than about 0.2.

[0251] Embodiment 53. The composition of any one of embodiments 1-52, wherein the poly dispersity index of the phospholipid content of the particles in the composition is less than about 0.2.

[0252] Embodiment 54. The composition of any one of embodiments 1-53, wherein the composition is a pharmaceutical composition.

[0253] Embodiment 55. A method of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated, the method comprises: 1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles.

[0254] Embodiment 56. The method of embodiment 55, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipidpolymer saturation ratio Ns.

[0255] Embodiment 57. The method of embodiment 55 or 56, wherein the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than or equal to the molar ratio of the phospholipid to the amphipathic polymer in the composition.

[0256] Embodiment 58. The method of embodiment 57, wherein the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than about 95% of the molar ratio of the phospholipid to the amphipathic polymer in the composition.

[0257] Embodiment 59. The method of any one of embodiments 55-58, wherein the detergent or organic compound comprises a cholate.

[0258] Embodiment 60. The method of any one of embodiments 55-59, wherein the amphipathic polymer is provided in a solution comprising a buffer.

[0259] Embodiment 61. The method of any one of embodiments 55-60, wherein the concentration of the amphipathic polymer in the solution is about 1 mg / ml to about 5 mg / mL.

[0260] Embodiment 62. The method of any one of embodiments 55-61, wherein the concentration of the amphipathic polymer in the solution is about 2.5 mg / ml to about 4 mg / mL.

[0261] Embodiment 63. The method of any one of embodiments 55-62, wherein the method further comprises isolating the particles.

[0262] Embodiment 64. The method of embodiment 63, wherein the particles are isolated by chromatography.

[0263] Embodiment 65. The method of any one of embodiments 55-64, wherein the method further comprises subjecting the particles to one or more freeze thaw cycles with or without a cryoprotectant.

[0264] Embodiment 66. The method of any one of embodiments 55-65, wherein the amphipathic polymer is an amphipathic polypeptide.

[0265] Embodiment 67. The method of embodiment 66, wherein the amphipathic polypeptide comprises one or more amphipathic helix sequences of about 9 to about 30 amino acids.

[0266] Embodiment 68. The method of embodiment 66 or 67, wherein the amphipathic polypeptide is selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, and a variant thereof.

[0267] Embodiment 69. The method of embodiment 68, wherein the amphipathic polypeptide is a variant of ApoA-I.

[0268] Embodiment 70. The method of embodiment 68 or 69, wherein the amphipathic polypeptide comprises one or more amphipathic helix sequences derived from ApoA-I.

[0269] Embodiment 71. The method of embodiment 69 or 70, wherein the amphipathic polypeptide comprises an N-terminus truncation relative to ApoA-I.

[0270] Embodiment 72. The method of any one of embodiments 66-71, wherein the amphipathic polypeptide is selected from the group consisting of MSP1D1, MSPE1D1, MSP1E2D1, MSP1E3D1, MSP2N2, and a variant thereof.

[0271] Embodiment 73. The method of embodiment 72, wherein the amphipathic polypeptide is a variant of any ofMSPIDl, MSP1E1D1, MSP1E2D1, MSP1E3D1, and MSP2N2, wherein the variant does not comprise the N-terminal His-tag portion (his-tag removed variant).

[0272] Embodiment 74. The method of embodiment 73, wherein the amphipathic polypeptide is (i) an MSP ID 1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 11; or (ii) an MSP1E3D1 His-tag removed variant comprising the amino acid sequence of SEQ ID NO: 12.

[0273] Embodiment 75. The method of any one of embodiments 55-74, wherein the phospholipid comprises one or more acyl chains, optionally with lengths of about 10 to about 22 carbon units.

[0274] Embodiment 76. The method of any one of embodiments 55-75, wherein the phospholipid has a glycerol backbone.

[0275] Embodiment 77. The method of any one of embodiments 55-76, wherein the phospholipid is cationic, anionic, zwitterionic, or any combination thereof.

[0276] Embodiment 78. The method of any one of embodiments 55-77, wherein the phospholipid comprises DMPC, DPPC, DMPS, POPC, POPS, or PiP2.

[0277] Embodiment 79. The method of any one of embodiments 55-78, wherein the amphipathic polymer is MSP ID 1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 65:1 to about 75:1.

[0278] Embodiment 80. The method of embodiment 79, wherein the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than about 65:1.

[0279] Embodiment 81. The method of embodiment 79 or 80, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is about 65: 1 to about 75: 1, and the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is about 55:1.

[0280] Embodiment 82. The method of any one of embodiments 55-78, wherein the amphipathic polymer is MSP ID 1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 46:1 to about 55:1.

[0281] Embodiment 83. The method of embodiment 82, wherein the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than about 45:1.

[0282] Embodiment 84. The method of embodiment 82 or 83, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is about 46: 1 to about 55:1, and the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is about 40: 1 or about 30:1.

[0283] Embodiment 85. The method of any one of embodiments 55-78, wherein the amphipathic polymer is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 85:1 to about 100:1.

[0284] Embodiment 86. The method of embodiment 85, wherein the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than or equal to about 90:1.

[0285] Embodiment 87. The method of embodiment 85 or 86, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 87: 1 to about 96: 1, and the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is about 90:1.

[0286] Embodiment 88. The method of any one of embodiments 55-78, wherein the amphipathic polymer is MSP1E3D1 or His-tag removed variant thereof, the phospholipid is DMPC, and the molar ratio of the phospholipid to the amphipathic polymer in the composition is from about 55:1 to about 80:1.

[0287] Embodiment 89. The method of embodiment 88, wherein the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than or equal to about 60:1.

[0288] Embodiment 90. The method of embodiment 88 or 89, wherein the molar ratio of the phospholipid to the amphipathic 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 amphipathic polymer in the preparation mixture is about 60: 1.

[0289] Embodiment 91. The method of embodiment 88 or 89, wherein the molar ratio of the phospholipid to the amphipathic 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 amphipathic polymer in the preparation mixture is about 50: 1.

[0290] Embodiment 92. A composition comprising the particles prepared according to the method of any one of embodiments 51-85.

[0291] Embodiment 93. A method of treating or preventing a disease associated with lipid accumulation in an individual, comprising administering an effective amount of the composition of any one of embodiments 1-54 and 92 to the individual.

[0292] Embodiment 94. The method of embodiment 93, wherein the disease is associated with lipid accumulation in the eye.

[0293] Embodiment 95. The method of embodiment 93 or 94, wherein the disease is age- related macular degeneration (AMD), photoreceptor neurodegeneration, optic nerve atrophy, loss of acuity, hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, or any combination thereof.

[0294] Embodiment 96. The method of any one of embodiments 93-95, wherein the disease is AMD.

[0295] Embodiment 97. The method of embodiment 96, wherein the AMD is dry AMD.

[0296] Embodiment 98. The method of embodiment 97, wherein the dry AMD is geographic atrophy (GA).

