Nanoparticles that encapsulate small molecules

Abstract

The present invention relates to a lipoprotein cage for intracellular delivery of cargo, the lipoprotein cage comprising: (i) at least one polypeptide comprising an amino acid sequence I consisting of SEQ ID NO: 1, the protein cage having a positively charged interior, and (ii) a surfactant composition comprising one or more amphiphiles, the one or more amphiphiles being selected such that the net charge of the composition is negative, the surfactant composition being encapsulated in the assembled protein cage. The present invention further relates to a complex comprising the lipoprotein cage of the present invention and one or more cargo molecules, and a method for producing the lipoprotein cage of the present invention, the method comprising the steps of self-assembling the protein cage from at least one polypeptide comprising amino acid sequence I, preferably from 24 polypeptides each comprising amino acid sequence I, and encapsulating an anionic amphiphile of the present invention in the protein cage without disassembling the protein cage.

Description

[Technical Field]

[0001] The present invention relates to the field of nanoparticles. In particular, the present invention relates to a lipoprotein cage comprising a protein cage and a surfactant composition for intracellular delivery of cargo. [Background technology]

[0002] Well-defined nanoparticles may be well suited for the protected transport of molecules within biological systems.

[0003] Protein cages have recently emerged as an important platform for nanotechnology development. Among naturally occurring protein cages, viruses are among the most efficient nanomachines, achieving replication and efficient self-assembly of components in complex biological environments. An artificial system capable of performing the most fundamental steps of virus particle assembly in vivo has been designed based on a patchwork cage formed from Aquifex aeolicus lumazine synthase and a circularly permuted variant with an appended cationic peptide. These two-component protein containers self-assemble in vivo and size-selectively capture endogenous RNA molecules (Azuma et al., Modular Protein Cages for Size-Selective RNA Packaging in Vivo, J. Am. Chem. Soc. 2018, vol. 140, 566-569).

[0004] In a further approach, synthetic genes were used to assemble nanoparticle phospholipid bilayer discs from a class of phospholipids and amphipathic helical proteins. The self-assembly process begins with a mixture of phospholipids and proteins in the presence of detergent. Upon removal of the detergent, particles containing saturated or unsaturated phospholipids are formed (Bayburt et al., Self-Assembly of Discoidal Phospholipid Bilayer Nanoparticles with Membrane Scaffold Proteins, Nano Letters 2002, vol. 2(8), pp. 853-85).

[0005] The emergence of de novo designed protein cages offers an alternative strategy for creating cargo transport vehicles. Designed non-functional protein cages have been transformed into nucleic acid delivery vehicles capable of encapsulating oligonucleotides in vitro with high binding affinity (Edwardson et al., Rational Engineering of a Designed Protein Cage for siRNA Delivery. J. Am. Chem. Soc. 2018, vol. 140, pp. 10439-10442).

[0006] However, protein compartments are limited in the types of cargo they can carry. The incorporation of molecules other than proteins and nucleic acids requires the development of additional sets of self-assembly rules. The development of protein cages by incorporating other molecular species has the potential to expand applicability to new fields, one of which is the transport of the smallest possible molecules when using the present invention. Summary of the Invention

[0007] The present invention provides porous lipoprotein cages (also referred to herein as lipoprotein scaffolds) that can encapsulate poorly water-soluble molecular cargo. In the lipoprotein cages of the present invention, hydrophobic compartmentalization is achieved by combining an amphiphile, which creates the hydrophobic interior of the cage, with an engineered protein that stabilizes the cage and acts as a biorecognizable barcode for the cargo delivered by the cage through specific pores (Figure 1a).

[0008] In the two-layer host-guest approach, a designed protein cage with a highly positively charged inner cavity is used to nucleate anionic surfactant molecules into micellar aggregates within the inner cavity at concentrations well below their critical aggregation concentration. Electrostatic attraction promotes the encapsulation of the anionic surfactant, which phase-separates due to its high effective concentration to form micellar aggregates within the protein cage. The nonpolar core of this stable protein-surfactant complex can then sequester small molecules through hydrophobic effects.

[0009] The protein cage is highly stable and acts as a template for the formation of a lipid / micelle core within its internal cavity, meaning that no prior formulation steps are necessary. Thus, the amphiphile does not need to form stable particles by itself before the addition of the protein.

[0010] Through their unique structure, the resulting lipoprotein cages of the present invention can recruit and sequester small, preferably small hydrophobic, molecules through the hydrophobic core of the protein scaffold. We have shown that these lipoprotein cages are stable and monodisperse, protecting their cargo from sequestration by serum proteins and thus enhancing the cellular uptake of poorly soluble fluorescent probes and cytotoxic drugs. These findings demonstrate the beneficial combination of electrostatically driven and amphiphilic self-assembly within stable protein compartments using proteins and surfactant molecules.

[0011] Due to the generality of the hydrophobic effect, this system can be used to encapsulate any kind of small, preferably hydrophobic, molecule, including hormones, hydrophobic peptides, luminescent metal complexes, therapeutic agents, and vitamins.

[0012] Thus, in a first aspect, the present invention provides a lipoprotein cage for intracellular delivery of a cargo, the lipoprotein cage comprising: (i) a protein cage comprising at least one polypeptide comprising an amino acid sequence I consisting of: MX 13 QAIGILELX1SIAAGMELGDAMLKSAX 14 VX 15 LLVSKTISX2GKFLLMLGGDIX8AIX9X 12 AIX 10 TGTX 11 QAGX3LLVDSLVLAX 16 IHPSVLPAIX 17 GX 18 NX 19 VX 20 X7X 21 QAVGIVETX4SVAACISAADX 22 AVX 23 GSX 24 VTLVRVHMAX5GIGGKCYMVVAGDVSDVALAVTVASSSAGAYGX6LVYASLIPX 25 PHX 26 AMWX 27 QMVX 28 GX 29 E (SEQ ID NO: 1) Here, X1~X 29 are each independently an amino acid, provided that at least three of X1 to X6 are each independently a positively charged amino acid (positively charged amino acid), and X1 to X in SEQ ID NO: 1 29 Up to five amino acids at positions other than those indicated may be replaced by any amino acid, The protein cage has a positively charged interior. a protein cage comprising at least one polypeptide comprising the amino acid sequence I; (ii) a surfactant composition comprising one or more amphiphiles, the one or more amphiphiles being selected such that the net charge of the composition is negative, the one or more amphiphiles comprising a hydrophilic group and a hydrophobic group, the hydrophobic group comprising at least one hydrocarbon moiety selected from the group consisting of C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, C4-C30 alkoxy, or C5-C30 cycloalkyl; The surfactant composition comprises a surfactant composition comprising one or more amphiphiles encapsulated in assembled protein cages; Includes:

[0013] In a further aspect, the present invention relates to a complex comprising a lipoprotein cage of the present invention and one or more cargo molecules.

[0014] In a further aspect, the present invention relates to a method for producing a lipoprotein cage of the present invention, comprising the steps of self-assembling a protein cage from at least one polypeptide comprising amino acid sequence I, preferably 24 polypeptides each comprising amino acid sequence I, and encapsulating a surfactant composition of the present invention in the protein cage without disassembling the protein cage.

[0015] In a further aspect, the present invention provides a method for producing a complex of the present invention, which comprises the step of mixing a lipoprotein cage of the present invention with one or more cargo molecules, wherein the cargo is encapsulated in the lipoprotein cage of the present invention without degrading the lipoprotein cage.

[0016] In a further aspect, the present invention provides a method of treating a cell with a complex of the present invention, the method comprising the step of contacting said cell with a complex of the present invention. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a cartoon illustration of the self-assembly of a lipoprotein-mimetic cage. [Figure 1a] FIG. 1 is a diagram of a high density lipoprotein (HDL) particle showing charged phospholipids, proteins and hydrophobic cargo molecules. [Figure 1b] Surface representation of the OP protein cage viewed along the 2-fold (left) and 4-fold (center) axes of symmetry, and a cartoon cutaway of the positively charged interior (right). [Figure 1c] Cartoon illustration of a lipoprotein particle with a positively charged porous protein scaffold, an encapsulated anionic surfactant, driven by electrostatic attraction to form a nonpolar core on a stable protein-surfactant complex, which can then sequester small nonpolar molecules by hydrophobic effects. [Figure 2] FIG. 1 is a diagram of capsid-templated micelle formation. [Figure 2a] FIG. 1 is a cartoon illustration of protein cage-templated micellization of sodium dodecyl sulfate (SDS) molecules. [Figure 2b] Native gel electrophoresis of OP cages in the presence of increasing molar equivalents of SDS. [Figure 2c] FIG. 11: Size exclusion chromatography of OP cages before and after incubation with 800 equivalents of SDS. [Figure 2d] Transmission electron micrograph of an empty OP cage, scale bar 30 nm. [Figure 2e] Transmission electron micrograph of OP cages in the presence of SDS (800 equiv.), scale bar 30 nm. [Figure 2f] Figure 1. Encapsulation of Atto488-labeled ssDNA visualized by native gel electrophoresis. Without preincubation with SDS (-), the OP cage quantitatively internalizes the ssDNA probe. After internalization of an SDS molecule (+), the OP cage can no longer encapsulate the oligonucleotide probe. [Figure 2g] 2-D projection, central slice, 3-D reconstruction and subtracted volume of an empty OP capsid determined by cryo-EM. [Figure 2h]2-D projection, central slice, 3-D reconstruction and subtracted volume determined by cryo-EM of an OP capsid containing SDS. [Figure 2i] 2-D projections, central slices, 3-D reconstructions, and subtracted volumes of OP capsids containing SDS:CS, as determined by cryo-EM. Additional density from the surfactant can be seen in the central slice and in the density obtained by subtracting empty OP from OP:SDS (shown as solid surfaces, right column). [Figure 3] Figure 1 shows hydrophobic core formation, cargo capacity and kinetics. [Figure 3a] FIG. 1 shows the steady-state fluorescence emission spectra of Nile Red in the presence of OP, SDS, and OP:SDS complex relative to PBS buffer. [Figure 3b] FIG. 1 shows the steady-state fluorescence emission spectra of Nile Red in the presence of OP with increasing molar equivalents of SDS. [Figure 3c] FIG. 10 is a diagram of the normalized fluorescence emission of Nile Red at 620 nm versus the molar equivalent of SDS relative to the OP cage. [Figure 3d] FIG. 10 shows normalized fluorescence emission at 620 nm versus molar equivalent of Nile Red added to the OP:SDS complex. [Figure 4] FIG. 1. Determination of biological activity, sodium dodecyl sulfate (SDS), cholesterol sulfate (CS). [Figure 4a] Flow cytometry data set (right panel) of HeLa cells treated with either free Nile red or Nile red packaged in an OP:SDS complex with five Nile red molecules per capsid (n=3). [Figure 4b] Confocal fluorescence micrographs of HeLa cells treated with Nile Red as a free molecule (left panel) or packaged in OP:SDS complexes (right panel). Hoechst 33342 (circular nucleus shape), Nile Red (cytosol surrounding the nucleus). Scale bar 30 μm. [Figure 4c]Confocal fluorescence micrographs of HeLa cells treated with OP-S38C:SDS:CS:Nile Red complex (1:600:200:5) to label the OP-S38C cage with Atto425. Hoechst 33342 (round nuclei, i), Nile Red (ii), Atto425 (OP-S38C; iii), and an overlay of Hoechst 33342, Nile Red, and Atto425 (iv). Scale bar is 30 μm. [Figure 4d] FIG. 11 is a flow cytometry comparison of HeLa cells treated with Nile Red (−) or Nile Red packaged in OP:SDS:CS complexes (+) at a ratio of OP:SDS:CS:Nile Red of 1:600:200:5. [Figure 4e] Figure 1 shows the viability of HeLa cells after treatment with 2.5 μM free lapatinib (−) or lapatinib encapsulated in OP:SDS:CS complexes (+). Control samples are PBS buffer, Triton X-100 (TX), and OP:SDS:CS complexes without encapsulated drug (OP). [Figure 4f] FIG. 10: Dose-response comparison of cell viability after treatment with free lapatinib (black) or lapatinib encapsulated in OP:SDS:CS complexes (grey). [Figure 5] FIG. 10 shows further characterization of OP:SDS complexes. [Figure 5a] A replicate of the native gel shown in Figure 2b, where lanes 1–11 are increments of 200 equivalents of SDS added to the OP cage, covering the range 0–2000, and lane 12 is the fully denatured protein, obtained by treatment with 65 mM SDS, corresponding to 130,000 equivalents. [Figure 5b] Dynamic light scattering of empty OP cages and OP:SDS complexes shows negligible change in external diameter and aggregation in solution. [Figure 5c]Native agarose gel stained with Coomassie blue for proteins (left) and visualized by Atto488 fluorescence for labeled ssDNA probes (right). Lane 1 - OP; Lane 2 - Atto488-labeled DNA; Lane 2 - DNA + OP; Lane 3 - DNA + preformed OP:SDS complex. Empty OP cages can quantitatively internalize DNA, while SDS-filled cages cannot encapsulate DNA probes. [Figure 6] 10A-10C are further TEM images of OP and OP:SDS complexes. [Figure 6a] Negative staining transmission electron micrographs of OP. [Figure 6b] OP:SDS negative stain transmission electron micrograph. Scale bar is 50 nm. [Figure 7] FIG. 1 shows cryo-EM of empty OP capsids. [Figure 7a] Representative cryo-electron micrographs of OP particles in vitreous ice. [Figure 7b] The best classes from three successive rounds of 2D classification (22 classes, 4,123 particles) were refined into 3D models with imposed octahedral symmetry. [Figure 7c] Reconstruction as a projection (c). [Figure 7d] The reconstruction is shown as a slice through the center (d). [Figure 7e] The reconstruction is shown as a 3D view (e). [Figure 7f] Reconstruction after post-processing (f). [Figure 7g] 3D reconstruction (g) that overlaps well with the reported crystal structure of OP (PDB-ID: 6FDB). [Figure 8] FIG. 1 shows cryo-EM of the OP:SDS complex. [Figure 8a] Representative cryo-electron micrographs of OP:SDS particles in vitreous ice. [Figure 8b]The best classes from three successive rounds of 2D classification (13 classes, 1,967 particles) were refined into 3D models with imposed octahedral symmetry. [Figure 8c] Reconstruction as a projection (c). [Figure 8d] The reconstruction is shown as a slice through the center (d). [Figure 8e] The reconstruction is shown as a 3D view (e). [Figure 8f] Reconstruction after post-processing (f). [Figure 8g] 3D reconstruction (g) that overlaps well with the reported crystal structure of OP (PDB-ID: 6FDB). [Figure 9] Representative flow cytometry data set. [Figure 9a] All sample gating and resulting Nile Red fluorescence histogram for treatment of HeLa cells with PBS buffer (blank). [Figure 9b] All sample gating and resulting histogram of Nile Red fluorescence for treatment of HeLa cells with Nile Red (500 nM). [Figure 9c] Figure 1 shows all sample gating and the resulting histogram of Nile red fluorescence for treatment of HeLa cells with OP:SDS:Nile red (1:800:5, at 500 nM Nile red). Population gating based on the PBS control reveals that 58% of cells are Nile red positive when treated with free dye, but this percentage of cells increases to 90% when treated with OP:SDS:Nile red. A minimum of 5,000 cells were analyzed for each sample replicate. [Figure 10]Additional confocal fluorescence micrographs are shown. In all cases, cells were exposed to 500 nM Nile Red. For detergent-loaded OP cages, the molar ratios were OP:SDS:NR = 1:800:5, OP:SDS:CS:NR = 1:600:200:5, and OP-S38C-A425:SDS:CS:NR = 1:600:200:5. Atto425-labeled OP-S38C capsids have an average of 1.9 dyes per capsid. All scale bars are 30 μm. n / a indicates not applicable. [Figure 11] Characterization of OP:SDS:CS complexes. [Figure 11a] Size exclusion chromatogram of OP and OP:SDS:CS complex. The negative and positive peaks at 21 mL are due to the elution of DMSO not present in the running buffer. [Figure 11b] Dynamic light scattering of the empty OP cage and the OP:SDS:CS complex shows negligible change in external diameter or aggregation in solution. [Figure 11c] Native agarose gel stained with Coomassie blue for proteins (left) and visualized by Atto488 fluorescence for the labeled ssDNA probe (right). Lane 1: Atto488-labeled DNA only; Lane 2: OP; Lane 3: DNA + OP; Lane 4: DNA + preformed OP:SDS:CS complex. While the empty OP cage can quantitatively internalize DNA, the SDS:CS-filled cage cannot encapsulate the DNA probe. [Figure 11d] Figure 1 shows the fluorescence spectra of Nile Red in buffer and in the presence of OP:SDS and OP:SDS:CS cages. The increase in fluorescence and blue shift of the Nile Red emission maximum indicate that CS is incorporated into the protein-scaffold micelle complex. [Figure 11e] Negative staining transmission electron micrograph of the OP:SDS:CS complex. Scale bar is 50 nm. [Figure 12] FIG. 1 shows cryo-EM of the OP:SDS:CS complex. [Figure 12a]Representative cryo-electron micrographs of OP:SDS:CS particles in vitreous ice. [Figure 12b] The best classes from three successive rounds of 2D classification (17 classes, 3,359 particles) were refined into 3D models with imposed octahedral symmetry. [Figure 12c] Reconstruction as a projection (c). [Figure 12d] The reconstruction is shown as a slice through the center (d). [Figure 12e] The reconstruction is shown as a 3D view (e). [Figure 12f] Reconstruction after post-processing (f). [Figure 12g] 3D reconstruction (g) that overlaps well with the reported crystal structure of OP (PDB-ID: 6FDB). [Figure 13] FIG. 1 is a diagram of Lapatini loading. [Figure 13a] Native agarose gel of OP:SDS:CS complexes visualized with Coomassie blue for protein (left) and lapatinib fluorescence (right). Lane 1 - OP:SDS:CS (1:600:200); Lane 2 - OP:SDS:CS:lapatinib (1:600:200:10). [Figure 13b] FIG. 1 shows that in the presence of OP:SDS:CS complexes, lapatinib fluorescence is significantly increased and the emission maximum shifts to lower wavelengths, consistent with encapsulation in a non-polar environment. [Figure 13c] Fluorescence spectra of lapatinib-loaded OP:SDS:CS complexes dialyzed against bovine serum albumin (BSA)-containing medium for 24, 48, or 72 hours compared to non-dialyzed controls. [Figure 13d]Native agarose gel of OP:SDS:CS complex and BSA visualized with Coomassie blue for protein (left) and lapatinib fluorescence (right). Lane 1 - OP:SDS:CS:lapatinib (1:600:200:10); Lane 2 - BSA:lapatinib (1:1); Lane 3 - OP:SDS:CS:lapatinib + BSA. These data demonstrate that lapatinib is stably encapsulated by the OP:SDS:CS complex after incubation with equimolar concentrations of BSA, confirming the results from the fluorescence experiment shown in Figure 13c. [Figure 14] FIG. 1 is a diagram of the formation of protein cage-micelle complexes in two chemically modified protein cage variants. [Figure 14a] Transmission electron micrograph of the OP-K93C protein cage, scale bar 100 nm. [Figure 14b] Transmission electron micrograph of the OP-S38C protein cage, scale bar 100 nm. [Figure 14c] Steady-state fluorescence emission spectra of protein-conjugated Atto495 and Nile Red in the presence of the OP-K93C protein cage with and without SDS in PBS. In both cases, samples were excited at 490 nm, and only in the presence of SDS was Förster resonance energy transfer (FRET) observed between the Atto495 and Nile Red fluorophores, consistent with internalization in the protein-micelle complex. [Figure 14d]Steady-state fluorescence emission spectra of protein-conjugated Atto495 and Nile Red in the presence of the OP-S38C protein cage with and without SDS in PBS. Because the S38C mutation is on the luminal surface of the protein cage, internalization of SDS causes a change in the fluorescence of the Atto495 fluorophore conjugated at that position. Furthermore, stronger FRET was observed between Atto495 and Nile Red in the OP-S38C:SDS complex than in OP-K93C:SDS, consistent with the closer proximity of the S38C position to the micelle core compared to the externally displayed K93C position. For each measurement, 800 equivalents of SDS and 4 equivalents of Nile Red were used relative to the OP capsid (120 nM). [Figure 15] FIG. 10. Formation of protein cage-micelle complex with C-terminal peptide tag. [Figure 15a] Size-exclusion chromatogram of a protein cage with a C-terminal modification. The major peak at approximately 15 mL corresponds to a 24-mer cage with a size difference consistent with the length of the peptide appendage. Additional smaller peaks are impurities removed during this purification step. [Figure 15b] Transmission electron micrograph of the OP-ZEGFR protein cage. Scale bar is 100 nm. [Figure 15c] Transmission electron micrograph of the OP-SP94 protein cage, scale bar 100 nm. [Figure 15d] Transmission electron micrograph of the OP-ZHER2 protein cage. Scale bar is 100 nm. [Figure 15e] Transmission electron micrograph of OP-96 protein cages, scale bar 100 nm. [Figure 15f]Steady-state fluorescence spectra of Nile Red in the presence of a 1:800:5 molar ratio of capsid:SDS:Nile Red protein-micelle complex at a concentration of 120 nM capsid. All protein variants induce an increase in Nile Red fluorescence and a blue shift in the emission maximum, consistent with the formation of a protein-micelle complex and the encapsulation of Nile Red within the hydrophobic core. The original OP:SDS complex is also included for comparison. Control samples of PBS buffer, SDS alone, and OP-ZHER2 capsid alone show negligible fluorescence increases. [Figure 16] Formation of a protein cage-micelle complex with an N-terminally modified protein. [Figure 16a] Size-exclusion chromatogram of the protein cage formed by OP-93. The major peak at 14 mL corresponds to the formation of a 24-mer cage structure similar in size to OP. Additional smaller peaks are impurities removed during this purification step. [Figure 16b] Steady-state fluorescence spectra of Nile Red in the presence of a 1:800:5 molar ratio capsid:SDS:Nile Red protein-micelle complex at a concentration of 120 nM capsid. The OP-93 protein variants cause an increase in Nile Red fluorescence and a blue shift in the emission maximum, consistent with the formation of a protein-micelle complex and the encapsulation of Nile Red within the hydrophobic core. The original OP:SDS complex is also included for comparison. Control samples of PBS buffer, SDS alone, and OP-93 capsid alone show negligible fluorescence increases. [Figure 17] Alteration of surfactant composition and small molecule cargo. [Figure 17a]Native agarose gel electrophoresis showing the encapsulation of three different drug molecules in protein-micelle complexes using a 1:1 SDS:SDBS detergent mixture, containing a total of 800 equivalents of detergent per capsid. Lane 1—OP; Lane 2—OP:SDS:SDBS:curcumin; Lane 3—OP:SDS:SDBS:lapatinib; Lane 4—OP:SDS:SDBS:daunorubicin. The top and bottom panels are the same gel lanes visualized by UV illumination or stained with Coomassie blue for protein visualization. [Figure 17b] Flow cytometry dataset of HeLa cells treated with either free curcumin (-) or curcumin packaged in OP:SDS:SDBS complexes (+). Surf and OP refer to cells treated with the same concentration of curcumin in the presence of the surfactant mixture or empty OP cages, respectively. The ratio of OP:SDS:SDBS:curcumin is 1:400:400:8. Error bars indicate the standard deviation of triplicate measurements. [Figure 17c] Native agarose gel electrophoresis showing increasing concentrations (0.7, 1.4, and 2.1 uM) of OP:SDS:CS:curcumin complexes containing 23 equivalents of curcumin per capsid. [Figure 17d] Flow cytometry dataset of HeLa cells treated with either free curcumin (-) or curcumin packaged in an OP:SDS:CS complex (+). PBS refers to untreated cells, while Surf and OP refer to cells treated with the same concentration of curcumin in the presence of the surfactant mixture or empty OP cages, respectively. Eight equivalents of curcumin per cage were used, and error bars indicate the standard deviation of triplicate measurements. [Figure 17e] Confocal microscopy of HeLa cells treated with free laurdan (left) or laurdan packaged in OP:Surf complexes (right), frames are 184 μm 2 . DETAILED DESCRIPTION OF THE INVENTION