[0297] Embodiment 99. The method of any one of embodiments 93-98, wherein the composition reduces lipid or prevent lipid accumulation in the individual by at least about 5%.EXAMPLESExample 1: Preparation of Exemplary Under-lipidated Particle Compositions

[0298] Compositions comprising a plurality of particles were prepared from a phospholipid, an amphipathic polymer, and an organic compound following the protocol provided below.Materials

[0299] MSPIDI(-) refers to MSP ID 1 with the N-terminal Histidine affinity tag removed, which was used for the preparation herein. MSPIDI(-) comprises the sequence of SEQ ID NO: 11. MSP1D1 refers to the N-terminal Histidine affinity tag not-removed format, comprising SEQ ID NO: 2. Concentration was measured by UV-Vis Spectroscopy, for which Extinction Coefficient at 280 nm = 18450 M'1(0.84 / mg / ml).

[0300] Phospholipid (e.g., DMPC, DPPC) was dissolved in chloroform and the concentration was measured by determination of total phosphorous (see, e.g., Chen, Toribara, and Warner (1956) Anal. Chem. 28: 1756-1758, the content of which is incorporated herein by reference in its entirety).

[0301] Other reagents used for 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.Preparation of DMPC / MSP ID 1 (-) Particle Composition

[0302] All manipulations were performed at room temperature (20 - 22°C).

[0303] The phospholipid-polymer saturation ratio Ns of a particle comprising DMPC and MSP1D1 (or MSP1D1(-)) is about 81 (see Table 2). DMPC / chloroform stock was added to a glass tube so that the final ratio of DMPC to MSPIDI(-) in the preparation mixture was 30: 1. DMPC was dried under a stream of nitrogen while rotating the glass tube, forming a thin film on the sides of the tube. The tube containing DMPC was then placed in a desiccator and vacuum was applied for a minimum of 4 hours, for instance, overnight.

[0304] DMPC was then hydrated in water. To determine the amount of water needed, the desired final sodium cholate concentration was first determined. The final cholate to lipid ratio was determined to be greater than 2. One exemplary concentration was 17 mM cholate. The amount of water and 200 mM sodium cholate were determined such that the final concentration of Sodium Cholate was 17 mM Sodium Cholate. The amount of protein was then determined such that the final protein concentration was about 3.5 mg / ml. Only water was added to the dried DMPC at this time.

[0305] The water / DMPC mixture was vortexed until the solution was milky white. 200 mM sodium cholate was then added to the hydrated DMPC to give a final cholate concentration of 17 mM in the assembly mixture. The sodium cholate / DMPC mixture was fully solubilized by placing the tube in a sonicating water bath for 5 min, followed by gentle heating at ~ 40°C, repeated for 2 times. MSPIDI(-) was then added to the sodium cholate / DMPC mixture and mixed well, arriving at a preparation mixture with a proteinconcentration of about 3.5 mg / ml. The preparation mixture was incubated for 10 - 20 minutes at room temperature.

[0306] The assembly of the particle composition was initiated by addition of Amberlite XAD-2 resin, where Amberlite absorbed the cholate detergent. The amount of packed Amberlite XAD-2 resin gave at least 50% of the volume of the assembly mixture, for example, for a 5 ml preparation mixture, Amberlite resin was added to the preparation mixture until the volume was 7.5 ml. The assembly mixture containing Amberlite resin was gently agitated by placing on a platform shaker or a tube rotator for at least four hours at room temperature. The supernatant containing assembled particles was recovered from the Amberlite XAD resin using a pipette and placed in a new tube. The Amberlite XAD resin was washed 2 times with PBS, and the washing solution containing residual assembled particles was added to a sample tube. Large particles were removed from the sample by passing through a 0.2 micron filter. Depending on the particle sample volume, this could also be done by centrifugal filters (Amicon Ultra Free), or a syringe equipped with a 0.2 micron filter.

[0307] Another under-lipidated particle composition comprising DMPC and MSPIDI(-) was similarly made with molar ratio of DMPC to MSPIDI(-) in the preparation mixture of about 55: 1. A third under-lipidated particle composition comprising DMPC and MSPIDI(-) was similarly made with molar ratio of DMPC to MSPIDI(-) in the preparation mixture of about 40: 1.

[0308] Under-lipidated particle composition comprising DMPC and MSP1E3D1(-) was similarly prepared with a different lipid to MSP molar ratio in the preparation mixture. The lipid to MSP molar ratios used for various DMPC / MSP1E3D1(-) compositions were also detailed in Example 6. MSP1E3D1(-) refers to MSP1E3D1 with the N-terminal Histidine affinity tag removed, which was used for the preparation herein. MSP1E3D1(-) comprises the sequence of SEQ ID NO: 12. MSP1E3D1 refers to the N-terminal Histidine affinity tag not- removed format, comprising SEQ ID NO: 5. The phospholipid-polymer saturation ratio Ns of a particle comprising DMPC and MSP1E3D1 (or MSP1E3D1(-)) is 167 (see Table 2). To make under-lipidated composition, the final ratio of DMPC to MSP1E3D1(-) in the preparation mixture was about 50: 1.

[0309] Another under-lipidated particle composition comprising DMPC and MSP1E3D1(-) was similarly made with molar ratio of DMPC to MSP1E3D1(-) in the preparation mixture of about 90: 1.

[0310] Particle compositions comprising other MSPs (or other phospholipid / MSP ratios were prepared following similar protocols detailed herein.

[0311] As controls, fully-lipidated particle compositions were also prepared. One fully- lipidated particle composition was made starting from a preparation mixture with DMPC to MSPIDI(-) molar ratio of about 80:1. Another fully-lipidated particle composition was made starting from a preparation mixture with DMPC to MSP ID 1 molar ratio of about 85:1. The method of making the control particle compositions was similar as the procedures detailed above with a different lipid to MSP molar ratio being used in the preparation mixture. For example, see Example 9.Example 2: Purification of Exemplary Under-lipidated Particle Compositions

[0312] The filtered samples containing DMPC / MSP1D1(-) particles or DMPC / MSP1E3D1(- ) particles from Example 1 were further purified by size exclusion chromatography (SEC). Control particle compositions were similarly purified.

[0313] For the column: Superdex S-200 increase 10 / 300 (~25 ml bed volume) was used. Buffer was PBS. Flow rate was 0.75 ml / min. Injection Volume was 0.5 mL.

[0314] The chromatogram of the preparative SEC of DMPC / MSP1D1(-) composition (preparation mixture ratio of about 30: 1) are shown in FIG. 1A. As shown in FIG. 1A, two peaks were isolated. The area on the right (dark grey) corresponding to a composition with more under-lipidated (less lipid to protein ratio) composition of particles containing approximately 51 lipids per MSPIDI(-). The phospholipid to MSP protein ratio in the final particle composition was measured by the method in Example 3 below. The peak positioned at 20 minutes (see upper panel of FIG. 1A) is the residual lipid-free protein.

[0315] The chromatograms of the preparative SEC of DMPC / MSP1E3D1(-) composition (preparation mixture ratio of about 50: 1) are shown in FIG. IB. As shown in FIG. IB, a single highlighted peak was collected, which comprises an under-lipidated particle composition with DMPC to MSP1E3D1(-) molar ratio of about 62: 1.Example 3: Measurement of the Phospholipid to MSP Ratio

[0316] This assay measured the phospholipid to MSP ratio by first extracting the phospholipid, followed by saponification and methylation of the fatty acids for rapid gas chromatography (GC) analysis. The measured quantity of fatty acids / MSP was twice of that of the starting phospholipid. Since phospholipid DPPC was used as standard, 2 fatty acids per phospholipid was already taken into account.

[0317] The phospholipid to MSP ratio in the particle composition was determined following the procedures detailed below.

[0318] The MSP concentration of the particle composition described herein was measured by UV-Vis spectroscopy. Particle composition sample was added to a 2 ml GC vial so that there was about 1 pg phospholipid. Each particle composition sample was run in triplicate. The particle composition sample was diluted to 100 pL with PBS. 10 pL of standard (DPPC) was added to each particle composition sample at a known concentration. The total phospholipid was extracted following the modified Bligh and Dyer technique described in conditions E and E’ in the phase diagrams of FIG. 7 in / / ? / . J. Mol. Sci. 2017, 7S(4), 708.

[0319] The extracted phospholipids were dried under a stream of nitrogen. The fatty acids were saponified and methylated simultaneously by dissolving the particle composition sample in 100 pL 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 at 45°C for overnight.

[0320] 0.5 ml water and 0.5 hexane were then added to the mixture. The mixture was vortexed, top hexane layer was recovered for GC analysis. A standard curve of known concentrations of DMPC in chloroform was prepared by serial dilution, 10 pL of DPPC standard was added to each dilution, and saponification was performed as described in Section 176. J Lipid Res. 2010; 51(3):635-40.

[0321] The DMPC content was determined from the standard curve by measuring the area ratio of the C 14 Fatty Acid Methyl Ester (DMPC) to that of C16 Fatty Acid Methyl Ester (DPPC), and compared to the standard curve.

[0322] From the UV-Vis spectroscopy-measured protein concentration and the DMPC concentration, a lipid to MSP ratio of the particle composition was determined.

[0323] An exemplary result generated by the GC method was shown in FIG. 2 using the DMPC / MSP1D1(-) particle composition (preparation mixture ratio of about 30: 1) from Example 2. In FIG. 2, DMPCfame is methyl myristate, and DPPCfame is methyl palmitate, i.e., the methylated fatty acids derived from DMPC and DPPC, respectively.

[0324] Based on the measurements, as detailed in FIG. 1A, starting from a preparation mixture with DMPC to MSPIDI(-) molar ratio of about 30: 1, an under-lipidated particle composition with DMPC to MSPIDI(-) molar ratio of about 72: 1 (hereinafter referred to as “DMPC:MSP1D1(-)=72: 1 particle composition”) and an under-lipidated particle composition with DMPC to MSPIDI(-) molar ratio of about 51 : 1 (hereinafter referred to as“DMPC:MSP1D1(-)=51:1 particle composition”) were obtained, wherein the yield of the DMPC:MSP1D1(-)=51:1 particle composition was optimized. Therefore, this condition was further used to prepare the DMPC:MSPlDl(-)=51 : 1 particle composition.

[0325] Starting from a preparation mixture with DMPC to MSPIDI(-) molar ratio of about 55:1, an under-lipidated particle composition with DMPC to MSPIDI(-) molar ratio of about 75:1 was obtained, hereinafter referred to as “DMPC:MSP1D1(-)=75:1 particle composition.” This condition was further used to prepare the DMPC:MSPlDl(-)=75: 1 particle composition.

[0326] Starting from a preparation mixture with DMPC to MSPIDI(-) molar ratio of about 40: 1, an under-lipidated particle composition with DMPC to MSPIDI(-) molar ratio of about 51:1 was obtained, hereinafter referred to as “DMPC:MSP1D1(-)=51:1 particle composition.” This condition was further used to prepare the DMPC:MSPlDl(-)=51 : 1 particle composition.

[0327] Starting from a preparation mixture with DMPC to MSP1E3D1(-) molar ratio of about 50:1, an under-lipidated particle composition with DMPC to MSP1E3D1(-) molar ratio of about 62:1 was obtained, hereinafter referred to as “DMPC:MSP1E3D1(-)=62:1 particle composition.”

[0328] Starting from a preparation mixture with DMPC to MSP1E3D1(-) molar ratio of about 60: 1, an under-lipidated particle composition with DMPC to MSP1E3D1(-) molar ratio of about 63:1 was obtained, hereinafter referred to as “DMPC:MSP1E3D1(-)=63:1 particle composition.”

[0329] Starting from a preparation mixture with DMPC to MSP1E3D1(-) molar ratio of about 90: 1, an under-lipidated particle composition with DMPC to MSP1E3D1(-) molar ratio of about 94:1 was obtained, hereinafter referred to as “DMPC:MSP1E3D1(-)=94:1 particle composition.”

[0330] For one control, starting from a preparation mixture with DMPC to MSP1D1 molar ratio of about 85:1, a particle composition (fully-lipidated) with DMPC to MSP1D1 molar ratio of about 83:1 was obtained, hereinafter referred to as “DMPC:MSP1D1=83:1 particle composition.”

[0331] For another control, starting from a preparation mixture with DMPC to MSPIDI(-) molar ratio of about 80: 1, a particle composition (fully-lipidated) with DMPC to MSPIDI(-) molar ratio of about 81:1 was obtained, hereinafter referred to as “DMPC:MSP1D1(-)=81 : 1 particle composition.”Example 4: Cholesterol Efflux Assay

[0332] The protocol for this assay was performed as described in the product information of Cholesterol Efflux Assay Kit (Cell-based) (ab 196985).

[0333] Briefly, J774 mouse macrophage cells were cultured and placed in a 96 well tissue culture plate. Cells were labeled with fluorescent cholesterol, rinsed, and incubated overnight. The cells were then treated with media containing test particles generated as described in Examples 1-3 for 4 hours. The media containing test particles was recovered, and the cholesterol fluorescence was measured in the media (Fmedia). The cells were lysed and the fluorescence remaining in the cells (F ceils) was also measured. % Cholesterol Efflux was defined by:% Efflux=Fmedia / (Fmedia + Fcells)

[0334] FIG. 3A and FIG. 3B show efflux assays performed on purified exemplary particle compositions as described in Examples 1-3. The lipid to MSP ratios were ratios measured from the final particle compositions. Besides fully-lipidated particle composition controls, MSPIDI(-) (protein only, lipid free), Apo- Al (protein only, lipid free), and high-density lipoprotein (HDL) also served as controls. A positive control from the Cholesterol Efflux Assay Kit (abl96985) was included. PBS served as negative control (not shown).