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0019] Throughout this specification and the claims that follow, unless the context requires otherwise, the words "comprise" or "include", and variations such as "comprises / includes" and "comprising / including", should be understood to imply the inclusion of elements, stated integers, steps or groups thereof, but not the exclusion of other elements, stated integers, steps or groups thereof.

[0020] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0021] The term "about" or "approximately," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 0-10% less than the stated numerical value and an upper limit of 0-10% greater than the stated numerical value. The term "about" or "approximately" preferably means ±10%, more preferably ±5%, even more preferably ±3%, or most preferably ±0%, each referring to a given numerical value. In embodiments of the present invention, the term "about" can be omitted. All ranges of values ​​disclosed herein refer to and include any and all values ​​falling within that range, including the values ​​defining the range.

[0022] In a first aspect, the present invention relates to a lipoprotein cage for intracellular delivery of cargo, the lipoprotein cage comprising: (i) a protein cage comprising at least one polypeptide comprising an amino acid sequence I consisting of: MX 13 QAIGILELX1SIAAGMELGDAMLKSAX 14 VX 15LLVSKTISX2GKFLLMLGGDIX8AIX9X 12 AIX 10 TGTX 11 QAGX3LLVDSLVLAX 16 IHPSVLPAIX 17 GX 18 NX 19 VX 20 X7X 21 QAVGIVETX4SVAACISAADX 22 AVX 23 GSX 24 VTLVRVHMAX5GIGGKCYMVVAGDVSDVALAVTVASSSAGAYGX6LVYASLIPX 25 PHX 26 AMWX 27 QMVX 28 GX 29 E (SEQ ID NO: 1) Here, X1~X 29 are each independently an amino acid, provided that at least three of X1 to X6 are each independently a positively charged amino acid, and 29 Up to five amino acids at positions other than those indicated may be replaced by any amino acid, The protein cage has a positively charged interior. a protein cage comprising at least one polypeptide comprising the amino acid sequence I; (ii) a surfactant composition comprising one or more amphiphiles, the one or more amphiphiles being selected so that the net charge of the composition is negative; the one or more amphiphiles comprise a hydrophilic group and a hydrophobic group, the hydrophobic group comprising at least one hydrocarbon moiety selected from the group consisting of C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, C4-C30 alkoxy, or C5-C30 cycloalkyl; The surfactant composition comprises a surfactant composition comprising one or more amphiphiles encapsulated in assembled protein cages; Includes:

[0023] As used herein, the terms protein cage and lipoprotein cage refer to cage-like nanoparticles. The protein cages and lipoprotein cages of the present invention are preferably in the nanometer size range (i.e., from about 1 nm to about 1000 nm). In a more preferred embodiment, the protein or lipoprotein cage has an outer diameter of up to about 50 nm. In a highly preferred embodiment, the lipoprotein cage has an outer diameter of about 13 nm.

[0024] The polypeptides of the present invention are selected so that the at least one polypeptide of the present invention can form a protein cage by self-assembly. In a preferred embodiment, the protein cage of the present invention comprises exactly 24 polypeptides of the present invention (i.e., the at least one polypeptide is defined as exactly 24 polypeptides of the present invention). In another preferred embodiment, the protein cage of the present invention has an octahedral shape (octahedral point group symmetry). In a preferred embodiment of the present invention, the protein cage has a quaternary structure of multiple subunits, preferably 8 subunits, each subunit comprising three polypeptides of the present invention.

[0025] In a preferred embodiment, the protein cage comprises (i) an outer protein scaffold and (ii) a central cavity (also referred to herein as the cavity, inner cavity, interior, or lumen). Preferably, the outer scaffold surrounds, i.e., is assembled around, the central cavity.

[0026] As used herein, the term "negatively charged" or "positively charged" includes and preferably refers to a molecule having a negatively or positively charged group. As used herein, the term "anion" refers to a negatively charged ion. More preferably, the anion or negatively charged molecule has a negatively charged group at neutral or physiological pH.

[0027] The protein cage of the present invention has a positively charged cavity. In a preferred embodiment, the protein cage is positively charged on the cavity surface. The positive charge comes from multiple positively charged amino acids, preferably arginine or lysine. Therefore, the protein cage of the present invention has a very strong affinity for encapsulating negatively charged molecules. In a preferred embodiment, the cavity of the protein cage has a diameter of about 6.5 nm to about 8 nm, preferably about 8 nm.

[0028] In a preferred embodiment, the protein cage has a porous structure, i.e., the protein cage comprises pores. Preferably, the external scaffold of the protein cage comprises pores connected to the cavity of the protein cage. Pores are defined herein as openings or gaps in the protein cage or in the external scaffold of the protein cage.

[0029] In a preferred embodiment, the protein or lipoprotein cage comprises six pores. Preferably, the external scaffold comprises six pores connected to the cavity of the protein or lipoprotein cage. In another preferred embodiment, the pores have a diameter of about 3 nm to about 4 nm. In another preferred embodiment, the protein or lipoprotein cage comprises six pores having a diameter of 3 to 4 nm.

[0030] In another more preferred embodiment, the protein cage of the present invention comprises six pores with a diameter of about 3-4 nm, the inner cavity of the protein cage has a diameter of about 8 nm, and the outer diameter of the protein cage is about 13 nm.

[0031] The loading (or encapsulation) and unloading (or release) of cargo into the protein cage of a lipoprotein or surfactant composition operates through the pores of the protein cage, and the surfactant composition influences the loading and unloading of cargo. The terms loading or encapsulation refer to any incorporation of a cargo, composition, or amphiphile into the lipoprotein or protein cage. The term unloading (or release) refers to the partial or complete, preferably complete, release or transfer / exchange of cargo.

[0032] The surfactant composition of the present invention is encapsulated in the assembled protein cage, i.e., without disassembling the protein cage. In a preferred embodiment, the assembled protein cage of the present invention can be packed with the surfactant composition without disassembling the protein cage. In a preferred embodiment of the present invention, electrostatic attraction drives the encapsulation of the surfactant composition. Preferably, in the lipoprotein cage of the present invention, the surfactant composition phase separates due to its high effective concentration within the lipoprotein cage. Preferably, the surfactant composition can form micellar aggregates within the protein cage.

[0033] More preferably, the surfactant composition according to the present invention creates a hydrophobic core within the protein cage. In a preferred embodiment, the lipoprotein cage of the present invention is both loadable and unloadable with a cargo without disassembling the lipoprotein cage. Even more preferably, the lipoprotein cage of the present invention, particularly the non-polar core of the lipoprotein cage of the present invention, can sequester a cargo molecule loaded into the lipoprotein cage of the present invention. Preferably, the cargo is encapsulated within the lipoprotein cage by a non-covalent interaction. The non-covalent interaction is preferably a hydrophobic interaction (hydrophobic effect). In a preferred embodiment, the lipoprotein cage of the present invention can deliver a cargo into a cell. Preferably, the cargo is delivered into a cell without disassembling the lipoprotein cage. In a preferred embodiment, the lipoprotein cage of the present invention can encapsulate a cargo without disassembling the lipoprotein cage and can preferably release the encapsulated cargo into a cell without disassembling the lipoprotein cage. This is possible due to the porous structure of the lipoprotein cage. In a further preferred embodiment, the lipoprotein cage of the present invention is capable of loading cargo extracellularly without disassembling the lipoprotein cage, and is incapable of loading said cargo intracellularly.

[0034] In a preferred embodiment, the lipoprotein cage of the present invention is capable of encapsulating a cargo outside a cell, entering a cell with the encapsulated cargo, and releasing the encapsulated cargo into the cell, more preferably the cytoplasm of the cell, without degrading the lipoprotein cage at each step. In another preferred embodiment, the lipoprotein cage of the present invention is capable of encapsulating a cargo, entering a cell with the encapsulated cargo, and releasing the encapsulated hydrophobic cargo into the cell, without degrading the lipoprotein cage at each step, and the released cargo can escape into the cytoplasm of the cell.

[0035] Preferably, the cargo is a hydrophobic cargo, more preferably a non-polar cargo.

[0036] Preferably, the cargo is a small cargo. Preferably, small cargo has a size of 1000 Da or less. In a preferred embodiment, a size of 1000 Da or less means that the cargo has a size of 1000 Da or less, preferably 800 Da or less, more preferably 600 Da or less, again more preferably 500 Da or less, again more preferably 400 Da or less, again more preferably 300 Da or less, again more preferably 200 Da or less, and again more preferably 100 Da or less.

[0037] In another embodiment, the cargo is a small hydrophobic cargo having a size of 1000 Da or less, even more preferably a small non-polar cargo having a size of 1000 Da or less.