[0335] As shown in FIG. 3A, when 2.5 pg of samples were used to incubate with cells labeled with fluorescent cholesterol, the under-lipidated DMPC:MSP1D1(-)=51: 1 particle composition (e.g., particle composition shown in FIG. 6D) showed much greater cholesterol efflux rate compared to particle compositions with higher lipid content, e.g., under-lipidated DMPC:MSP1D1(-)=75: 1 particle composition (e.g., particle composition shown in FIG. 6B), and fully-lipidated DMPC:MSP1D1(-)=81 : 1 particle composition (e.g., particle composition shown in FIG. 6A). FIG. 3A further shows that removing N-terminal his-tag portion of the MSP protein did not affect cholesterol efflux (compare fully-lipidated DMPC:MSP1D1(- )=81 : 1 particle composition and fully-lipidated DMPC:MSP1D1=83: 1 particle composition). The phospholipid-polymer saturation ratio Ns of a particle comprising DMPC and MSP1E3D1 (or MSP1E3D10) is 167 (see Table 2). Hence the DMPC:MSPlE3Dl(-)=62: 1 particle composition and DMPC:MSP1E3D1(-)=94: 1 particle composition are very under- lipidated. As shown in FIG. 3A, DMPC:MSP1E3D1(-)=62: 1 particle composition also showed slightly better cholesterol efflux rate compared to DMPC:MSP1E3D1(-)=94: 1 particle composition (with higher lipid content). The under-lipidated DMPC:MSP1D1(- )=51 : 1 particle composition, DMPC:MSP1E3D1(-)=62: 1 particle composition, andDMPC:MSP1E3D1(-)=94: 1 particle composition all showed much greater cholesterol efflux rate compared to lipid-free MSPIDI(-) protein control, Apo- Al control, and HDL control. FIG. 3B shows a significant increase in cholesterol efflux capacity with increasing dose of particle compositions (10 pg vs. 2.5 pg). Surprisingly, 2.5 pg under-lipidated DMPC:MSP1D1(-)=51 : 1 particle composition even showed stronger cholesterol efflux capacity compared to 10 pg fully-lipidated DMPC:MSP1D1(-)=81: 1 particle composition, further demonstrating the technical superiority of the under-lipidated particle compositions.

[0336] These results demonstrated that, the under-lipidated particle compositions prepared herein had surprisingly superior cholesterol efflux capacity.Example 5: Stability of Under-Lipidated Particle Compositions to Freeze thaw Cycles

[0337] The stability of the prepared particle compositions from Examples 1-3 was tested. One exemplary DMPC / MSP1D1(-) particle composition tested was the right area portion of the chromatogram peak in FIG. 1A, corresponding to an under-lipidated DMPC:MSP1D1(- )=51 : 1 particle composition. The sample was frozen at -80°C with or without 10% glycerol as a cryoprotectant. Then the frozen samples were removed from -80°C storage and thawed rapidly. Samples were subjected to SEC and compared to the chromatograms taken prior to freezing. The results are shown in FIG. 4, indicating that the chromatograms of samples being freeze-and-thawed (without glycerol) and pre-freezing are nearly identical. Chromatograms were also nearly identical after a freeze-thaw cycle between samples frozen with or without cryoprotectant (data not shown). These results indicate that the prepared under-lapidated particle compositions had surprisingly high stability when subject to freeze- and-thaw cycles.

[0338] A freeze-thaw cycle on the under-particle compositions had no effect on cholesterol efflux efficiency either. The comparison of cholesterol efflux by the under-lipidated DMPC:MSP1D1(-)=51 : 1 particle composition before and after freeze-and-thaw is shown in FIG. 5. As shown in FIG. 5, the cholesterol efflux efficiency of the under-lipidated particle composition remained high after a freeze-and-thaw cycle (second column), and remained superior compared to a fully-lipidated particle composition control with higher DMPC content (DMPC:MSP1D1=83: 1 particle composition), as well as higher than lipid-free HDL and blank control PBS solution.Example 6: Correspondence between phospholipid / MSP!Dl(-) ratio in the preparation mixture and the final particle composition

[0339] The correspondence between the phospholipid / MSPlDl(-) ratio in the preparation mixture and the final particle composition was examined. Exemplified particle compositions comprising a plurality of DMPC / MSP1D1(-) particles were synthesized and purified following the procedures described in Examples 1 and 2, and measured for ratio as in Example 3. For the composition started with DMPC / MSP1D1(-) preparation mixture ratio of 80: 1, 55: 1, and 40: 1, the collected fraction from the first SEC was repurified with a second SEC (repurification). For the composition started with DMPC / MSP1D1(-) preparation mixture ratio of 40: 1, a third SEC purification (repurification 2) was further conducted. For the composition started with DMPC / MSP1D1(-) preparation mixture ratio of 30: 1, only one step of SEC was conducted. The lipid to protein ratio was measured in the final particle composition after the final purification step. The results are shown in FIGs. 6A-6D. As shown in FIG. 6A, a starting DMPC / MSP1D1(-) ratio of 80: 1 in the preparation mixture yielded a composition a plurality of particles wherein the DMPC / MSP1D1(-) ratio was about 81 : 1, such as 81±3 : 1. As shown in FIG. 6B, a starting DMPC / MSP1D1(-) ratio of 55: 1 in the preparation mixture yielded a composition a plurality of particles wherein the DMPC / MSP1D1(-) ratio was about 75: 1, such as 75±1.5 : 1. As shown in FIG. 6C, a starting DMPC / MSP1D1(-) ratio of 40: 1 in the preparation mixture yielded a composition a plurality of particles wherein the DMPC / MSP1D1(-) ratio was about 51 : 1, such as 51±1 : 1. As shown in FIG. 6D, a starting DMPC / MSP1D1(-) ratio of 30: 1 in the preparation mixture yielded a composition a plurality of particles wherein the DMPC / MSP1D1(-) ratio was about 51 : 1, such as 51±1 : 1. In addition, the 30: 1 (prep, ratio) DMPC to MSPIDI(-) was adequately isolated in a single purification step. These results collectively demonstrated that a low phospholipid / MSP ratio (e.g., a starting DMPC / MSP1D1(-) ratio of less than 81 : 1 (phospholipid-polymer saturation ratio Ns of DMPC and MSP1D1) in the preparation mixture) could yield surprisingly stable under-lipidated compositions with superior cholesterol efflux activity (see Example 4).Example 7: Correspondence between phospholipid / MSPlE3Dl(-) ratio in the preparation mixture and the final particle composition