[0038] Preferably, the cargo has low solubility in aqueous media. Preferably, the small cargo has a size of 1000 Da or less and has low solubility in aqueous media. More preferably, the low-solubility cargo is included in Class II or Class IV of the Biopharmaceutics Classification System (BCS). Even more preferably, the low-solubility cargo has lower solubility than a highly soluble cargo, the full-strength dose of which dissolves in 250 mL or less of aqueous media at 37±1° C. over a pH range of 1.0 to 7.5, more preferably over a pH range of 1.0 to 6.8. Preferred methods for determining solubility are a USP dissolution apparatus, a shake-flask method, or acid or base titration.

[0039] Preferably, the cargo is an active agent, preferably a therapeutic or diagnostic agent. More preferably, the cargo is selected from the group consisting of chemotherapeutic agents such as doxorubicin or paclitaxel, antifungal agents such as bifonazole or amphotericin B, antiviral agents such as indinavir or ritonavir, and antibiotics.

[0040] The surfactant composition for lipoproteins comprises one or more amphiphiles selected so that the net charge of the composition is negative. The net charge of the composition is the total charge contributed by all compounds contained in the composition. Preferably, the net charge of the composition is negative at physiological pH.

[0041] Based on the positive charges of the protein cages constructed according to the examples and the number of amphiphiles encapsulated therein, the inventors have found that at least 20 mol % of the compounds of the surfactant composition having at least one negative charge is sufficient for cargo encapsulation, especially for small cargo molecules having a size of 1000 Da or less, or hydrophobic cargo molecules, or poorly water-soluble cargo molecules included in BCS class II or class IV.

[0042] Therefore, in a further preferred embodiment, at least 20 mol% of the compounds contained in the surfactant composition have at least one negative charge. In a further preferred embodiment, at least 20 mol% of the amphiphiles contained in the surfactant composition have at least one negative charge. If a compound has multiple negative charges, i.e., N negative charges, the value of 20 mol% can be divided by N.

[0043] In a further preferred embodiment, at least 20 mol%, preferably at least 30 mol%, more preferably at least 40 mol%, even more preferably at least 50 mol%, even more preferably at least 60 mol%, even more preferably at least 70 mol%, even more preferably at least 80 mol%, even more preferably at least 90 mol%, even more preferably at least 100 mol% of the compounds or amphiphiles comprised in the surfactant composition carry at least one negative charge.

[0044] An amphiphile or amphiphilic compound (also referred to herein as a surfactant) is defined herein as an organic compound comprising at least one hydrophobic group and at least one hydrophilic group. In a preferred embodiment, the amphiphile is a diblock compound comprising a hydrophilic "head" group and a hydrophobic "tail" region. In a preferred embodiment, the amphiphile consists of at least one hydrophobic group and at least one hydrophilic group. In a preferred embodiment, the amphiphile is a diblock compound comprising, and preferably consisting of, a hydrophilic "head" group and a hydrophobic "tail" region.

[0045] A hydrophobic group comprises at least one hydrocarbon moiety. In a preferred embodiment, the hydrophobic group consists of at least one hydrocarbon moiety. As used herein, the term "hydrocarbon moiety" includes compounds consisting solely of hydrogen and carbon linked by covalent bonds. This term includes open-chain (aliphatic) hydrocarbons, including straight-chain (unbranched) and branched hydrocarbons, as well as saturated and mono- and polyunsaturated hydrocarbons. This term also includes hydrocarbons containing one or more cyclic or aromatic rings.

[0046] At least one hydrocarbon moiety is selected from the group consisting of linear or branched C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, C4-C30 alkoxy, and C5-C30 cycloalkyl. In a preferred embodiment, the hydrocarbon moiety is selected from the group consisting of linear ... or branched C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, C4-C30 alkoxy, and C5-C30 cycloalkyl. In a preferred embodiment, the hydrocarbon moiety is selected from the group consisting of C8-C20, preferably C10-18, more preferably C12-C18, and most preferably C12, C14, C16, C17, or C18, linear or branched alkyl, alkenyl, alkynyl, alkoxy, or cycloalkyl. In a preferred embodiment, the hydrocarbon moiety is selected from the group consisting of C8 to C20, preferably C10-18, more preferably C12 to C18, and most preferably C12, C14, C16, C17, or C18 straight chain alkyl, straight chain alkenyl, or cycloalkyl. In a preferred embodiment, the hydrocarbon moiety further comprises an aryl moiety in addition to the alkyl, alkenyl, alkynyl, alkoxy, and cycloalkyl moieties.

[0047] In a preferred embodiment, the hydrocarbon moiety is selected from the group consisting of C5 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16, C17, or C18 branched or straight chain alkyl, branched or straight chain alkenyl, or cycloalkyl. In a preferred embodiment, the anionic moiety is a sulfate moiety and the hydrocarbon moiety is a straight chain alkyl, straight chain alkyl ether, or cycloalkyl residue, wherein the straight chain alkyl, straight chain alkyl ether, or cycloalkyl residue is C5 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16, C17, or C18.

[0048] As used herein, the term "alkyl" or "alkyl residue" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having 4 to 30 carbon atoms (e.g., C4-C30 alkyl), and optionally, or typically, attached to the rest of the molecule by a single bond. Whenever it appears herein, a numerical range such as "4-30" refers to each integer within the given range. For example, "C4-C30" means that the alkyl group can consist of 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, etc., up to and including 30 carbon atoms, although this definition is also intended to cover occurrences of the term "alkyl" where no numerical range is specifically specified. Exemplary alkyl groups include, but are not limited to, alkyl ether, methyl, ethyl, n-propyl, 1-methylethyl (used interchangeably with isopropyl; abbreviated herein as iPr or Pri), n-butyl, isobutyl, sec-butyl, isobutyl, tertiary butyl (used interchangeably with 1,1-dimethylethyl or tert-butyl), n-pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, and decyl. Unless otherwise specifically indicated herein, alkyl groups are optionally substituted with one or more substituents that are independently alkenyl, alkoxy, carboxylic acid group (—COOH), heteroalkyl, heteroalkenyl, hydroxyl, phosphate group (—OP(O)(OH)O—), phosphonate group (—OP(O)O—), phenyl group (—CH) optionally substituted with halogen, preferably iodine, or carboxylic acid group. Preferably, as used herein, the term “alkyl” refers to unsubstituted alkyl as defined herein.

[0049] As used herein, the term "alkenyl" or "alkenyl residue" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having 4 to 30 carbon atoms (i.e., C4-C30 alkenyl), which may be, or typically is, attached to the remainder of the molecule by a single bond. Whenever it appears herein, a numerical range such as "4-30" refers to each integer within the given range; for example, "C4-C30" means that the alkenyl group can consist of 4 carbon atoms, 5 carbon atoms, etc., or contain up to 30 carbon atoms. Typical alkenyl groups include, but are not limited to, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, and penta-1,4-dienyl, alkenyl ethers. Each double bond may be in either the (E)- or (Z)-configuration. Thus, an alkenyl may contain, where applicable, the double bond in its (E)-configuration, the double bond in its (Z)-configuration, or mixtures thereof, in any ratio. Unless otherwise specifically indicated herein, an alkenyl group may be optionally substituted with one or more substituents that are independently alkenyl, alkoxy, carboxylic acid group (-COOH), heteroalkyl, heteroalkenyl, hydroxyl, phosphate group (-OP(O)(OH)O-), phosphonate group (-OP(O)O-), phenyl group (-CH) optionally substituted with halogen, preferably iodine, or carboxylic acid group. Preferably, the term "alkenyl" as used herein refers to unsubstituted alkenyl as defined herein.

[0050] As used herein, the term "alkynyl" or "alkynyl residue" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, and having 2 to 10 carbon atoms (i.e., C4-C30 alkynyl). Whenever it appears herein, a numerical range such as "4-30" refers to each integer within the given range; for example, "C4-C30" means that the alkynyl group can consist of 4 carbon atoms, 5 carbon atoms, etc., and can contain up to 30 carbon atoms. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl. Unless otherwise specifically stated in the specification, an alkynyl group is optionally substituted with one or more substituents which are independently alkenyl, a carboxylic acid group (-COOH), a heteroalkyl, a heteroalkenyl, a phosphate group (-OP(O)(OH)O-), a phosphonate group (-OP(O)O-), a phenyl group (-CH) optionally substituted with a halogen, preferably iodine, or a carboxylic acid group. Preferably, as used herein, the term "alkynyl" refers to an unsubstituted alkynyl as defined herein.

[0051] The term "alkoxy" or "alkoxy residue," as used herein, refers to an -O-alkyl group, including straight-chain, branched, and combinations thereof, of 4 to 30 carbon atoms, attached to the parent structure through an oxygen. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, and cyclohexyloxy. The term "alkoxy" includes substituted alkoxy, which refers to an alkoxy in which the alkyl moiety is substituted (i.e., -O-(substituted alkyl)). Unless otherwise specifically stated in the specification, the alkyl group of an alkoxy group may be optionally substituted with one or more substituents, independently, an alkenyl, a carboxylic acid group (-COOH), a heteroalkyl, a heteroalkenyl, a phosphate group (-OP(O)(OH)O-), a phosphonate group (-OP(O)O-), a phenyl group optionally substituted with a halogen (-CH), preferably iodine, or a carboxylic acid group.

[0052] As used herein, the term "aryl" or "aryl residue" refers to an aromatic radical having 6 to 10 ring atoms (e.g., C6-C10 aromatic or C6-C10 aryl) with at least one ring having a conjugated pi-electron system that is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl). Divalent radicals formed from substituted benzene derivatives and having free valences on ring atoms are called substituted phenylene radicals. Divalent radicals derived from monovalent polycyclic hydrocarbon radicals ending in "-yl" by removing a hydrogen atom from the carbon atom having the free valence are named by adding "-idene" to the name of the corresponding monovalent radical; for example, a naphthyl group having two points of attachment is called a naphthylidene. The term includes monocyclic or fused-ring polycyclic (i.e., rings sharing adjacent pairs of ring atoms) groups.

[0053] As used herein, the term "cycloalkyl" or "cycloalkyl residue" refers to a monocyclic or polycyclic radical containing only carbon and hydrogen and which may be saturated or partially unsaturated. Cycloalkyl groups include groups having 5 to 30 ring carbon atoms (i.e., C5-30 cycloalkyl). Whenever appearing herein, numerical ranges such as "5 to 30" refer to individual integers within the given range; for example, "C5-30 cycloalkyl" means that the cycloalkyl group may consist of up to 30 carbon atoms, such as 5 carbon atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, moieties such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, etc. The term "cycloalkyl" also refers to monocyclic or polycyclic radicals containing additional hydrocarbon moieties such as linear or branched alkyl, alkenyl, alkynyl, alkoxy, or aryl. The most preferred cycloalkyl is saturated C17 polycyclic cycloalkyl.

[0054] The term "amphiphile" as used herein includes, in preferred embodiments, amphiphilic compounds selected from the group consisting of phospholipids, sphingolipids, glycerolipids, saccharolipids, fatty acids, fatty acid esters, steroids, sterols, steroid esters, polyketides, amphiphilic block copolymers, peptides, amphiphiles comprising peptides or oligonucleotides, peptide nucleic acids, carboxylates, sulfates, sulfonates, boronates, phosphonates and phosphates.

[0055] In a preferred embodiment, the surfactant composition comprises a mixture of different amphiphiles.

[0056] In a preferred embodiment, the hydrophilic group of the amphiphile is anionic, i.e., comprises at least one anion. Preferably, the anionic hydrophilic group is such that the amphiphile has a negative net molecular charge (referred to herein as an anionic amphiphile).

[0057] In a preferred embodiment, the surfactant composition comprises at least one anionic amphiphile, i.e., an amphiphile having a negative net molecular charge. In another preferred embodiment, the surfactant composition comprises at least one anionic amphiphile having a negative net molecular charge and a cationic, uncharged and / or zwitterionic amphiphile. In a preferred embodiment, all amphiphiles in the surfactant composition are anionic amphiphiles.

[0058] In another preferred embodiment, the surfactant composition comprises a steroid. Preferably, the steroid is an anionic steroid.

[0059] Preferably, the anionic amphiphile comprises an anionic moiety and a positively charged counterion, such as an ammonium, alkali, or alkaline earth metal ion. More preferably, the positively charged counterion is a sodium ion, potassium ion, or ammonium ion, and even more preferably a sodium ion.

[0060] In preferred embodiments, the hydrophilic group of the amphiphile is selected from the group consisting of carboxylate, sulfate, sulfonate, boronate, phosphonate, phosphate, peptide, nucleic acid, amino acid moiety or peptide nucleic acid. In preferred embodiments, the hydrophilic group of the anionic amphiphile is selected from the group consisting of carboxylate, sulfate, sulfonate, boronate, phosphonate, phosphate, peptide, nucleic acid, amino acid moiety or peptide nucleic acid anion.

[0061] In a preferred embodiment, the amino acid moiety comprises, preferably consists of, one or two covalently bound amino acids. In a preferred embodiment, the peptide moiety comprises, preferably consists of, three or more amino acids. The amino acids of the hydrophilic group or of the peptide moiety include hydrophilic amino acids such as negatively charged Asp and Glu, or positively charged Lys, His and Arg, preferably negatively charged Asp and Glu.

[0062] In preferred embodiments, the nucleic acid moiety comprises, and preferably consists of, DNA, RNA, or analogs thereof, having a size of 5 to 500 base pairs, preferably 5 to 300 base pairs, more preferably 5 to 200 base pairs, or again more preferably 5 to 100 base pairs, again more preferably 5 to 50 base pairs. DNA or RNA analogs are structurally similar to natural (native) nucleic acids but differ (e.g., via chemical modifications) from natural nucleic acids in one or more of the nucleic acid backbone (e.g., phosphate in natural nucleic acids), nucleic acid sugar (e.g., deoxyribose in natural DNA and ribose in natural RNA), and nucleobase (e.g., adenosine, cytosine, guanine, thymidine, or purine in natural nucleic acids). Nucleic acid analogs and mimetics generally result from modifications of natural nucleic acids at the nucleobase (e.g., modified bases), sugar (e.g., fluorinated or deoxy sugars), and / or phosphodiester backbone (e.g., peptide or thioester backbone). Nucleic acid analogs and mimetics are known to those skilled in the art and include, for example, locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholinos.

[0063] In more preferred embodiments, the hydrophilic group is selected from the group consisting of carboxylate, sulfate, sulfonate, boronate, phosphonate, and phosphate moieties. In even more preferred embodiments, the hydrophilic group is selected from the group consisting of carboxylate, sulfate, sulfonate, and phosphate moieties. Even more preferably, the hydrophilic group is a sulfate or phosphate moiety. Even more preferably, the hydrophilic group is a sulfate moiety.

[0064] In another preferred embodiment, the hydrophilic group comprises an anionic moiety. In another preferred embodiment, the hydrophilic group comprises an anionic moiety selected from the group consisting of carboxylate, sulfate, sulfonate, phosphonate, boronate, phosphate, and an amino acid moiety. In another preferred embodiment, the hydrophilic group comprises an anionic moiety selected from the group consisting of carboxylate, sulfate, sulfonate, phosphonate, boronate, phosphate, and an amino acid moiety. Preferably, the anionic moiety is a sulfate.

[0065] In another preferred embodiment, at least one of the one or more amphiphiles included in the surfactant composition comprises (i) a hydrophilic group selected from the group consisting of a carboxylate, sulfate, sulfonate, boronate, phosphonate, phosphate moiety, and an amino acid, and (ii) a hydrophobic group comprising at least one hydrocarbon moiety selected from the group consisting of a linear or branched C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, C4-C30 alkoxy, or C5-C30 cycloalkyl.

[0066] As used herein, the term "carboxy moiety" or "carboxylate moiety" preferably refers to R-CO2 - As used herein, the term "sulfate moiety" refers to a group of R-SO4 -As used herein, the term "sulfonate moiety" preferably refers to a group of R-SO3 - As used herein, the term "phosphonate moiety" preferably refers to a group of R-PO - -R or R-PO3 2- As used herein, the term "phosphonate moiety" preferably refers to a group of R-PO4 - -R, R-PO4 2- As used herein, the term "boronate moiety" preferably refers to a group of R-BO2 2- Refers to the group of

[0067] In another preferred embodiment, the amphiphile is R-CO2 - , R-SO4 - , R-SO3 - , R-PO4 - -R, R-PO4 2- , R-PO3 - -R,R-PO3 2- , R-BO2 2- wherein R comprises a hydrocarbon moiety selected from the group consisting of linear or branched C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, C4-C30 alkoxy, or C5-C30 cycloalkyl. In a more preferred embodiment, the amphiphile is R-CO2 - , R-SO4 - , R-SO3 - , R-PO4 - , R-PO4 2- , R-PO3 - -R,R-PO3 2- , R-BO2 2- wherein R consists of a hydrocarbon moiety selected from the group consisting of straight chain C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, C4-C30 alkoxy, or C5-C30 cycloalkyl.

[0068] In a preferred embodiment, the hydrophilic group of at least one of the one or more amphiphiles included in the surfactant composition is a sulfate moiety, and in a preferred embodiment, the at least one of the one or more amphiphiles included in the surfactant composition is a sulfate salt.

[0069] In a preferred embodiment, the hydrophilic group of the amphiphile comprises a sulfate moiety and the hydrocarbon portion of the hydrophobic group is selected from the group consisting of linear or branched alkyl or alkyl ether residues, wherein the alkyl or alkyl ether is C6 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16, or C18, or a mixture thereof. In a preferred embodiment, the hydrophilic group comprises a sulfate moiety and the hydrocarbon portion is selected from the group consisting of linear or branched alkyl, alkyl ether, or alkenyl residues, wherein the alkyl or alkyl ether residue is C6 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16, or C18, or a mixture thereof. In a preferred embodiment, the hydrophilic group comprises a sulfate moiety and the hydrocarbon moiety is selected from the group consisting of straight chain alkyl, alkyl ether or alkenyl residues, wherein the straight chain alkyl or alkyl ether residue is C6 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16 or C18 or a mixture thereof.

[0070] In another preferred embodiment, the amphiphile is a salt of a primary or secondary alkyl sulfate (fatty alcohol) or alkyl ether sulfate (fatty alcohol ether).

[0071] In another preferred embodiment, the amphiphile is a salt of a primary or secondary alkyl sulfate (fatty alcohol) or alkyl ether sulfate (fatty alcohol ether), the sulfate comprising a sulfate moiety (as the hydrophilic group) and a linear alkyl residue, the linear alkyl residue being C6-C30, preferably C8-C20, more preferably C10-18, even more preferably C12-C18, most preferably C12, C14, C16 or C18 or a mixture thereof. In another preferred embodiment, the amphiphile is a salt of a primary or secondary alkyl sulfate (fatty alcohol) or alkyl ether sulfate (fatty alcohol ether), wherein the hydrophilic group of the amphiphile consists of a sulfate moiety and the hydrocarbon moiety is a linear alkyl residue, wherein the linear alkyl residue is C6-C30, preferably C8-C20, more preferably C10-18, even more preferably C12-C18, and most preferably C12, C14, C16, or C18, or a mixture thereof. The fatty alcohols and fatty alcohol ethers may be synthetic or derived from natural fats. Primary alkyl sulfate amphiphiles are defined herein as compounds with a sulfate moiety at the end of the carbon chain. Secondary alkyl sulfate amphiphiles are defined herein as compounds with sulfate moieties randomly distributed along the carbon chain.

[0072] In a preferred embodiment, at least one of the one or more amphiphiles comprised in the surfactant composition is a salt of a primary or secondary alkyl sulfate (a fatty alcohol comprising a sulfate moiety (as the hydrophilic moiety) and a linear alkyl residue), wherein the linear alkyl residue is C6-C30, preferably C8-C20, more preferably C10-18, even more preferably C12-C18, even more preferably C12, C14, C16 or C18, or a mixture thereof. In another preferred embodiment, the amphiphile is a salt of a primary alkyl sulfate (a fatty alcohol) comprising a sulfate moiety and a linear alkyl residue, wherein the linear alkyl residue is C6-C30, preferably C8-C20, more preferably C10-18, even more preferably C12-C18, even more preferably C12, C14, C16 or C18, or a mixture thereof. In another preferred embodiment, the amphiphile is a salt of a primary alkyl sulfate (fatty alcohol), the sulfate comprising a sulfate moiety and a linear alkyl residue, the linear alkyl residue being C6-C20, preferably C8-C16, more preferably C10-12, and even more preferably C12.

[0073] In a preferred embodiment, at least one of the one or more amphiphiles included in the surfactant composition is a salt of a primary or secondary alkyl sulfate (fatty alcohol), the sulfate comprising a sulfate moiety (as the hydrophilic moiety) and a linear alkyl residue, the linear alkyl residue being C6-C30, preferably C8-C20, more preferably C10-18, even more preferably C12-C18, even more preferably C12, C14, C16 or C18, and the salt of a steroid comprising an anionic moiety selected from the group consisting of sulfonate, sulfate, carboxylate, phosphonate, boronate, phosphate ester or amino acid. More preferably, the steroid comprises an anionic moiety selected from the group consisting of a sulfonate, sulfate, carboxylate, phosphonate, boronate or phosphate moiety and a steroid moiety. Even more preferably, the steroid comprises an anionic moiety selected from the group consisting of a sulfonate, sulfate, carboxylate, phosphonate, or phosphate moiety and a sterol, preferably a cholesterol, moiety.

[0074] Preferably, the amphiphile has the formula H3C-(CH2) n -CH2-O-SO3 - or H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 -wherein m is a value of 1 to 4, and n is a value of 4 to 20, preferably 6 to 20, more preferably 8 to 18, even more preferably 10 to 16, even more preferably 10 to 14, even more preferably 10 to 12, and most preferably 10. In another preferred embodiment, m is 2 or 3, and n is a value of 4 to 20, preferably 6 to 20, more preferably 8 to 18, even more preferably 10 to 16, even more preferably 10 to 14, even more preferably 10 to 12, and most preferably 10. In another preferred embodiment, m is 2, and n is a value of 4 to 20, preferably 6 to 20, more preferably 8 to 18, even more preferably 10 to 16, even more preferably 10 to 14, even more preferably 10 to 12, and most preferably 10.

[0075] Preferably, the amphiphile has the formula H3C-(CH2) n -CH2-O-SO3 - In the formula, n is a value of 4 to 20, preferably 6 to 20, more preferably 8 to 18, even more preferably 10 to 16, even more preferably 10 to 14, even more preferably 10 to 12, and most preferably 10.

[0076] In another preferred embodiment, the amphiphile has the formula H3C-(CH2) n -CH2-O-SO3 - where n is a value between 4 and 20, preferably between 6 and 20, more preferably between 8 and 18, even more preferably between 10 and 16, even more preferably between 10 and 14, even more preferably between 10 and 12, and most preferably 10, and is a steroid sulfonate, sulfate, or carboxylate, phosphonate, boronate, phosphate ester of a steroid. More preferably, the anionic steroid for these amphiphiles is a sulfonate, sulfate, carboxylate, phosphonate, boronate, or phosphate ester of cholesterol.

[0077] In another preferred embodiment, the amphiphile is selected from the group consisting of salts of lauryl sulfate, laureth sulfate, pareth sulfate, myreth sulfate, n-octyl sulfate, 8-hexadecyl sulfate, and tetradecyl sulfate. In another preferred embodiment, the amphiphile is selected from the group consisting of sodium lauryl sulfate, ammonium lauryl sulfate, potassium lauryl sulfate, sodium laureth sulfate, ammonium laureth sulfate, sodium pareth sulfate, sodium myreth sulfate, n-octyl sulfate, 8-hexadecyl sulfate, and sodium tetradecyl sulfate. In another preferred embodiment, the amphiphile is a salt of lauryl sulfate or laureth sulfate.

[0078] Preferably, the salt of the sulfate is selected from the ammonium, alkali or alkaline earth salts, more preferably the sodium, potassium or ammonium salts, even more preferably the sodium salt.

[0079] In preferred embodiments, the amphiphile is selected from the group consisting of sodium lauryl sulfate (sodium dodecyl sulfate, SLS, or SDS, typically referred to herein as SDS), ammonium lauryl sulfate, potassium lauryl sulfate, sodium laureth sulfate (sodium lauryl ether sulfate or SLES), ammonium laureth sulfate, sodium pareth sulfate, sodium myreth sulfate, sodium n-octyl sulfate, sodium 8-hexadecyl sulfate, sodium dodecylbenzenesulfonate (SDBS), and sodium tetradecyl sulfate.

[0080] In another preferred embodiment, the surfactant composition comprises an amphiphile selected from the group consisting of SDS, ammonium lauryl sulfate, potassium lauryl sulfate, SLES, ammonium laureth sulfate, sodium pareth sulfate, sodium myreth sulfate, sodium n-octyl sulfate, sodium 8-hexadecyl sulfate, and sodium tetradecyl sulfate in combination with a steroid containing an anionic moiety selected from the group consisting of sulfonate, sulfate, carboxylate, phosphonate, boronate, and phosphate ester. More preferably, the steroid is a sulfonate, sulfate, carboxylate, phosphonate, boronate, or phosphate ester of cholesterol.

[0081] In a preferred embodiment, the amphiphile is an ammonium, alkali, or alkaline earth salt of lauryl sulfate or laureth sulfate. In a more preferred embodiment, the amphiphile is a sodium, potassium, or ammonium salt of lauryl sulfate or laureth sulfate. In another preferred embodiment, the amphiphile is a sodium salt of lauryl sulfate or laureth sulfate.

[0082] In a preferred embodiment, the amphiphile is an ammonium, alkali, or alkaline earth salt of lauryl sulfate (sodium dodecyl sulfate, SLS, or SDS). In a more preferred embodiment, the amphiphile is a sodium, potassium, or ammonium salt of lauryl sulfate. Most preferably, the amphiphile is sodium lauryl sulfate.

[0083] In a preferred embodiment, the hydrophilic group of at least one of the one or more amphiphiles included in the surfactant composition is a sulfonate, boronate, or phosphonate moiety, and in a preferred embodiment, the at least one of the one or more amphiphiles included in the surfactant composition is a salt of a sulfonate, boronate, or phosphonate.

[0084] In a preferred embodiment, the hydrophilic group of at least one of the amphiphiles in the surfactant composition is a sulfonate, boronate, or phosphonate moiety, and the hydrocarbon portion of the hydrophobic group is selected from the group consisting of alkyl, alkyl-benzene, benzene-alkyl, alkyl-ester, alkenyl, alkyl-succinate, alkyl-acetate, and alkyl-tauride, where the alkyl or alkenyl is linear or branched, preferably linear C6-C30.The hydrophilic group of at least one of the amphiphiles in the surfactant composition is a sulfonate, boronate, or phosphonate moiety, and the hydrocarbon portion is selected from the group consisting of alkyl, alkyl-benzene, benzene-alkyl, alkyl-ester, alkenyl, alkyl-succinate, alkyl-acetate, and alkyl-tauride, where the alkyl or alkenyl is linear or branched, preferably linear C8-C20. The hydrophilic group of at least one of the amphiphiles in the surfactant composition is a sulfonate, boronate, or phosphonate moiety, and the hydrocarbon moiety is selected from the group consisting of alkyl, alkyl-benzene, benzene-alkyl, alkyl ester, alkenyl, alkyl-succinate, alkyl-acetate, and alkyl-tauride, where the alkyl or alkenyl is linear or branched, preferably linear C10-18. The hydrophilic group of at least one of the amphiphiles in the surfactant composition is a sulfonate, boronate, or phosphonate moiety, and the hydrocarbon moiety is selected from the group consisting of alkyl, alkyl-benzene, benzene-alkyl, alkyl ester, alkenyl, alkyl-succinate, alkyl-acetate, and alkyl-tauride, where the alkyl or alkenyl is linear or branched, preferably linear C12-C18, or a mixture thereof.The hydrophilic group of at least one of the amphiphiles in the surfactant composition is a sulfonate, boronate, or phosphonate moiety, and the hydrocarbon moiety is selected from the group consisting of alkyl, alkyl-benzene, benzene-alkyl, alkyl ester, alkenyl, alkyl-succinate, alkyl-acetate, and alkyl-tauride, where the alkyl or alkenyl is linear or branched, preferably linear C12, C14, C16, or C18, or a mixture thereof. The hydrophilic group of at least one of the amphiphiles in the surfactant composition is a sulfonate, boronate, or phosphonate moiety, and the hydrocarbon moiety is selected from the group consisting of alkyl, alkyl-benzene, benzene-alkyl, alkyl ester, alkenyl, alkyl-succinate, alkyl-acetate, and alkyl-tauride, where the alkyl or alkenyl is linear or branched, preferably linear C12 or C14, or a mixture thereof.

[0085] In a preferred embodiment, at least one of the one or more amphiphiles in the surfactant composition is a salt of a sulfonate, the sulfonate being selected from the group consisting of primary alkyl sulfonates, secondary alkyl sulfonates, sulfonated alkyl esters, alkyl ester sulfonates, alpha olefin sulfonates, aryl alkyl sulfonates, alkyl aryl sulfonates, alkyl benzene sulfonates (ABS), benzene alkyl sulfonates, alkyl sulfoacetates, alkyl sulfosuccinates, alkyl taurides, sulfolipids, and sulfoglycolipids. In another preferred embodiment, the amphiphile is a salt of a sulfonate selected from the group consisting of primary alkyl sulfonates, secondary alkyl sulfonates, sulfonated alkyl esters, alkyl ester sulfonates, aryl alkyl sulfonates, alkyl aryl sulfonates, alkyl benzene sulfonates, benzene alkyl sulfonates, alkyl sulfoacetates, alkyl sulfosuccinates, and alkyl taurides. In more preferred embodiments, the amphiphile is selected from the group consisting of primary alkyl sulfonates, secondary alkyl sulfonates, sulfonated alkyl esters, alkyl ester sulfonates, alpha olefin sulfonates, alkyl benzene sulfonates, benzene alkyl sulfonates, alkyl sulfoacetates, alkyl sulfosuccinates, and alkyl taurides. In even more preferred embodiments, the amphiphile is a salt of a primary alkyl sulfonate or a secondary alkyl sulfonate.

[0086] In a preferred embodiment, the amphiphile is a salt of a sulfonate comprising a linear or branched, preferably linear, alkyl or alkenyl residue, the salt of the sulfonate being selected from the group consisting of primary alkyl sulfonates, secondary alkyl sulfonates, sulfonated alkyl esters, alkyl ester sulfonates, alpha olefin (alkenyl) sulfonates, alkyl benzene sulfonates (ABS), benzene alkyl sulfonates, alkyl sulfoacetates, alkyl sulfosuccinates, and alkyl taurides, wherein the alkyl residue is C6-C30, preferably C8-C20, more preferably C10-18, even more preferably C12-C18, and most preferably C12, C14, C16, or C18, or a mixture thereof. In another preferred embodiment, the amphiphile is a salt of a primary or secondary alkylsulfonate containing a linear or branched alkyl residue of C6 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, most preferably C12, C14, C16 or C18, or a mixture thereof. In another preferred embodiment, the amphiphile is a salt of a primary or secondary alkylsulfonate containing a linear or branched alkyl residue of C6 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, most preferably C12, C14, C16 or C18, or a mixture thereof.

[0087] Preferably, said salts of sulfates, sulfonates, boronates or phosphonates are selected from ammonium, alkali or alkaline earth salts, more preferably sodium, potassium or ammonium salts, even more preferably sodium salts.

[0088] In a preferred embodiment, at least one of the one or more amphiphiles included in the surfactant composition is a salt of a primary or secondary alkyl sulfate or a salt of a primary or secondary alkyl sulfonate, wherein the alkyl residue of the alkyl sulfate or alkyl sulfonate is a linear C6-C30 alkyl group or has the formula H3C-(CH2) n-CH2-(O-CH2CH2) m -O-SO3 - wherein m is a value from 1 to 4 and n is a value from 4 to 20. Preferably, the amphiphile is a salt of a primary or secondary alkyl sulfate or a salt of a primary or secondary alkyl sulfonate, the alkyl residue of which is a linear C8-C20 alkyl sulfate or alkyl sulfonate of the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - wherein m is a value from 1 to 4 and n is a value from 6 to 18. Preferably, the amphiphile is a salt of a primary or secondary alkyl sulfate or a salt of a primary or secondary alkyl sulfonate, the alkyl residue of which is a linear C10-C18 alkyl sulfate or alkyl sulfonate, or of the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - where m is a value of 2 or 3 and n is a value of 8 to 16. Preferably, the amphiphile is a salt of a primary or secondary alkyl sulfate or a salt of a primary or secondary alkyl sulfonate, where the alkyl sulfate or alkyl residue of the alkyl sulfonate is a linear C10 to C16 or has the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - where m is 2 or 3 and n is 8 to 14. Preferably, the amphiphile is a salt of a primary or secondary alkyl sulfate or a salt of a primary or secondary alkyl sulfonate, the alkyl residue of which is a linear C10-C14 alkyl sulfate or alkyl sulfonate, or of the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 -where m is 2 or 3 and n is 8 to 12. Preferably, the amphiphile is a salt of a primary or secondary alkyl sulfate or a salt of a primary or secondary alkyl sulfonate, the alkyl residue of which is a linear C12 or has the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - wherein m is 2 or 3 and n is 10. Preferably, the salt of the sulfate or sulfonate is selected from the ammonium, alkali or alkaline earth salts, more preferably the sodium, potassium or ammonium salts, even more preferably the sodium salt.