[0340] The correspondence between the phospholipid / MSPlE3Dl(-) ratio in the preparation mixture and the final particle composition was examined. Exemplified particle compositions comprising a plurality of DMPC / MSP1E3D1(-) particles were synthesized and purifiedfollowing the procedures described in Examples 1 and 2, and measured for ratio as in Example 3. Only one step of SEC was conducted. The lipid to protein ratio was measured in the final particle composition after the final purification step. The results are shown in FIGs. 7A-7C. As shown in FIG. 7A, a starting DMPC / MSP1E3D1(-) ratio of 90: 1 in the preparation mixture yielded a composition a plurality of particles wherein the DMPC / MSP1D1(-) ratio was about 94: 1, such as 94±4 : 1. As shown in FIG. 7B, a starting DMPC / MSP1E3D1(-) ratio of 60: 1 in the preparation mixture yielded a composition a plurality of particles wherein the DMPC / MSP1D1(-) ratio was about 63: 1, such as 63±4 : 1. As shown in FIG. 7C, a starting DMPC / MSP1D1(-) ratio of 50: 1 in the preparation mixture yielded a composition a plurality of particles wherein the DMPC / MSP1D1(-) ratio was about 62: 1, such as 62±0.5 : 1. These results collectively demonstrated that a low phospholipid / MSP ratio (e.g., a starting DMPC / MSP1E3D1(-) ratio of less than 167: 1 (phospholipid-polymer saturation ratio Ns of DMPC and MSP1E3D1) in the preparation mixture) could yield surprisingly stable under-lipidated compositions with superior cholesterol efflux activity (see Example 4).Example 8: Cholesterol binding capacity assay

[0341] Large Unilamellar Vesicles (LUVs) have a single phospholipid bilayer and can be loaded with a high amount of hydrophilic materials. Cholesterol-containing LUVs can be used to measure the equilibrium binding (or partition coefficient) of cholesterol to under- lipidated particle compositions described herein. Such assay represents the ability of under- lipidated particle compositions to passively remove cholesterol from the cholesterol- containing LUVs. The cholesterol binding “capacity” measured by this assay depends on the ability of the under-lipidated particle compositions to bind cholesterol as well as the conditions used in the assay, and it does not reflect the maximum cholesterol binding capacity of the under-lipidated particle compositions. The cholesterol binding “capacity” number is an equilibrium value where the passive diffusion rate of cholesterol from the LUVs to the under-lipidated particle compositions equals the diffusion rate of cholesterol from the under-lipidated particle compositions back to the LUVs. The conditions of the assay, such as LUV concentration, mol% of cholesterol in the LUV, and MSP concentration, can be readily adjusted for in vitro assessment of cholesterol binding capacity of various under-lipidated particle compositions.

[0342] The under-lipidated DMPC:MSP1D1(-)=51 : 1 particle composition prepared from Examples 1-3 (starting from a preparation mixture with DMPC to MSPIDI(-) molar ratio ofabout 30: 1), and an exemplary LUV comprising 70% POPC and 30% cholesterol, were used to demonstrate the cholesterol binding capacity at equilibrium using the LUV-based assay. Preparation of exemplary LUVs

[0343] POPC and cholesterol in chloroform stocks were combined to reach a final ratio of 7:3 POPC : cholesterol. Solvent was removed by drying the composition under a stream of nitrogen gas, followed by drying in a vacuum desiccator for 4 hours. The lipids (POPC and cholesterol) were then hydrated in phosphate buffered saline (PBS) and vortexed until a milky white suspension was achieved. LUVs were formed by extruding the suspension through 100 nm filters (Whatman Nucl epore Hydrophilic Membrane, 0.1 pm Pore Size) 9 times. The clarified LUV sample was subjected to Gas Chromatography -Mass Spectrometry (GC-MS) to measure the total cholesterol concentration prior to experiments.Cholesterol binding capacity measurement

[0344] The under-lipidated DMPC:MSP1D1(-)=51 : 1 particle composition in PBS was combined with the obtained LUVs (70% POPC: 30% cholesterol) at 37°C to arrive at a final concentration of 1 mM cholesterol and 0.02 mM MSPIDI(-) protein (given the 51 : 1 ratio in the particle composition, the DMPC was at about 1.02 mM in the final mixture). At various time points after the mixing, 50 pl of the reaction was removed and the LUVs were separated from the DMPC:MSP1D1(-)=51 : 1 particle composition by size exclusion chromatography (SEC) and detected by monitoring the absorbance at 280 nm. The fraction containing the DMPC:MSP1D1(-)=51 : 1 particle composition was collected, and the MSPIDI(-) protein concentration was determined by the integrated area of the SEC chromatogram. Cholesterol content in the fraction containing DMPC:MSP1D1(-)=51 : 1 particle composition was measured by GC-MS.

[0345] FIG. 8 shows the cholesterol : MSPIDI(-) ratio in the fraction containing DMPC:MSP1D1(-)=51 : 1 particle composition as a function of time. FIG. 8 shows that equilibrium under the assay condition was obtained at about 8-10 hours of post-mixing at 37°C, which represented about 3.5 mols of cholesterol bound per 1 mol of MSPIDI(-). Since the under-lipidated DMPC:MSP1D1(-)=51: 1 particle composition contains two MSPIDI(-) proteins per particle, this result indicates about 7 mols of cholesterol bound per 1 mol of under-lipidated DMPC:MSP1D1(-)=51 : 1 particle composition at equilibrium.Example 9: Preparation of exemplary fully-lipidated particle compositions

[0346] Fully-lipidated particle compositions were assembled from a phospholipid, an MSP, and a detergent, as controls. See Sligar Lab, Nanodisc Technology: Protocols for Preparationof Nanodiscs (2008) University of Illinois at Urbana Champaign as an exemplary method, the content of which is incorporated herein by reference in its entirety.

[0347] Briefly, a frozen solution containing MSP in 20 mM tromethamine (Tris) (pH 7.4), 0.1 M NaCl, 0.5 mM EDTA, 0.01% NaNs was thawed and filtered through a 0.22 micron filter. The concentration of MSP was determined spectrophotometrically using the molar extinction coefficient s280=21,000 M^cm’1.

[0348] Phospholipid stocks were prepared in chloroform at 50-100 mM for long-term storage at -20°C in 4 ml glass vials with Teflon-lined screw caps. Concentration of the stock solution was determined by phosphate analysis, the desired amount of chloroform lipid stock was dispensed into a disposable glass culture tube, and the solvent was dried up using a gentle stream of nitrogen gas in a fume hood. The tube was rotated at an angle to obtain a thin film on the lower walls. Residual solvent was removed by placing the tube in a vacuum desiccator under high vacuum for at least 4 hours.

[0349] Buffer comprising a detergent and sodium cholate was added to the tube so that the ratio of cholate to lipid in the mixture was 2: 1. The tube was vortexed, heated under hot tap water, and sonicated in an ultrasonic bath until the solution was clear and no lipid remained on the walls of the tube. MSP was added to the solution and incubated for at least 15 minutes.

[0350] To remove the cholate and initiate self-assembly of the particle compositions, 0.5-0.8 g of Amberlite XAD 2 beads (Sigma) were added per every ml of the reconstitution mixture. The suspension was placed on the orbital shaker and incubated overnight. The optimal lipid to MSP ratios and incubation temperature for various lipids, and the optimized incubation temperatures and minimum incubation times are shown in Table 3.Table 3

[0351] Samples were removed from Amberlite XAD 2 beads, filtered through a 0.22 micron filter, and fractionated on a Superdex 200 increase 10 / 300 GL column (GE Healthcare) in MSP Standard Buffer with a flow rate of 0.75 ml / min. Samples were filtered prior to injection and fractions were collected every minute. FIG. 18 displays an example chromatogram (MSP1D1+DMPC) of the reaction.