[0089] In a preferred embodiment, the hydrophilic group of at least one of the one or more amphiphiles included in the surfactant composition is a carboxyl moiety, and in a preferred embodiment, at least one of the one or more amphiphiles included in the surfactant composition is a carboxylate salt.

[0090] In a preferred embodiment, the hydrophilic group is a carboxy moiety and the hydrocarbon portion of the hydrophobic group is selected from the group consisting of alkyl, alkynyl, alkenyl or fatty acid residues, preferably straight chain alkyl, alkenyl or fatty acid residues. In a preferred embodiment, the hydrophilic group is a carboxy moiety and the hydrocarbon portion of the hydrophobic group is selected from the group consisting of straight chain alkyl, alkynyl, alkenyl or fatty acid residues, wherein the alkyl or alkenyl residue is C6-C30, preferably C8-C20, more preferably C10-18, even more preferably C12-C18, and most preferably C12, C14, C16 or C18 or a mixture thereof.

[0091] In a preferred embodiment, the hydrophilic group is a carboxy moiety and the hydrocarbon portion of the hydrophobic group is a straight chain alkyl residue, the straight chain alkyl residue being C6 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16 or C18 or a mixture thereof.

[0092] In a preferred embodiment, the hydrophilic group is a carboxy moiety and the hydrocarbon portion of the hydrophobic group is a linear or branched moiety selected from alkyl, alkynyl, alkenyl, or a fatty acid, alkyl ester, alkyl ether, alkyl polyglycol, or alkyl sarcosinate, where alkyl or alkenyl is C6 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16, or C18, or a mixture thereof. In a preferred embodiment, the hydrophilic group is a carboxy moiety and the hydrocarbon portion of the hydrophobic group is a linear alkyl residue, where the linear alkyl residue is C6 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16, or C18, or a mixture thereof.

[0093] In a preferred embodiment, at least one of the one or more amphiphiles contained in the surfactant composition is an amphiphile selected from the group consisting of primary or secondary alkyl sulfates, primary or secondary alkyl sulfonates, alkyl sarcosinates or alkyl carboxylates, wherein the alkyl residue of the primary or secondary alkyl sulfates, alkyl sulfonates, alkyl sarcosinates or alkyl carboxylates is linear or branched, preferably linear C6-C30, or of the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 -wherein m is a value from 1 to 4 and n is a value from 4 to 20. More preferably, the amphiphile is a salt selected from the group consisting of primary or secondary alkyl sulfates, primary or secondary alkyl sulfonates, alkyl sarcosinates or alkyl carboxylates, wherein the alkyl residue of the primary or secondary alkyl sulfate, alkyl sulfonate, alkyl sarcosinate or alkyl carboxylate is linear or branched, preferably linear C8 to C20, or alkyl of the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - where m is 2 or 3 and n is a value from 6 to 18. More preferably, the amphiphile is a salt selected from the group consisting of primary or secondary alkyl sulfates, primary or secondary alkyl sulfonates, alkyl sarcosinates or alkyl carboxylates, wherein the alkyl residue of the primary or secondary alkyl sulfate, alkyl sulfonate, alkyl sarcosinate or alkyl carboxylate is linear or branched, preferably linear C10 to C18, or alkyl of the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - where m is 2 or 3 and n is a value from 8 to 16. More preferably, the amphiphile is a salt selected from the group consisting of primary or secondary alkyl sulfates, primary or secondary alkyl sulfonates, alkyl sarcosinates or alkyl carboxylates, wherein the alkyl residue of the primary or secondary alkyl sulfate, alkyl sulfonate, alkyl sarcosinate or alkyl carboxylate is linear or branched, preferably linear C12 to C18, or alkyl of the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 -wherein m is 2 or 3 and n is a value from 10 to 16. More preferably, the amphiphile is a salt selected from the group consisting of primary or secondary alkyl sulfates, primary or secondary alkyl sulfonates, alkyl sarcosinates or alkyl carboxylates, wherein the alkyl residue of the primary or secondary alkyl sulfate, alkyl sulfonate, alkyl sarcosinate or alkyl carboxylate is linear or branched, preferably linear C12 to C16, or alkyl of the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - (wherein m is 2 or 3, and n is a value of 10 to 14).

[0094] In a preferred embodiment, at least one of the one or more amphiphilic substances included in the surfactant composition is an amphiphilic lipid. As used herein, the term "amphiphilic lipid" refers to an amphiphilic compound that includes both a hydrophilic portion and a hydrophobic portion containing a hydrocarbon selected from oils, fats (such as fatty acids and glycerides), sterols, steroids, and derivative forms of these compounds. Suitable amphiphilic lipids include portions derived from fatty acids and their derivatives, hydrocarbons and their derivatives, and sterols such as cholesterol. In a preferred embodiment, the amphiphilic lipid is selected from the group consisting of phospholipids, sphingolipids, glycerolipids, and saccharolipids.

[0095] In a preferred embodiment, the hydrophilic group of at least one of the one or more amphiphiles included in the surfactant composition is a phosphate moiety, and in a preferred embodiment, at least one of the one or more amphiphiles included in the surfactant composition is a phosphate salt.

[0096] In a preferred embodiment, the hydrophilic group is a phosphate moiety and the hydrocarbon portion of the hydrophobic group is selected from the group consisting of a fatty acid, a fatty acid ester, an alkyl, an alkenyl, or a glyceride moiety. Preferably, the fatty acid, fatty acid ester, alkyl, or alkenyl is linear. Preferably, the hydrophobic group is selected from the group consisting of a saturated fatty acid, a fatty acid ester, a linear alkyl, or a linear alkenyl.

[0097] The term "fatty acid" as used herein refers to a hydrocarbon chain terminating in a carboxylic acid group, which hydrocarbon chain is typically and preferably either alkyl or alkenyl, typically 3 to 32 carbons in length, and therefore saturated or unsaturated, and which contains one or more, preferably one, carboxylic acid groups (-COOH), one or more, preferably one C alkyl, one or more, preferably one phosphate groups (HOP(O)(OH)O-), one or more, preferably one phosphonate groups (HOP(O)O-), one or more, preferably one may be substituted with one thiophosphate group (HOP(O)(SH)O-), one or more, preferably one dithiophosphate group (HOP(S)(SH)O-), one or more, preferably one diphosphate group (HO-P(O)(OH)-OP(O)(OH)-O-), one or more, preferably one triphosphate group (HO-P(O)(OH)-OP(O)(OH)-OP(O)(OH)-O-), one or more phenyl groups (-CH), one or more phenyl groups substituted with halogen, preferably iodine, or a carboxylic acid group. When the fatty acid contains one or more double bonds and is therefore unsaturated, it can be either cis or trans geometric isomerism. The term "fatty acid moiety," as used herein, refers to a moiety derived from a fatty acid, as defined herein, in which one carboxylic acid group (-COOH) of the fatty acid becomes a -C(O)- group of the fatty acid moiety, which is linked to the oligonucleotide, either directly or via a spacer according to the invention. The term "fatty acid" includes fatty acid diacids, which refer to fatty acids as defined herein, but with an additional carboxylic acid group at the omega position. In a preferred embodiment, the amphiphile is a phosphate salt comprising a phosphate moiety and at least one linear or branched, preferably linear C6-C30, chain of a fatty acid, fatty acid ester, alkyl, or alkenyl. In a further preferred embodiment, the amphiphile is a phosphate salt comprising a phosphate moiety and at least one C8-C20 chain of a fatty acid, alkyl, or alkenyl.In a further preferred embodiment, the amphiphile is a phosphate salt comprising a phosphate moiety and at least one C10-C18 chain of a fatty acid, alkyl, or alkenyl. In a further preferred embodiment, the amphiphile is a phosphate salt comprising a phosphate moiety and at least one C12-C18 chain of a fatty acid, alkyl, or alkenyl. In a further preferred embodiment, the amphiphile is a phosphate salt comprising a phosphate moiety and at least one C12-C18 chain of a fatty acid, alkyl, or alkenyl. In a further preferred embodiment, the amphiphile is a phosphate salt comprising a phosphate moiety and at least one C12, C14, C16, or C18 chain of a fatty acid, alkyl, or alkenyl, or a mixture thereof. Preferably, the alkyl or alkenyl is linear. More preferably, the alkyl or alkenyl is linear and the fatty acid is unsaturated.

[0098] In a preferred embodiment, at least one of the one or more amphiphiles included in the surfactant composition is a phosphate salt selected from the group consisting of monoalkyl phosphate ester salts, dialkyl phosphate ester salts, monoalkenyl phosphate ester salts, dialkenyl phosphate ester salts, and phospholipids.

[0099] In a preferred embodiment, at least one of the one or more amphiphiles contained in the surfactant composition is a phospholipid. Preferably, the phospholipid consists of at least two hydrophobic fatty acid moieties, preferably exactly two fatty acid moieties, and a hydrophilic phosphate moiety. The moieties are preferably covalently linked by a glycerol moiety. The phosphate moiety is preferably modified with a simple organic molecule such as choline, ethanolamine, or serine.

[0100] In a preferred embodiment, at least one of the one or more amphiphiles included in the surfactant composition is a phosphate salt selected from the group consisting of monoalkyl phosphate ester salts, dialkyl phosphate ester salts, monoalkenyl phosphate ester salts, dialkenyl phosphate ester salts, and phospholipids, and the alkyl or alkenyl of the fatty acid of the phosphate ester salt or phospholipid is a linear C6 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16, or C18, or a mixture thereof.

[0101] In a preferred embodiment, at least one of the one or more amphiphiles in the surfactant composition is a salt selected from the group consisting of a primary or secondary alkyl sulfate, a primary or secondary alkyl sulfonate, an alkyl phosphonate, an alkyl boronate, an alkyl sarcosinate, an alkyl carboxylate, a monoalkyl phosphate ester, or a dialkyl phosphate ester, wherein the alkyl residue of the alkyl sulfate, alkyl sulfonate, alkyl phosphonate, alkyl boronate, alkyl sarcosinate, or alkyl carboxylate or phosphate ester is a linear C6-C30 alkyl residue or has the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - wherein m is a value from 1 to 4 and n is a value from 4 to 20. More preferably, the amphiphile is a salt selected from the group consisting of a primary or secondary alkyl sulfate, a primary or secondary alkyl sulfonate, an alkyl phosphonate, an alkyl boronate, an alkyl sarcosinate or an alkyl carboxylate, a monoalkyl phosphate ester, or a dialkyl phosphate ester, wherein the alkyl residue of the alkyl sulfate, alkyl sulfonate, alkyl phosphonate, alkyl boronate, alkyl sarcosinate or alkyl carboxylate or phosphate ester is a linear C8 to C30 alkyl residue or has the formula H3C-(CH2)n -CH2-(O-CH2CH2) m -O-SO3 - wherein m is 2 or 3 and n is a value from 6 to 18. More preferably, the amphiphile is a salt selected from the group consisting of primary or secondary alkyl sulfates, primary or secondary alkyl sulfonates, alkyl phosphonates, alkyl boronates, alkyl sarcosinates or alkyl carboxylates, monoalkyl phosphate esters, or dialkyl phosphate esters, wherein the alkyl residue of the alkyl sulfate, alkyl sulfonate, alkyl phosphonate, alkyl boronate, alkyl sarcosinate or alkyl carboxylate or phosphate ester is a linear C10-C18 or has the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - wherein m is 2 or 3 and n is a value from 8 to 16. More preferably, the amphiphile is a salt selected from the group consisting of a primary or secondary alkyl sulfate, a primary or secondary alkyl sulfonate, an alkyl phosphonate, an alkyl boronate, an alkyl sarcosinate or an alkyl carboxylate, a monoalkyl phosphate ester, or a dialkyl phosphate ester, wherein the alkyl residue of the primary or secondary alkyl sulfate, alkyl sulfonate, alkyl phosphonate, alkyl boronate, alkyl sarcosinate or alkyl carboxylate or phosphate ester is a linear C12 to C18 or has the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 -wherein m is 2 or 3 and n is a value from 10 to 16. More preferably, the amphiphile is a salt selected from the group consisting of primary or secondary alkyl sulfates, primary or secondary alkyl sulfonates, alkyl phosphonates, alkyl boronates, alkyl sarcosinates or alkyl carboxylates, monoalkyl phosphate esters, or dialkyl phosphate esters, wherein the alkyl residue of the primary or secondary alkyl sulfates, alkyl sulfonates, alkyl phosphonates, alkyl boronates, alkyl sarcosinates or alkyl carboxylates or phosphate esters is a linear C12 to C16 or has the formula H3C-(CH2) n -CH2-(O-CH2CH2) m -O-SO3 - (wherein m is 2 or 3, and n is a value of 10 to 14).

[0102] In a preferred embodiment, the molar ratio of total surfactant molecules (i.e., anionic amphiphile and optional anionic steroid) to protein cages encapsulated in the assembled protein cage is up to about 1000: 1. In a preferred embodiment, the molar ratio of total surfactant molecules to protein cages is about 800: 1.

[0103] In another preferred embodiment, the anionic amphiphile is a salt of dodecyl sulfate, preferably sodium dodecyl sulfate, and the molar ratio of dodecyl sulfate molecules encapsulated in the assembled protein cages to the protein cages is up to about 1000: 1. In a preferred embodiment, the anionic amphiphile is a salt of dodecyl sulfate, preferably sodium dodecyl sulfate (SDS), and the molar ratio of dodecyl sulfate molecules encapsulated in the assembled protein cages to the protein cages is up to about 800: 1. This is based on data showing that the change in the quaternary structure of the protein cages of the present invention does not occur at a concentration of about 800 equivalents of SDS, but above 1000 equivalents, a band of increased mobility appears, potentially due to the external association of SDS molecules with the protein cages.

[0104] In preferred embodiments, the hydrocarbon moiety is selected from the group consisting of fatty acids, fatty acid esters, steroids, sterols, and steroid esters.

[0105] In another preferred embodiment, the one or more amphiphiles in the surfactant composition comprise an amphiphilic steroid. In another preferred embodiment, the one or more amphiphiles in the surfactant composition comprise an anionic amphiphile steroid. In another preferred embodiment, the one or more amphiphiles in the surfactant composition comprise an anionic amphiphile and an anionic steroid. In another preferred embodiment, the one or more amphiphiles in the surfactant composition comprise an anionic amphiphile and an anionic steroid, and the hydrophilic groups of the anionic amphiphile and the anionic steroid are selected from the group consisting of sulfonate, sulfate, carboxylate, phosphonate, boronate, and phosphate moieties. More preferably, in another preferred embodiment, the one or more amphiphiles in the surfactant composition comprise an anionic amphiphile and an anionic steroid, and the hydrophilic groups of the anionic amphiphile and the anionic steroid are sulfate moieties.

[0106] The term "steroid" as used herein preferably includes steroids, steroid esters and sterols, more preferably anionic steroids, steroid esters and sterols.

[0107] Preferably, the anionic steroid is selected from the group consisting of sulfonates, sulfates, carboxylates, phosphonates, boronates, phosphates, phosphate esters, or steroids containing hydrophilic amino acids. More preferably, the anionic steroid is selected from the group consisting of steroid sulfonates, steroid sulfates, and steroid phosphate esters. More preferably, the anionic steroid comprises an anionic moiety selected from the group consisting of sulfonates, sulfates, carboxylates, phosphonates, boronates, and phosphates; and a steroid moiety selected from the group consisting of estradiol, estriol, diethylstilbestrol, dehydroepiandrosterone, cholesterol, pregnenolone, DHEA, androstenediol, androsterone, estrone, and testosterone. In another preferred embodiment, the anionic steroid is selected from the group consisting of estriol sulfate, estradiol sulfate, estradiol disulfate (EDS), diethylstilbestrol disulfate, dehydroepiandrosterone sulfate, cholesterol sulfate, pregnenolone sulfate, DHEA sulfate, androstenediol sulfate, androsterone sulfate, estrone sulfate, estradiol sulfate, and testosterone sulfate. Most preferably, the anionic steroid is cholesterol sulfate (CS).

[0108] In a preferred embodiment, the anionic amphiphile of the surfactant composition of the present invention is sodium dodecyl sulfate (SDS) and the anionic steroid is cholesterol sulfate ((CS).

[0109] In a preferred embodiment, the surfactant composition comprises an anionic amphiphile and an anionic steroid encapsulated in an assembled protein cage, the molar ratio of anionic amphiphile and anionic steroid in the surfactant composition being between 50% anionic amphiphile / 50% anionic steroid and 100% anionic amphiphile / 0% anionic steroid, more preferably between 75% anionic amphiphile / 25% anionic steroid and 100% anionic amphiphile / 0% anionic steroid. In a preferred embodiment, the anionic steroid is used for encapsulation of large planar cargo molecules, preferably the surfactant composition comprises, expressed as a molar ratio, 75% anionic amphiphile and 25% anionic steroid.

[0110] In a preferred embodiment, the anionic amphiphile is SDS and the anionic steroid is CS, and SDS is contained in the surfactant composition in an amount of 100% to 50%, preferably 100% to 75%, relative to CS, expressed as a molar ratio.

[0111] In a preferred embodiment, the surfactant composition comprises SDS and CS. In a preferred embodiment, the surfactant composition consists of SDS and CS. In a preferred embodiment, the surfactant composition comprises SDS and CS, and the molar ratio of the SDS to CS is 4:1 to 2:1. In a preferred embodiment, the surfactant composition consists of SDS and CS, and the molar ratio of the SDS to CS is 4:1 to 2:1. In a preferred embodiment, the surfactant composition comprises SDS and CS, and the molar ratio of the SDS to CS is 3:1. In a preferred embodiment, the surfactant composition comprises SDS and CS, and the molar ratio of the SDS to CS is 3:1.

[0112] A protein cage comprising at least one polypeptide comprising an amino acid sequence I consisting of: MX 13 QAIGILELX1SIAAGMELGDAMLKSAX 14 VX 15LLVSKTISX2GKFLLMLGGDIX8AIX9X 12 AIX 10 TGTX 11 QAGX3LLVDSLVLAX 16 IHPSVLPAIX 17 GX 18 NX 19 VX 20 X7X 21 QAVGIVETX4SVAACISAADX 22 AVX 23 GSX 24 VTLVRVHMAX5GIGGKCYMVVAGDVSDVALAVTVASSSAGAYGX6LVYASLIPX 25 PHX 26 AMWX 27 QMVX 28 GX 29 E (SEQ ID NO: 1) Here, X1~X 29 are each independently an amino acid, provided that at least three of X1 to X6 are each independently a positively charged amino acid, and 29 Up to five amino acids at positions other than those indicated may be replaced by any amino acid.

[0113] The term "polypeptide" as used herein refers to any peptide-linked polymer of amino acids, regardless of size, length, secondary and tertiary structure, number of subunits, or post-translational modifications. Thus, the term "polypeptide" should be understood to encompass the terms "peptide," "protein," "amino acid chain," and "amino acid sequence." Polypeptides according to the present invention may be open linear peptide chains or cyclic peptides. Alternatively, or in addition, peptides of the present invention may contain at least one chemical modification, such as lipidation, glycosylation, and phosphorylation. Peptides as understood herein, particularly peptides of the present invention, may be isolated or, preferably, produced by chemical synthesis, RNA translation, and / or recombinant processes.

[0114] As used herein, the term "amino acid" refers to an organic compound containing the functional groups amine (-NH) and carboxylic acid (-COOH) and their zwitterions, typically and preferably with a side chain specific to each amino acid. The term "amino acid" typically and preferably includes naturally occurring amino acids, such as proteinogenic amino acids (produced by RNA translation), non-proteinogenic amino acids (produced by other metabolic mechanisms, e.g., post-translational modification), standard or canonical amino acids (directly encoded by codons in the genetic code), and non-standard or non-canonical amino acids (not directly encoded by the genetic code). Naturally occurring amino acids include proteinogenic amino acids and non-proteinogenic amino acids. As used herein, the term "amino acid" also includes chemically synthesized non-natural amino acids. Furthermore, the term encompasses alpha- (α-), beta- (β-), gamma- (γ-), and delta- (δ-) amino acids, as well as mixtures thereof in any ratio, and, where applicable, any isomeric form of an amino acid, i.e., its D- and L-stereoisomers (alternatively designated by the (R) and (S) nomenclature), as well as mixtures thereof in any ratio, preferably a 1:1 racemic ratio. Amino acids in the present invention are typically preferably in the L-configuration. The terms "D-stereoisomer," "L-stereoisomer," "D-amino acid," or "L-amino acid" refer to the chiral alpha carbon of an amino acid. Amino acids can include modified and / or conjugated compounds and residues, e.g., residues used in peptide synthesis, such as Boc, Fmoc, or both.

[0115] "X n ~X m " and "X n-m The terms "A" and "B" are used interchangeably herein to refer to specific amino acid positions in SEQ ID NO:1.

[0116] X in SEQ ID NO:1 1-29Up to five amino acids at positions not indicated with may be replaced with any amino acid. The terms "amino acid exchange" or "replaced by any amino acid", as used interchangeably herein, include or preferably refer to a deletion of one amino acid or a replacement (addition) of a single amino acid with one or more amino acids, more preferably with one, two or three amino acids. Most preferably, the term "amino acid exchange" refers to a deletion of a single amino acid or a replacement of a single amino acid with one, two or three amino acids. In a preferred embodiment, the amino acid exchange is a substitution. In another preferred embodiment, the amino acid exchange is a conservative amino acid substitution.

[0117] The term "conservative substitution" refers to an amino acid substitution that changes a given amino acid to a different amino acid with similar biochemical properties. Conservative substitutions include, and preferably refer to, isosteric substitutions and substitutions that maintain the charged, polar, aromatic, aliphatic, or hydrophobic nature of the amino acid. Conservative substitutions refer to substitutions that maintain the ability of the polypeptides of the present invention to self-assemble into the protein cages of the present invention.

[0118] As used herein, the term "positively charged" includes and preferably refers to molecules that have a positively charged group. More preferably, the positively charged molecule has a positively charged group at neutral or physiological pH.

[0119] In a preferred embodiment of the present invention, X in SEQ ID NO: 1 1-29 For positions not indicated with , optionally up to four amino acids, more preferably up to three amino acids, even more preferably up to two amino acids, and most preferably one amino acid are replaced by any amino acid.

[0120] In a preferred embodiment, the polypeptide has a length of about 500 amino acids or less, preferably about 400 amino acids or less, more preferably 300 amino acids or less, even more preferably 250 amino acids or less, even more preferably 200 amino acids or less, even more preferably 188 to 230 amino acids, and most preferably 192 amino acids.

[0121] In another preferred embodiment, the polypeptide is an isolated polypeptide.

[0122] In another preferred embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 1. In another preferred embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO: 1, and X of SEQ ID NO: 1 1-29 Up to five amino acids at positions not indicated with a may be replaced by any amino acid.

[0123] In another preferred embodiment, the at least three positively charged amino acids X1 to X6 are, independently of one another, arginine or conservative substitutions for arginine. In another preferred embodiment, the at least three positively charged amino acids X1 to X6 are, independently of one another, selected from the group consisting of arginine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, thialysine, and histidine.

[0124] In another preferred embodiment, the at least three positively charged amino acids X1 to X6 are independently selected from the group consisting of arginine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, and thialysine.

[0125] In another more preferred embodiment, the at least three positively charged amino acids X1 to X6 are, independently of one another, histidine, arginine, or lysine. In another more preferred embodiment, the at least three positively charged amino acids X1 to X6 are, independently of one another, arginine or lysine. In another more preferred embodiment, the at least three positively charged amino acids X1 to X6 are independent of each other. In one embodiment, each of the positively charged amino acids X1 to X6 is arginine. In one embodiment, the positively charged amino acids X1 to X6 are lysine.

[0126] X7 is a cysteine, a conservative substitution thereof, or a positively charged amino acid.

[0127] In preferred embodiments, the conservative substitution for cysteine ​​is selected from the group consisting of methionine, homocysteine, selenocysteine, serine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-L-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-L-norleucine, S-(2-hydroxyethyl)-L-cysteine, phosphoserine, 4-hydroxy-L-threonine, threonine, and phosphothreonine. In a preferred embodiment, the conservative substitution for cysteine ​​is selected from the group consisting of homocysteine, selenocysteine, serine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-L-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-L-norleucine, S-(2-hydroxyethyl)-L-cysteine, phosphoserine, 4-hydroxy-L-threonine, threonine, and phosphothreonine. More preferably, the conservative substitution for cysteine ​​is selected from the group consisting of homocysteine, selenocysteine, and serine. Even more preferably, the conservative substitution for cysteine ​​is homocysteine ​​or selenocysteine.

[0128] In another preferred embodiment, X7 is selected from the group consisting of homocysteine, selenocysteine, cysteine, arginine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, and thialysine.

[0129] In another preferred embodiment, X7 is selected from the group consisting of homocysteine, selenocysteine, serine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-L-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-L-norleucine, S-(2-hydroxyethyl)-L-cysteine, phosphoserine, 4-hydroxy-L-threonine, threonine, and phosphothreonine, arginine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, ... In another preferred embodiment, X7 is selected from the group consisting of arginine, lysine, serine, homocysteine, or cysteine. In another even more preferred embodiment, X7 is selected from the group consisting of arginine, lysine, homocysteine, or cysteine.

[0130] When X7 is a cysteine ​​or a conservative substitution of cysteine, it can be used to add a positively charged group to the polypeptides of the invention via disulfide formation (e.g., cysteamine, 1-(3-mercaptopropyl)guanidine, etc.).

[0131] In one embodiment, the at least three of X1 to X6 are independently lysine or arginine, and X7 is selected from arginine, lysine, serine, homocysteine, or cysteine.

[0132] In the amino acid sequence I consisting of SEQ ID NO: 1, at least three of the amino acids X1 to X6 are, independently of one another, positively charged amino acids. In a preferred embodiment, at least four, more preferably at least five, and even more preferably six, i.e., each of the amino acids X1 to X6 is, independently of one another, a positively charged amino acid.

[0133] In a preferred embodiment, at least four, preferably at least five, more preferably six, i.e., the amino acids X1 to X6, are each independently a positively charged amino acid, and the positively charged amino acid is arginine or lysine. In a preferred embodiment, at least four, preferably at least five, more preferably, the amino acids X1 to X6 are each independently a positively charged amino acid, and the positively charged amino acid is arginine. In a preferred embodiment, at least four, preferably at least five, more preferably, six, of the amino acids X1 to X6 are each independently a positively charged amino acid, and the positively charged amino acid is lysine.

[0134] In a preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, X1 to X3. In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, X 1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 X2 and X6.

[0135] In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 2、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X2、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 2、 X3 and X6.

[0136] In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 3、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 3、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 3、 X4 and X1.

[0137] In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 4、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 4、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 4、 X5 and X2.

[0138] In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 5、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 5、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 5、 X6 and X3.

[0139] In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 2、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 X4 and X6.

[0140] In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 2、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 2、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, X3 to X6. In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, X 2、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 2、 X3, X4, and X6.

[0141] In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1-5 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1-4 and X6. In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1-3 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X 1、 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X1 and X 3-6 In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, X2 to X6.

[0142] In another preferred embodiment, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X1 to X6 are, independently of one another, positively charged amino acids.

[0143] More preferably, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X1 to X6, or X4 to X6, or X1, X2 and X5, or X 1、X2, X4 and X5, or X1, X3, X4 and X6, or X1 and X4 to X6, or X1 to X3 and X6, or X1 to X5. More preferably, the at least three X1 to X6 are, independently of one another, positively charged amino acids, and X1 to X6, or X4 to X6, or X1, X2 and X5, or X 1、 X2, X4 and X5, or X1, X3, X4 and X6, or X1 and X4 to X6, or X1 to X3 and X6, or X1 to X5.

[0144] In a preferred embodiment, X4 to X6 are each independently lysine or arginine. In another preferred embodiment, X1, X2, and X4 are each independently lysine or arginine. In another preferred embodiment, X1, X2, X4, and X5 are each independently lysine or arginine. In another preferred embodiment, X1, X3, X4, and X6 are each independently lysine or arginine. In another preferred embodiment, X1, and X3 to X6 are each independently lysine or arginine. In another preferred embodiment, X 1-3 , X5, and X6 are each independently lysine or arginine. 1-5 are each independently lysine or arginine. In another preferred embodiment, X1 to X6 are each independently lysine or arginine.

[0145] In a preferred embodiment, X1 to X6 are each independently arginine or lysine. In a preferred embodiment, X1 to X6 are each independently arginine. In a preferred embodiment, X1 to X6 are each independently lysine.

[0146] In another preferred embodiment, X1 to X6 are each independently lysine or arginine, and X7 is selected from arginine, lysine, or cysteine.

[0147] The inventors have determined that positions X8 to X 12 It has been found that the amino acids at positions X8 to X9 define the pore size of the protein cage formed through self-assembly by the polypeptide of the present invention.12 Larger amino acids at these positions decreased the pore size, whereas smaller amino acids at these positions increased the pore size.

[0148] Thus, in a preferred embodiment, positions X8 to X9 in the polypeptide of the invention 12 The amino acids in the X to X groups of the polypeptide of the present invention are selected so that the surfactant composition can be loaded into the protein cage, released from the protein cage, and transported to / from the lipoprotein cage without degradation. 12 The amino acid at position is selected so that the cargo can be loaded extracellularly into the protein cage and released intracellularly, preferably into the cytoplasm of the cell, without degradation.

[0149] Preferably, the cargo is a small cargo. Preferably, small cargo has a size of 1000 Da or less. In a preferred embodiment, a size of 1000 Da or less means that the cargo has a size of 1000 Da or less, preferably 800 Da or less, more preferably 600 Da or less, again more preferably 500 Da or less, again more preferably 400 Da or less, again more preferably 300 Da or less, again more preferably 200 Da or less, and again more preferably 100 Da or less.

[0150] Preferably, the cargo is a hydrophobic cargo, more preferably a non-polar cargo. Preferably, the cargo has low solubility in aqueous media. Preferably, the small cargo has a size of 1000 Da or less and has low solubility in aqueous media. More preferably, the low-solubility cargo is included in Class II or Class IV of the Biopharmaceutics Classification System (BCS). Even more preferably, the low-solubility cargo has lower solubility than a highly soluble cargo, in which a full-strength dose is soluble in 250 mL or less of aqueous media over a pH range of 1.0 to 7.5, more preferably 1.0 to 6.8, at 37±1°C.

[0151] In a preferred embodiment, X8 is glycine or a conservative substitution thereof. Preferably, the conservative substitution for glycine is selected from the group consisting of alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline. More preferably, the conservative substitution for glycine is selected from the group consisting of alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline. In a further preferred embodiment, X8 is selected from the group consisting of glycine, alanine, leucine, and valine. In a preferred embodiment, X8 is glycine.

[0152] In another preferred embodiment, the X and X 12 are each independently glutamine or a conservative substitution thereof. Preferably, the conservative substitution for glutamine is selected from the group consisting of asparagine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, and n-methyl-asparagine. In another preferred embodiment, the X and X are 12 are independently glutamine or asparagine.

[0153] In another preferred embodiment, the X and X 12 In another preferred embodiment, X and X are both glutamine. 12 and X are both asparagine. 12 In another preferred embodiment, X is glutamine. In another preferred embodiment, X is glutamine. 12 is glutamine.

[0154] In another preferred embodiment, the X 10is glutamate or a conservative substitution thereof. Preferably, the conservative substitution for glutamate is selected from the group consisting of glutamate, aspartate, (2S,4R)-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, and 3-methyl-aspartic acid. In a further preferred embodiment, the X 10 is glutamate or aspartate. 10 is glutamate.

[0155] In another preferred embodiment, the X 11is selected from the group consisting of serine or a conservative substitution thereof. Preferred conservative substitutions for serine are selected from the group consisting of cysteine, methionine, homocysteine, selenocysteine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-L-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-L-norleucine, S-(2-hydroxyethyl)-L-cysteine, phosphoserine, 4-hydroxy-L-threonine, threonine, and phosphothreonine. More preferred such conservative substitutions for serine are selected from the group consisting of cysteine, homocysteine, selenocysteine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-L-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-L-norleucine, S-(2-hydroxyethyl)-L-cysteine, phosphoserine, 4-hydroxy-L-threonine, threonine, and phosphothreonine. Even more preferred such conservative substitutions for serine are selected from the group consisting of hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-L-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-L-norleucine, S-(2-hydroxyethyl)-L-cysteine, phosphoserine, 4-hydroxy-L-threonine, threonine, and phosphothreonine.Even more preferably, the conservative substitution for serine is selected from the group consisting of homoserine, threonine, 4-hydroxy-L-threonine, 6-hydroxy-L-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine.