[0352] To construct exemplary particle compositions, MSP ID 1 was added to the sodium cholate lipid mixture at a lipid to MSP ratio of 10: 1, 32.5: 1, 50: 1, and 65: 1 for POPC lipids, and 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, and 85: 1 for DMPC lipids. No lipid addition served as control (lipid free). As shown in FIG. 9A, a POPC to MSP ID 1 ratio of 65: 1 resulted in a fully-lipidated (10 nm diameter) particle compositions. As shown in FIG. 9B, at a molar ratio of 85: 1 DMPC to MSP ID 1, a fully-lipidated particle compositions was formed.Example 10: Exemplary particle compositions alter ability to efflux cholesterol from macrophages in vitro

[0353] Particle compositions containing DMPC (Lipid A) or POPC (Lipid B) at different lipid concentrations were formed with MSP ID 1 using the methods of Example 9. J774 cells derived from a murine macrophage cell line were plated and radiolabeled with 74 kBq of3H- cholesterol per millimeter. ABCA1 was up-regulated by means of a 6-hour incubation with 0.3 mM 8-(4-chlorophenylthio)-cAMP. Subsequently, the particle compositions and efflux medium were added and incubated for 4 hours.

[0354] All steps were performed in the presence of the acyl-coenzyme A cholesterol acyltransferase inhibitor CPI 13,818 (2 pg / mL). Liquid scintillation counting was used to quantify the efflux of radioactive cholesterol from the cells. The quantity of radioactive cholesterol incorporated into cellular lipids was calculated by means of isopropanol extraction of control wells not exposed to particle compositions / media. The percentage of cholesterol efflux was calculated by the following formula: 100

[0355] All experiments were performed in duplicate, and performed according to published 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 content of which is incorporated herein by reference in its entirety).

[0356] As shown in FIG. 10, the POPC (Lipid B) containing particle compositions with lipid to MSP1D1 ratios of 50:1 and 32.5: 1 (both under-lipidated) had higher cholesterol efflux percentage than fully-lipidated POPC (Lipid B) particle compositions with a lipid to MSP ID 1 ratio of 65: 1. Particle compositions containing POPC at lipid to MSP ID 1 ratios from 32.5: 1 to 65: 1 all demonstrated better cholesterol efflux properties compared to fully- lipidated particle composition containing DMPC at lipid to MSP ID 1 ratio of 85: 1. Further,all tested particle compositions demonstrated at least about 2-fold cholesterol efflux of that achieved by MSP ID 1 protein per se.

[0357] As shown in FIG. 11, the percent of cholesterol efflux with the exemplary fully- lipidated particle composition with a DMPC to MSP ID 1 ratio of 85: 1 was more than two times that of naturally occurring ApoA-I.Example 11: Exemplary fully-lipidated particle compositions remove cholesterol from human retinal pigment epithelial cells in vitro

[0358] Fully-lipidated particle composition with a POPC to MSP1D1 ratio of 65: 1 (MSP1D1 Lipid B) and fully-lipidated particle composition with a DMPC to MSP ID 1 ratio of 85: 1 (MSP ID 1 Lipid A) were formed using the methods of Example 9. The methods of measuring cholesterol efflux in Example 10 were conducted on human retinal pigment epithelial (RPE) cells (Neuromics) to measure cholesterol efflux at 2, 4 and 6 hours of incubation. Passive efflux (no protein or particle composition added) and MSP1D1 protein alone were used for comparison.

[0359] FIGs. 12A-12C show percent cholesterol efflux from RPE cells after incubation for 2 hours (FIG. 12A), 4 hours (FIG. 12B), and 6 hours (FIG. 12C). These results collectively demonstrate that fully-lipidated particle composition with POPC to MSP ID 1 ratio of 65: 1 and fully-lipidated particle composition with DMPC to MSP ID 1 ratio of 85: 1 are both capable of continuously promoting cholesterol efflux from RPE cells for at least 6 hours, which were significantly more than that achieved by MSP ID 1 protein alone. Further, fully- lipidated particle composition with DMPC to MSP1D1 ratio of 85: 1 seemed to efflux cholesterol faster than fully-lipidated particle composition with POPC to MSP ID 1 ratio of 65: 1 during 2-4 hour incubation window, but fully-lipidated particle composition with POPC to MSP1D1 ratio of 65: 1 caught up speed by 6 hour.

[0360] Under-lipidated particle composition comprising DMPC and MSP ID 1 and under- lipidated particle composition comprising POPC and MSP ID 1 may be able to promote cholesterol efflux from RPE cells better than their fully-lipidated version.Example 12: In vivo delivery of exemplary fully-lipidated particle composition in murine eyes

[0361] To determine the penetrability and persistence of the exemplary fully-lipidated particle composition in the eye in vivo, a fully-lipidated particle composition comprising MSP1D1 and DMPC in a molar ratio of 1 :85 was prepared, with the MSP1D1 linked to agreen fluorescent label via a cysteine residue engineered into the MSP ID 1 protein. The fully- lipidated particle composition was prepared the day before the procedure and stored at 4°C.

[0362] 6-week old C57B1 / 6J mice were anesthetized with 87.6mg / kg Ketamine plus lOmg / kg Xylazine cocktail via IP injection and administered 0.5% proparacaine hydrochloride ophthalmic drops for pain management. The prepared fully-lipidated particle composition was administered at a dose of 7 pg / eye in a volume of 1.14 pl via intravitreal injection. A subset of mice had the left eye dilated with 1% Tropicamide ophthalmic solution, followed by application of 2.5% Gonak hypromellose demulcent solution and noninvasive fundus imaging using Phoenix Micron III imaging system at various time points. At times indicated in FIGs. 13A-13B, mice were sacrificed and eyes were enucleated. Eyes were fixed in 10% Neutral buffered formalin for 2 hrs at room temperature (RT) before being transferred to graded sucrose (10% followed by 30%) overnight. The fixed, cryopreserved eyes were mounted in optimal cutting temperature (O.C.T.) and slides were prepared by cutting sections (20 pM) on a Lieca 1850 cryostat. Coverslips were applied using Vectasheild antifade mounting medium with Dapi to counterstain nuclei and to avoid additional processing steps associated with other counterstaining methods. The prepared slides were imaged on an Olympus F VI 000 confocal microscope using system-optimized z step distances for each respective objective. This allowed for direct visualization of fluorescently labeled fully- lipidated particle composition following the successful removal of cholesterol from the retina.

[0363] As indicated by FIGs. 13A-13B, the fully-lipidated particle composition was successfully delivered to the murine eye via intravitreal injection, and the fully-lipidated particle composition was able to enter from the vitreous, got to the retinal space, into the RPE, and into the ocular muscle by 6 hours. By 24 hours, the fully-lipidated particle composition appeared to have left the eye. These results indicate that penetrability of the exemplary fully-lipidated particle composition is excellent in vivo, and it does not dwell in the eye for an extended period of time (> 24 hours).