[0156] In a further preferred embodiment, the X 11 is selected from the group consisting of serine, homoserine, and threonine. 11 is serine.

[0157] In a preferred embodiment, X8 is selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline; and X9 and X 12 are each independently selected from the group consisting of glutamine, asparagine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid and n-methyl-asparagine; 10 is selected from the group consisting of glutamate, aspartate (2S,4R)-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methyl-aspartic acid, and X 11is selected from the group consisting of serine, homoserine, threonine, 4-hydroxy-L-threonine, 6-hydroxy-L-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine.

[0158] In a further preferred embodiment, X8 is selected from the group consisting of glycine, alanine, leucine, and valine, and X9 and X 12 are, independently of each other, glutamine or asparagine, and X 10 is glutamate or aspartate, and X 11 is selected from the group consisting of serine, threonine, and homoserine.

[0159] In a further preferred embodiment, X8 is glycine, and X9 and X 12 is glutamine and X 10 is glutamate and X 11 is serine.

[0160] In a preferred embodiment, X8, X9, X 10 , X 11 and X 12are glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline, glutamine, asparagine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine, glutamate, aspartate (2S,4R)-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β 4,5-Dihydroxy-L-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine.

[0161] In a further preferred embodiment, X8, X9, X 10 , X 11 and X 12 are each independently selected from the group consisting of glycine, alanine, leucine, valine, glutamate, aspartate, glutamine, asparagine, serine, threonine, and homoserine.

[0162] In a further preferred embodiment, X8, X9, X 10 , X 11 and X 12 are each independently selected from the group consisting of serine, glycine, glutamine, and glutamate.

[0163] In a preferred embodiment, in sequence I, X 1-6is lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8, X9, X 10 , X 11 and X 12 are glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline, glutamine, asparagine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine, glutamate, aspartate (2S,4R)-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β In a preferred embodiment, the amino acid sequence I is preferably at the C-terminus of the amino acid sequence I, and in sequence I, X is independently selected from the group consisting of X-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid, serine, homoserine, threonine, 4-hydroxy-L-threonine, 6-hydroxy-L-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine. 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8, X9, X 10 , X 11 and X 12are glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline, glutamine, asparagine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine, glutamate, aspartate (2S,4R)-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β 4,5-Dihydroxy-L-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine.

[0164] In another preferred embodiment, X 1-6 at least three of which are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; X8 is selected from the group consisting of glycine, alanine, leucine, and valine; and X9 and X 12 are, independently of each other, glutamine or asparagine, and X 10 is glutamate or aspartate, and X 11 is selected from the group consisting of serine, threonine, and homoserine. 1-6 at least three of the following are independently lysine or arginine, X8 is glycine, and X9 and X 12 are glutamines independently of each other, and X 10 is glutamate and X11 is serine. In another preferred embodiment, X 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is glycine, X9 and X 12 are glutamines independently of each other, and X 10 is glutamate and X 11 is serine. In another preferred embodiment, X 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is glycine, X9 and X 12 are glutamines independently of each other, and X 10 is glutamate and X 11 is serine.

[0165] In one embodiment, the X 1-6 are independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is glycine, and X9 and X 12 are glutamines independently of each other, and X 10 is glutamate and X 11 is serine.

[0166] In another preferred embodiment, X1 to X6 are each independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is glycine, and X9 and X 12 are glutamines independently of each other, and X 10 is glutamate and X 11 is serine and the polypeptide.

[0167] In a preferred embodiment, in sequence I, X 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is glycine, X9 and X 12 are glutamines independently of each other, and X 10 is glutamate and X 11is serine. In a preferred embodiment, in sequence I, X1 to X6 are lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is glycine, X9 and X12 are each independently glutamine, X10 is glutamate, and X11 is serine, and the protein cage or the complex of the present invention comprising the polypeptide of the present invention is capable of endosomal escape.

[0168] When the polypeptide of the present invention forms a protein cage by self-assembly, the amino acid X 13 ~X 29 are exposed on the outer surface of the protein cage and are therefore most likely to tolerate mutations while still maintaining the ability to self-assemble into a cage-like protein cage.

[0169] Thus, in a preferred embodiment, the amino acid X exposed on the outer surface of the protein cage 13 ~X 29 is any amino acid.

[0170] In a preferred embodiment, X 13 , X 17 and X 19are each independently serine or a conservative substitution thereof. Preferred conservative substitutions for serine are selected from the group consisting of cysteine, methionine, homocysteine, selenocysteine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-L-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-L-norleucine, S-(2-hydroxyethyl)-L-cysteine, phosphoserine, 4-hydroxy-L-threonine, threonine, and phosphothreonine. More preferred such conservative substitutions for serine are selected from the group consisting of cysteine, homocysteine, selenocysteine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-L-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-L-norleucine, S-(2-hydroxyethyl)-L-cysteine, phosphoserine, 4-hydroxy-L-threonine, threonine, and phosphothreonine. Even more preferred such conservative substitutions for serine are selected from the group consisting of hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-L-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-L-norleucine, S-(2-hydroxyethyl)-L-cysteine, phosphoserine, 4-hydroxy-L-threonine, threonine, and phosphothreonine.Even more preferably, the conservative substitution for serine is selected from the group consisting of homoserine, threonine, 4-hydroxy-L-threonine, 6-hydroxy-L-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine.

[0171] In another preferred embodiment, X 13 , X 17 and X 19 are each independently selected from the group consisting of serine, homoserine, and threonine. 13 , X 17 and X 19 are independently serine.

[0172] In another preferred embodiment, X 14 , X 16 and X 24 are, independently of each other, asparagine or a conservative substitution thereof. Preferably, the conservative substitution for asparagine is selected from the group consisting of glutamine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, and n-methyl-asparagine.

[0173] In another preferred embodiment, X 14 , X 16 and X 24 are each independently asparagine. 14 , X 16 and X 24 are each independently asparagine or glutamine.

[0174] In another preferred embodiment, X 15 , X 20 , X 26and X 28 are, independently of one another, aspartate, glutamate or a conservative substitution thereof. Preferably, the conservative substitution for aspartate or glutamate is selected from the group consisting of (2S,4R)-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methyl-aspartic acid.

[0175] In another preferred embodiment, X 15 and X 20 are each independently aspartate. 26 and X 28 are independently glutamates.

[0176] In another preferred embodiment, X 15 , X 20 , X 26 and X 28 are, independently of each other, aspartate or glutamate. 15 , X 20 , X 26 and X 28 are independently selected from the group consisting of glutamate, aspartate, 2S,4R-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methyl-aspartic acid.

[0177] In another preferred embodiment, X 18 and X 29are, independently of each other, leucine or a conservative substitution thereof. Preferably, the conservative substitution for leucine is selected from the group consisting of glycine, alanine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline.

[0178] In another preferred embodiment, X 18 and X 29 are each independently leucine. 18 and X 29 are each independently selected from the group consisting of glycine, alanine, leucine, and valine. 18 and X 29 are independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline.

[0179] In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27 are, independently of each other, positively charged amino acids.

[0180] In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27are, independently of each other, arginine, lysine, or a conservative substitution thereof. Preferably, the conservative substitution for arginine or leucine is selected from the group consisting of histidine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, and thialysine. More preferably, the conservative substitution for arginine or leucine is selected from the group consisting of 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, and thialysine.

[0181] In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27 are, independently of each other, histidine, arginine or lysine. 21 , X 22 , X 23 , X 25 and X 27 are, independently of each other, arginine or lysine. 21 , X 22 , X 23 , X 25 and X 27 are, independently of each other, arginine. 21 , X 22 , X 23 , X 25 and X 27 are, independently of each other, lysine.

[0182] In another preferred embodiment, X 21 , X22 , X 25 and X 27 are, independently of each other, arginine. 23 is lysine. In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27 are independently selected from the group consisting of arginine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, thialysine, and histidine.

[0183] In another preferred embodiment, X 13 , X 17 and X 19 are serine independently of each other, and X 14 , X 16 and X 24 are independently asparagine, and X 15 and X 20 are aspartates independently of each other, and X 18 and X 29 are independently leucine, and X 26 and X 28 are glutamates independently of each other, and X 21 , X 22 , X 25 and X 27 are independently arginine, and X 23 is lysine.

[0184] In another preferred embodiment, X 13 , X 17 and X 19 are each independently selected from the group consisting of serine, homoserine, and threonine; 14 , X 16 and X 24are, independently of each other, asparagine or glutamine, and X 15 , X 20 , X 26 and X 28 are each independently aspartate or glutamate, and the X 18 and X 29 are each independently selected from the group consisting of glycine, alanine, leucine, and valine; 21 , X 22 , X 23 , X 25 and X 27 are, independently of each other, arginine or lysine.

[0185] In another preferred embodiment, X 13 , X 17 and X 19 are independently selected from the group consisting of serine, homoserine, threonine, 4-hydroxy-L-threonine, 6-hydroxy-L-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine; X 14 , X 16 and X 24 are independently selected from the group consisting of glutamine, asparagine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, and n-methyl-asparagine; X 15 , X 20 , X 26 and X 28are independently selected from the group consisting of glutamate, aspartate, (2S,4R-4)-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methyl-aspartic acid; X 18 and X 29 are independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline; X 21 , X 22 , X 23 , X 25 and X 27 are independently selected from the group consisting of arginine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, thialysine, and histidine.

[0186] In another preferred embodiment, X8 is glycine or a conservative substitution thereof, preferably wherein the conservative substitution for glycine is selected from the group consisting of alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline; X9 and X 12 are, independently of each other, glutamine or a conservative substitution thereof, and preferably, the conservative substitution for glutamine is selected from the group consisting of asparagine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, and n-methyl-asparagine; X 10 is glutamate or a conservative substitution thereof, preferably the conservative substitution of glutamate is selected from the group consisting of aspartate (2S,4R)-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methyl-aspartic acid; X 11 is serine or a conservative substitution thereof, preferably the conservative substitution for serine is selected from the group consisting of homoserine, threonine, 4-hydroxy-L-threonine, 6-hydroxy-L-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine, more preferably X8 is glycine, X9 and X 12 are independently glutamine or asparagine, and X 10 is glutamate or aspartate, and X 11 is serine.

[0187] In another preferred embodiment, X 13 , X 17 , and X 19are each independently serine or a conservative substitution thereof, and preferably the conservative substitution for serine is selected from the group consisting of homoserine, threonine, 4-hydroxy-L-threonine, 6-hydroxy-L-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine; X 14 , X 16 , and X 24 are, independently of each other, asparagine, glutamine or a conservative substitution thereof, and preferably, the conservative substitution for asparagine or glutamine is selected from the group consisting of β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, and n-methyl-asparagine; X 15 , X 20 , X 26 and X 28 are, independently of one another, aspartate, glutamate or a conservative substitution thereof, and preferably the conservative substitution of aspartate or glutamate is selected from the group consisting of (2S,4R)-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methyl-aspartic acid; X 18 and X 29 are, independently of each other, leucine or a conservative substitution thereof, and preferably, the conservative substitution for leucine is selected from the group consisting of glycine, alanine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline; X 21, X 22 , X 23 , X 25 and X 27 are, independently of each other, arginine, lysine or a conservative substitution thereof, and preferably, the conservative substitution for arginine or leucine is selected from the group consisting of histidine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine and thialysine.

[0188] In a preferred embodiment, X8 is glycine or a conservative substitution thereof, X9 and X12 are each independently glutamine, asparagine, or a conservative substitution thereof, X10 is glutamate, aspartate, or a conservative substitution thereof, X11 is serine or a conservative substitution thereof, X13, X17, and X19 are each independently serine or a conservative substitution thereof, X14, X16, and X24 are each independently asparagine, glutamine, or a conservative substitution thereof, X15, X20, X26, and X28 are each independently aspartate, glutamate, or a conservative substitution thereof, X18 and X29 are each independently leucine or a conservative substitution thereof, and X21, X22, X23, X25, and X27 are each independently arginine, lysine, or a conservative substitution thereof.

[0189] In another preferred embodiment, the at least three positively charged amino acids X1 to X6 are independently selected from the group consisting of arginine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, and thialysine; X7 is cysteine, a conservative substitution thereof or a positively charged amino acid, preferably X7 is selected from the group consisting of homocysteine, cysteine, selenocysteine, arginine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, thialysine and serine; X8 is glycine or a conservative substitution thereof and is selected from the group consisting of alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline; X9 and X 12 are, independently of each other, glutamine or a conservative substitution thereof and are selected from the group consisting of asparagine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, and n-methyl-asparagine; X 10 is glutamate or a conservative substitution thereof and is selected from the group consisting of aspartate (2S,4R)-4-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methyl-aspartic acid; X 11 is serine or a conservative substitution thereof and is selected from the group consisting of homoserine, threonine, 4-hydroxy-L-threonine, 6-hydroxy-L-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine; X 13 , X 17 and X 19 are independently selected from the group consisting of serine, homoserine, threonine, 4-hydroxy-L-threonine, 6-hydroxy-L-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-L-valine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2S,3R)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, and allo-threonine; X 14 , X 16 and X 24 are independently selected from the group consisting of glutamine, asparagine, β-hydroxyasparagine, 3-methyl-L-glutamine, (2S,4S)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, and n-methyl-asparagine; X 15 , X 20 , X 26 and X 28 are independently selected from the group consisting of glutamate, aspartate, (2S,4R-4)-methylglutamate, (3S)-3-methyl-L-glutamic acid, (3R)-3-methyl-L-glutamic acid, 5-O-methyl-glutamic acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanedioic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methyl-aspartic acid; X 18 and X 29 are independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine 2-aminobutyric acid, diethylalanine, norleucine, and norvaline; X 21 , X 22 , X 23 , X 25 and X 27are independently selected from the group consisting of arginine, 5-methyl-arginine, gamma-hydroxyarginine, 2-amino-4-guanidinobutryric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropanoic acid, (2S)-2,8-diaminooctanoic acid, ornithine, thialysine, and histidine.

[0190] In another preferred embodiment, the at least three positively charged amino acids of X1 to X6 are, independently of one another, arginine or lysine; X7 is selected from the group consisting of homocysteine, cysteine, arginine, and lysine; X8 is selected from the group consisting of glycine, alanine, leucine, and valine; and X9 and X10 are selected from the group consisting of glycine, alanine, leucine, and valine. 12 are, independently of each other, glutamine or asparagine, and X 10 is glutamate or aspartate, and X 11 , X 13 , X 17 and X 19 are each independently selected from the group consisting of serine, threonine, and homoserine; X 14 , X 16 and X 24 are, independently of each other, asparagine or glutamine, and X 15 , X 20 , X 26 and X 28 are, independently of each other, aspartate or glutamate, and X 18 and X 29 are each independently selected from the group consisting of leucine, glycine, alanine, and valine; X 21 , X 22 , X 23 , X 25 and X 27 are, independently of each other, arginine or lysine.

[0191] In another preferred embodiment, the at least three positively charged amino acids X1 to X6 are, independently of one another, arginine or lysine; X7 is selected from the group consisting of homocysteine, cysteine, arginine, and lysine; X8 is glycine; and X9 and X10 are each independently of one another. 12 are independently glutamine or asparagine, and X 10 is glutamate or aspartate, and X 11 is serine and X 13 , X 17 , and X 19 are serine independently of each other, and X 14 , X 16 , and X 24 are independently asparagine, and X 15 , X 20 , X 26 and X 28 are independently aspartate or glutamate, and X 18 and X 29 are independently leucine, and X 21 , X 22 , X 23 , X 25 and X 27 are independently arginine or lysine.

[0192] In another preferred embodiment, X 1-6 at least three of which are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; X8 is selected from the group consisting of glycine, alanine, leucine, and valine; and X9 and X 12 are, independently of each other, glutamine or asparagine, and X 10 is glutamate or aspartate, and X 11 is selected from the group consisting of serine, threonine, and homoserine; X 18 and X 29 are independently leucine, and X 15 and X 20 are aspartates independently of each other, and X 26 and X 28 are glutamates independently of each other, and X14 , X 16 , and X 24 are independently asparagine, and X 13 , X 17 and X 19 are serine independently of each other, and X 21 , X 22 , X 25 and X 27 are independently arginine, and X 23 is lysine.