[0364] Under-lipidated particle compositions may be able to achieve similar effects compared their corresponding fully-lipidated particle compositions.Example 13: In vivo safety and efficacy of exemplary fully-lipidated particle composition in murine eyes

[0365] The fully-lipidated DMPC:MSP1D1=85: 1 (molar ratio) particle composition from above examples was further tested to exemplify in vivo safety and efficacy in the eye. TheMSP ID 1 was linked to a green fluorescent label via a cysteine residue engineered into the MSP ID 1 protein.

[0366] The fluorescence-labelled exemplary fully-lipidated particle composition was intravitreally delivered into the eyes of anesthetized mice, and its path and rate of diffusion within the eye was visualized by imaging post-mortem retinal tissue along a 24-hour postinjection time course (FIG. 14A). As shown in FIG. 14B, the fluorescence signal was detectable within 15 minutes of injection and diffused rapidly across the retina in a vitreous- to-RPE (retinal pigment epithelium) gradient, indicating that the particle compositions can be delivered into the eye and traffic through the retina effectively to target tissues. These results collectively demonstrated that the exemplary particle composition can be distributed to RPE.

[0367] Next, a model of ‘wet’ AMD mice was used, which had a laser-induced injury of the Bruch’s membrane to cause choroidal neovascularization (CNV). The exemplary fully- lipidated particle composition was intravitreally injected in tandem with, or 3 days after (‘+d3 ’), laser injury to examine the resulting CNV area size (FIG. 15A). Importantly, the mean CNV area was similar across control and exemplary particle composition-treated mice, suggesting that the exemplary particle composition does not exacerbate prominent features of AMD in the mouse (FIGs. 15B-15C). These studies were key in showing that the exemplary fully-lipidated particle composition can be delivered efficiently to the retina and does not exacerbate features of neovascular AMD in the mouse, unlike the currently approved complement inhibitors for the treatment of geographic atrophy (GA).

[0368] To determine whether the exemplary fully-lipidated particle composition could reduce lipid burden in the eye, a transgenic mouse species was implemented, which allows for specific deletion of Abcal and Abcgl cholesterol efflux transporters in rod photoreceptors (Abcal / gl-rod / -rod), resulting in lipid accumulation in the RPE and Bruch’s membrane and retinal neurodegeneration similar to patients with early to intermediate dry AMD. To accelerate lipid accumulation, the mice was provided with high-fat diet food for the duration of the study. As shown in FIG. 16A, for the first cohort of mice (Study 1), baseline (TO) optical coherence tomography (OCT) fundus images of the mouse retina were collected prior to high-fat diet induction and intravitreal delivery of the exemplary fully-lipidated particle composition (‘Disc’ in FIGs. 16B-16C) or a blank injection (‘Vehicle’ in FIGs. 16B-16C). A second intravitreal injection was administered 3 weeks later. After 6 weeks of high-fat diet exposure, follow-up OCT fundus imaging (Tl) and blood sample collection were performed followed by electroretinography (ERG) visual function testing and tissue harvest. First, fullfield scotopic and light bleach recovery ERG diagnostics that measure photoreceptor andRPE function, respectively, revealed no differences between vehicle and exemplary particle composition (“Disc”)-injected mice, demonstrating safety of the exemplary fully-lipidated particle composition on retinal electrical signaling (FIGs. 16B-16C). Further confirmation of safety was demonstrated by examination of retinal histology where retinal inflammation or differences in retinal integrity among mouse groups was the same (FIG. 16D). Additionally, retina section immunostaining revealed complement 3 (C3) staining was detectable in the mouse retina but similarly expressed across vehicle and exemplary particle composition (“Disc”)-treated mice, indicating that the exemplary fully-lipidated particle composition did not exacerbate expected complement signaling in this dry AMD model (FIG. 16E). RPE disruption and hypertransmission, ellipsoid zone abnormalities, inner retinal subsidence, and hyperreflective foci are primary anatomical changes linked to AMD progression are identified with noninvasive imaging. Thus, follow-up (Tl) optical coherence tomography (OCT) images were examined for potential structural changes resulting from the genetic mutation and high-fat diet. As expected, numerous incidences of inner segment / outer segment (IS / OS) junction abnormalities and RPE disruptions including pigment epithelial detachments (PED) were found in images of vehicle-injected mice following 6 weeks of high-fat diet absent in baseline TO images (FIG. 16F). However, these abnormalities were rarely detected or absent in exemplary particle composition (“Disc”)-treated mouse eyes, indicating ameliorating features of AMD on OCT imaging.

[0369] To replicate this study, and to further accentuate the high-fat diet-induced phenotype, a second cohort of mice (Study 2) on a high-fat diet for 6 weeks was tested but did not perform injections of the exemplary fully-lipidated particle composition (“Disc”) or vehicle until 2- and 4-weeks after diet induction (FIG. 17A). At 6 weeks, visual function and again was evaluated, and no differences was found in full-field scotopic and light-bleach recovery responses between vehicle and exemplary particle composition (“Disc”)-treated mouse groups, confirming the safety of the fully-lipidated particle composition (FIGs. 17B-17C). In analyzing follow-up OCT imaging, several more lesions in the IS / OS junction and RPE were observed in vehicle-injected mice, but far fewer lesions were observed in mice injected with the exemplary fully-lipidated particle composition. Furthermore, greater numbers of lesions in both groups were observed compared to the first study, suggesting that exposing Abcal / gl-rod / -rod mice to a high-fat diet for 2 weeks prior to initial intravitreal injection worsened the disease (Table 4 below, which summarizes cumulative incidence of retinal lesions in vehicle and exemplary particle composition-treated mice via OCT imaging examination across two studies). Regardless, treatment of mice with the exemplary fully-lipidated particle composition resulted in reduced retinal lesions in both studies by nearly four times compared to vehicle.Table 4 Cumulative incidence of retinal lesions

[0370] Lastly, lipidomics analyses were performed on retinal samples to examine potential changes in individual lipid species in the mouse eye following treatment with the exemplary fully-lipidated particle composition. Cholesteryl ester (CE) composition, a major component of drusen-causing AMD, was found to decrease in the retinas of particle composition (“Disc”)-treated mice (FIG. 17D), suggesting that the treatment reduces cholesterol load in diseased mice with features of AMD. Additionally, taken together with the improvement in OCT lesions, this reduction in cholesteryl ester, the main lipid species found in drusen, provides strong support of both the mechanism of action as well as scientific promise of the particle compositions provided herein.

[0371] Under-lipidated particle compositions provided herein may be able to achieve similar or even better results than their corresponding fully-lipidated particle compositions.

Claims

1. A composition comprising a plurality of particles comprising an amphipathic polymerand a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated.

2. The composition of claim 1, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns.

3. The composition of claim 2, wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than about 95% of the phospholipidpolymer saturation ratio Ns.

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

5. The composition of claim 4, wherein the amphipathic polypeptide is selected from the group consisting of MSP1D1, MSP1E1D1, MSP1E2D1, MSP1E3D1, MSP2N2, MSPIDI(-), MSPIEIDI(-), MSP1E2D1(-), MSP1E3D1(-), MSP2N2(-), and a variant thereof.

6. The composition of claim 5, wherein the amphipathic polypeptide is MSP ID 1, MSP ID 1(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 81.

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

8. The composition of claim 5, wherein the amphipathic polypeptide is MSP1D1, MSP ID 1(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 81.

9. The composition of claim 8, wherein the molar ratio of DPPC to the amphipathic polypeptide in the composition is less than about 75:1.

10. The composition of claim 5, wherein the amphipathic polypeptide is MSP ID 1, MSP ID 1(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 62.

11. The composition of claim 10, wherein the molar ratio of POPC to the amphipathic polypeptide in the composition is less than about 58:1.

12. The composition of claim 5, wherein the amphipathic polypeptide is MSP1E1D1, MSPIEIDI(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 107.

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

14. The composition of claim 5, wherein the amphipathic polypeptide is MSP1E1D1, MSPIEIDI(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 107.

15. The composition of claim 14, wherein the molar ratio of DPPC to the amphipathic polypeptide in the composition is less than about 100:1.

16. The composition of claim 5, wherein the amphipathic polypeptide is MSP1E1D1, MSPIEIDI(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 80.

17. The composition of claim 16, wherein the molar ratio of POPC to the amphipathic polypeptide in the composition is less than about 76:1.

18. The composition of claim 5, wherein the amphipathic polypeptide is MSP1E2D1, MSP 1E2D 1(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 135.

19. The composition of claim 18, wherein the molar ratio of DMPC to the amphipathic polypeptide in the composition is less than about 128:1.

20. The composition of claim 5, wherein the amphipathic polypeptide is MSP1E2D1, MSP 1E2D 1(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 135.

21. The composition of claim 20, wherein the molar ratio of DPPC to the amphipathic polypeptide in the composition is less than about 128:1.

22. The composition of claim 5, wherein the amphipathic polypeptide is MSP1E2D1, MSP 1E2D 1(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 102.

23. The composition of claim 22, wherein the molar ratio of POPC to the amphipathic polypeptide in the composition is less than about 96:1.

24. The composition of claim 5, wherein the amphipathic polypeptide is MSP1E3D1, MSP 1E3D 1(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 167.

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

26. The composition of claim 5, wherein the amphipathic polypeptide is MSP1E3D1, MSP 1E3D 1(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 167.

27. The composition of claim 26, wherein the molar ratio of DPPC to the amphipathic polypeptide in the composition is less than about 158:1.

28. The composition of claim 5, wherein the amphipathic polypeptide is MSP1E3D1, MSP 1E3D 1(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 126.

29. The composition of claim 28, wherein the molar ratio of POPC to the amphipathic polypeptide in the composition is less than about 118:1.

30. The composition of claim 5, wherein the amphipathic polypeptide is MSP2N2, MSP2N2(-), or a variant thereof, the phospholipid is DMPC, and the Ns is about 387.

31. The composition of claim 30, wherein the molar ratio of DMPC to the amphipathic polypeptide in the composition is less than about 367:1.

32. The composition of claim 5, wherein the amphipathic polypeptide is MSP2N2, MSP2N2(-), or a variant thereof, the phospholipid is DPPC, and the Ns is about 387.

33. The composition of claim 32, wherein the molar ratio of DPPC to the amphipathic polypeptide in the composition is less than about 367:1.

34. The composition of claim 5, wherein the amphipathic polypeptide is MSP2N2, MSP2N2(-), or a variant thereof, the phospholipid is POPC, and the Ns is about 291.

35. The composition of claim 34, wherein the molar ratio of POPC to the amphipathic polypeptide in the composition is less than about 276:1.

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

37. A method of preparing a composition comprising a plurality of particles comprising an amphipathic polymer and a phospholipid, wherein at least about 85% of the plurality of particles are under-lipidated, the method comprises:1) incubating the amphipathic polymer and the phospholipid to obtain a preparation mixture, wherein the phospholipid is solubilized by a detergent or organic compound prior to the incubation; and2) removing the detergent or organic compound from the preparation mixture, thereby obtaining the particles.

38. The method of claim 37,(i) wherein the molar ratio of the phospholipid to the amphipathic polymer in the composition is less than a phospholipid-polymer saturation ratio Ns;(ii) wherein the molar ratio of the phospholipid to the amphipathic polymer in the preparation mixture is less than or equal to the molar ratio of the phospholipid to the amphipathic polymer in the composition;(iii) wherein the method further comprises isolating the particles; and / or(iv) wherein the detergent or organic compound comprises a cholate.

39. The method of claim 37 or 38,(i) wherein the amphipathic polymer is an amphipathic polypeptide selected from the group consisting of apolipoprotein A-I (ApoA-I), ApoA-II, ApoC, ApoE, and a variant thereof; and / or(ii) wherein the phospholipid comprises DMPC, DPPC, DMPS, POPC, POPS, or PiP2.

40. The method of claim 39, wherein the amphipathic polypeptide is selected from the group consisting of MSP1D1, MSPE1D1, MSP1E2D1, MSP1E3D1, MSP2N2, MSPIDI(-), MSPIEIDI(-), MSP1E2D1(-), MSP1E3D1(-), MSP2N2(-), and a variant thereof.

41. The method of claim 40, wherein the amphipathic polypeptide is MSP ID 1, MSP ID 1(-), or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphipathic polypeptide in the composition is from about 65:1 to about 75:1; and wherein the molar ratio of DMPC to the amphipathic 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 amphipathic polypeptide is MSP ID 1, MSP ID 1(-), or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphipathic polypeptide in the composition is from about 46:1 to about 55:1; and wherein the molar ratio of DMPC to the amphipathic 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 amphipathic polypeptide is MSP1E3D1, MSP 1E3D 1(-), or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphipathic polypeptide in the composition is from about 85:1 to about 100:1; and wherein the molar ratio of DMPC to the amphipathic 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 amphipathic polypeptide in the composition is about 87:1 to about 98:1, and the molar ratio of DMPC to the amphipathic polypeptide in the preparation mixture is about 90:1.

45. The method of claim 40, wherein the amphipathic polypeptide is MSP1E3D1, MSP 1E3D 1(-), or a variant thereof, the phospholipid is DMPC, and the molar ratio of DMPC to the amphipathic polypeptide in the composition is from about 55:1 to about 80:1; and wherein the molar ratio of DMPC to the amphipathic polypeptide in the preparation mixture is less than or equal to about 60:1.

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

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

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

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 nerve atrophy, loss of acuity, hemianopia, visual agnosia, strabismus, retinal neurovascular disorder, lipid keratopathy, corneal lipidosis, or any combination thereof.

Images