[0193] In another preferred embodiment, X 1-6 at least three of which are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; X8 is selected from the group consisting of glycine, alanine, leucine, and valine; and X9 and X 12 are, independently of each other, glutamate or aspartate, and X 10 is glutamate or aspartate, and X 11 is selected from the group consisting of serine, threonine, and homoserine; X 13 , X 17 and X 19 are each independently selected from the group consisting of serine, homoserine, and threonine; 14 , X 16 and X 24 are, independently of each other, asparagine or glutamine, and X 15 , X 20 , X 26 and X 28 are each independently aspartate or glutamate, and the X 18 and X 29 are each independently selected from the group consisting of glycine, alanine, leucine, and valine; X 21 , X 22 , X 23 , X 25 and X 27 are, independently of each other, arginine or lysine.

[0194] In another preferred embodiment, X 1-6are each independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is selected from the group consisting of glycine, alanine, leucine, and valine, X9 and X 12 are, independently of each other, glutamate or aspartate, and X 10 is glutamate or aspartate, and X 11 is selected from the group consisting of serine, threonine, and homoserine; X 13 , X 17 and X 19 are each independently selected from the group consisting of serine, homoserine, and threonine; 14 , X 16 and X 24 are, independently of each other, asparagine or glutamine, and X 15 , X 20 , X 26 and X 28 are each independently aspartate or glutamate, and the X 18 and X 29 are each independently selected from the group consisting of glycine, alanine, leucine, and valine; X 21 , X 22 , X 23 , X 25 and X 27 are, independently of each other, arginine or lysine.

[0195] In another preferred embodiment, X 1-6 are each independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is selected from the group consisting of glycine, alanine, leucine, and valine, X9 and X 12 are, independently of each other, glutamine or asparagine, and X 10 is glutamate or aspartate, and X 11 is selected from the group consisting of serine, threonine, and homoserine; X 13 , X 17 and X 19 are serine independently of each other, and X 14 , X 16 and X24 are independently asparagine, and X 15 and X 20 are aspartates independently of each other, and X 18 and X 29 are independently leucine, and X 26 and X 28 are glutamates independently of each other, and X 21 , X 22 , X 25 and X 27 are independently arginine, and X 23 is lysine.

[0196] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 16 and 20 to 27. In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 16 and 20 to 27.

[0197] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and 10 to 16. In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and 10 to 16.

[0198] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, and 22. In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, and 20 to 22.

[0199] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, 20, and 21. In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, 20, and 21. In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, and 22. In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, 20, 21, and 22.

[0200] In a preferred embodiment, the amino acid sequence I is SEQ ID NO: 2. In a preferred embodiment, the amino acid sequence I is SEQ ID NO: 3. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 4. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 5. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 10. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 11. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 12. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 13. In a preferred embodiment, the amino acid sequence I is SEQ ID NO: 14. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 15. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 16. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 20. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 21. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 22.

[0201] In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 2. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 3. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 4. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 5. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 10. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 11. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 12. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 13. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 14. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 15. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 16. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 20. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 21. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 22. In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 3, and the polypeptide of the present invention comprises a histidine tag (His tag) consisting of three or more consecutive histidines, and the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of amino acid sequence I.

[0202] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 16 and 20 to 27, and the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, and the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1.

[0203] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and 10 to 16, and the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, and the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1.

[0204] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, and 22, and the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, and the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1.

[0205] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and SEQ ID NOs: 10 to 16, 20 and 21, and the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, and the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1.

[0206] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, and 22, and the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, and the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1.

[0207] In a preferred embodiment, the amino acid sequence I is SEQ ID NO: 2, and the detergent composition comprises, and preferably consists of, SDS and CS, wherein the molar ratio of SDS to CS is 4:1 to 2:1, and preferably the molar ratio of SDS to CS is 3:1. In a preferred embodiment, the amino acid sequence I is SEQ ID NO: 3, and the detergent composition comprises, and preferably consists of SDS and CS, wherein the molar ratio of SDS to CS is 4:1 to 2:1, and preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 4, and the detergent composition comprises, and preferably consists of SDS and CS, wherein the molar ratio of SDS to CS is 4:1 to 2:1, and preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 5, the detergent composition comprises SDS and CS, preferably consists of SDS and CS, the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 10, the detergent composition comprises SDS and CS, preferably consists of SDS and CS, the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 11, the detergent composition comprises SDS and CS, preferably consists of SDS and CS, the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 12, and the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, and the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1.In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 13, and the detergent composition comprises, and preferably consists of, SDS and CS, wherein the molar ratio of SDS to CS is 4:1 to 2:1, and preferably the molar ratio of SDS to CS is 3:1. In a preferred embodiment, the amino acid sequence I is SEQ ID NO: 14, and the detergent composition comprises, and preferably consists of SDS and CS, wherein the molar ratio of SDS to CS is 4:1 to 2:1, and preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 15, and the detergent composition comprises, and preferably consists of SDS and CS, wherein the molar ratio of SDS to CS is 4:1 to 2:1, and preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 16, the detergent composition comprises SDS and CS, preferably consists of SDS and CS, the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 20, the detergent composition comprises SDS and CS, preferably consists of SDS and CS, the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 21, the detergent composition comprises SDS and CS, preferably consists of SDS and CS, the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1. In another preferred embodiment, the amino acid sequence I is SEQ ID NO: 22, and the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, and the molar ratio of SDS to CS is 4:1 to 2:1, preferably the molar ratio of SDS to CS is 3:1.

[0208] In a preferred embodiment, the polypeptide of the present invention comprises a tag, i.e., a peptide or non-peptide tag, preferably a peptide tag (i.e., a functional amino acid sequence). In a preferred embodiment, the tag is located at the C-terminus or N-terminus of the polypeptide of the present invention. In a preferred embodiment, the tag is a non-peptide tag, preferably polyethylene glycol (PEG). Preferably, the PEG is coupled to the amino acid sequence I via the amino acid serine or cysteine. PEG coupled to the polypeptide of the present invention increases stability and reduces immunogenicity.

[0209] In a preferred embodiment, the polypeptide of the present invention comprises a tag, preferably a peptide tag, which is located at the C- or N-terminus, preferably the C-terminus, of the polypeptide, preferably the amino acid sequence I, and which is preferably fused to the C- or N-terminus of the polypeptide, preferably the amino acid sequence I.

[0210] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 16 and 20 to 27, and the polypeptide of the present invention comprises a tag, preferably a peptide tag, which is located at the C- or N-terminus, preferably the C-terminus, of the polypeptide, preferably the amino acid sequence I, and which is preferably fused to the C- or N-terminus of the polypeptide, preferably the amino acid sequence I.

[0211] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and 10 to 16, and the polypeptide of the present invention comprises a tag, preferably a peptide tag, which is located at the C- or N-terminus, preferably the C-terminus, of the polypeptide, preferably the amino acid sequence I, and which is preferably fused to the C- or N-terminus of the polypeptide, preferably the amino acid sequence I.

[0212] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, and 20 to 22, and the polypeptide of the present invention comprises a tag, preferably a peptide tag, which is located at the C- or N-terminus, preferably the C-terminus, of the polypeptide, preferably the amino acid sequence I, and which is preferably fused to the C- or N-terminus of the polypeptide, preferably the amino acid sequence I.

[0213] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 16 and 20 to 27, the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, the molar ratio of SDS to CS is 4:1 or 2:1, preferably the molar ratio of SDS to CS is 3:1, the polypeptide of the present invention comprises a tag, preferably a peptide tag, the tag is located at the C- or N-terminus, preferably the C-terminus, of the polypeptide, preferably the amino acid sequence I, and the tag is preferably fused to the C- or N-terminus of the polypeptide, preferably the amino acid sequence I.

[0214] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and 10 to 16, the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, the molar ratio of SDS to CS is 4:1 or 2:1, preferably the molar ratio of SDS to CS is 3:1, the polypeptide of the present invention comprises a tag, preferably a peptide tag, the tag is located at the C- or N-terminus, preferably the C-terminus, of the polypeptide, preferably the amino acid sequence I, and the tag is preferably fused to the C- or N-terminus of the polypeptide, preferably the amino acid sequence I.

[0215] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, and 20 to 22, the surfactant composition comprises SDS and CS, preferably consists of SDS and CS, the molar ratio of SDS to CS is 4:1 or 2:1, preferably the molar ratio of SDS to CS is 3:1, the polypeptide of the present invention comprises a tag, preferably a peptide tag, the tag is located at the C- or N-terminus, preferably the C-terminus, of the polypeptide, preferably the amino acid sequence I, and the tag is preferably fused to the C- or N-terminus of the polypeptide, preferably the amino acid sequence I.

[0216] In another preferred embodiment, the tag is selected from the group consisting of polyhistidine (His tag, i.e., an amino acid sequence consisting of two or more consecutively linked histidines), a degradation tag, a targeting tag, a cell-penetrating tag, and an endosomal escape tag. In a preferred embodiment, the additional tag contained in the polypeptide of the present invention is attached via a releasable bond, such as a photocleavable bond, or via reversible coupling.

[0217] The targeting tag preferably binds to a cancer target, i.e., the targeting tag is a cancer targeting tag, which includes receptors with increased expression levels in / on specific tumor cells and tumor antigens.

[0218] In a preferred embodiment, the targeting tag is a peptide ligand, a peptidomimetic, an affibody, an antibody binding domain, or an antibody. The targeting tag is preferably folic acid.

[0219] In a preferred embodiment, the His tag, preferably the His6 tag, comprises a halogenated histidine, preferably a fluorinated histidine. In a preferred embodiment, the His tag comprises a fluorophore. In a preferred embodiment, the polypeptide of the invention comprises a fluorophore.

[0220] In a preferred embodiment, the polypeptide of the present invention comprises a degradation tag. Preferably, the degradation tag is functional in mammalian cells. In a preferred embodiment, the degradation tag is ornithine decarboxylase (cODC), preferably the C-terminal sequence of SEQ ID NO: 17, or a (poly)ubiquitin comprising or consisting of at least two consecutively linked ubiquitins. In another preferred embodiment, the degradation tag consists of cODC of SEQ ID NO: 17 (EFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV). In another preferred embodiment, the degradation tag comprises at least two consecutively linked ubiquitins. More preferably, the degradation tag consists of at least two consecutively linked ubiquitins.

[0221] In a preferred embodiment, the polypeptide of the present invention comprises polyhistidine (His tag). In a preferred embodiment, the polyhistidine is an amino acid sequence comprising two or more histidines. In another preferred embodiment, the polyhistidine is an amino acid sequence comprising two or more consecutively linked histidines. In another preferred embodiment, the polyhistidine is an amino acid sequence comprising three or more consecutively linked histidines. In another preferred embodiment, the polyhistidine is an amino acid sequence comprising 3 to 9 consecutively linked histidines. In a preferred embodiment, the His tag is bound to the C-terminus or N-terminus of amino acid sequence I, preferably the C-terminus.

[0222] In a further embodiment, the polypeptide of the present invention comprises an endosomal escape peptide or cell-penetrating peptide (CPP). Endosomal escape peptides are, for example, dimerized disulfide-linked TAT or thiol groups. As used herein, the term "cell-penetrating peptide" or CPP refers to a group of peptides capable of penetrating the plasma membrane for delivery of cargo into cells. Preferably, the CPP used in the polypeptide of the present invention is a hydrophilic or cationic peptide. In another embodiment, the CPP is selected from amphipathic, anionic, or hydrophobic peptides. A database of over 1,600 CPPs has been described by Agrawal et al. (Agrawal P, Bhalla S, Usmani SS, Singh S, CHaudhary K, Raghava GPS, et al. CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides. Nucl Acids Res. 2016, 44: D1098-D103).

[0223] In a preferred embodiment, the additional tag included in the polypeptide of the present invention is attached via a releasable bond, such as a photocleavable bond, or via reversible coupling.

[0224] In a preferred embodiment, the polypeptide of the present invention comprises an amino acid sequence I selected from the group consisting of SEQ ID NOs: 2 to 27, and the lipoprotein cage is capable of loading and unloading cargo without degradation of the lipoprotein cage.

[0225] In a preferred embodiment, the polypeptide of the present invention comprises an amino acid sequence I selected from the group consisting of SEQ ID NOs: 2-5, 10-16, and 18-22, and the lipoprotein cage is capable of loading and unloading cargo without degradation of the lipoprotein cage.

[0226] In a preferred embodiment, the polypeptide of the present invention comprises an amino acid sequence I selected from the group consisting of SEQ ID NOs: 2-5, 10-16, 18 or 19, and the lipoprotein cage is capable of loading and unloading cargo without degradation of the lipoprotein cage.

[0227] In a preferred embodiment, the polypeptides of the invention are modified after production, either genetically (for direct fusion of peptides) or chemically.

[0228] In a preferred embodiment, the lipoprotein cages of the present invention are comprised in a composition, preferably a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier.

[0229] In a further aspect, the present invention relates to a complex comprising a lipoprotein cage of the present invention and one or more cargo molecules.

[0230] Preferably, the cargo is a hydrophobic cargo, more preferably a non-polar cargo. Preferably, the cargo is a small cargo. Preferably, the small cargo has a size of 1000 Da or less.

[0231] In a preferred embodiment, a size of 1000 Da or less means that the cargo has a size of 1000 Da or less, preferably 800 Da or less, more preferably 600 Da or less, again more preferably 500 Da or less, again more preferably 400 Da or less, again more preferably 300 Da or less, again more preferably 200 Da or less, and again more preferably 100 Da or less. In another embodiment, the cargo is a small hydrophobic cargo having a size of 1000 Da or less, even more preferably a small non-polar cargo having a size of 1000 Da or less. Preferably, the cargo has low solubility in aqueous media. Preferably, the small cargo has a size of 1000 Da or less and has low solubility in aqueous media. More preferably, the cargo with low solubility is included in Class II or Class IV of the Biopharmaceutics Classification System (BCS). Even more preferably, the poorly soluble cargo has a lower solubility than a highly soluble cargo, the full strength dose of which is soluble in 250 mL or less of aqueous medium over a pH range of 1.0 to 7.5, more preferably 1.0 to 6.8, at 37±1° C. Preferred methods for determining solubility are a USP dissolution apparatus, a shake flask method, or an acid or base titration method.

[0232] Preferably, the cargo is an active agent, preferably a therapeutically and / or diagnostically active agent. In a preferred embodiment, the cargo is an imaging agent, such as a fluorescent agent. More preferably, the cargo is selected from the group consisting of chemotherapeutic agents, antifungals such as bifonazole or amphotericin B, antivirals such as indinavir or ritonavir, and antibiotics. Preferably, the cargo is included in Class II or Class IV of the Biopharmaceutics Classification System (BCS). Preferably, the chemotherapeutic agent is a small molecule cargo molecule selected from the group consisting of doxorubicin, paclitaxel, dasatinib, imatinib, lapatinib, camptothecin, daunorubicin, buparlisib, amsacrine, bifonazole, glibenclamide, bicalutamide, celecoxib, fenofibrate, and danazol.

[0233] Treatment of cells with the complex of the present invention results in intracellular delivery of the complex and release of its cargo into the cytosol of the treated cell without degradation of the lipoprotein cage. Thus, in a preferred embodiment, the complex of the present invention comprises a lipoprotein cage of the present invention and one or more cargo molecules, which are encapsulated in the lipoprotein cage without degradation of the lipoprotein cage. In a further preferred embodiment, the complex of the present invention comprises a lipoprotein cage of the present invention and one or more cargo molecules, which are encapsulated in the lipoprotein cage extracellularly without degradation of the lipoprotein cage, and the lipoprotein cage is capable of releasing the cargo into the cell without degradation of the lipoprotein cage. In a further preferred embodiment, the complex of the present invention comprises a lipoprotein cage of the present invention and one or more cargo molecules, which are encapsulated in the lipoprotein cage extracellularly without degradation of the lipoprotein cage, and the lipoprotein cage is capable of being taken up by the cell and releasing the encapsulated cargo molecules into the cell, preferably into the cytosol of the cell, without degradation of the lipoprotein cage.

[0234] Although the encapsulation of cargo into the lipoprotein cages of the present invention is reversible in the presence of competing host molecules or environments, the lipoprotein cages and complexes of the present invention are stable and not degraded extracellularly. Thus, in a further preferred embodiment, the lipoprotein cages of the present invention can reversibly encapsulate and release cargo molecules without degradation of the lipoprotein cage. Cargo release is triggered by a protein that can bind to the cargo more strongly than the surfactant composition can bind to the cargo, or by a lipid bilayer into which the cargo preferentially partitions.

[0235] In a further aspect, the present invention provides a method for producing a lipoprotein cage of the present invention, comprising the steps of: 1. Self-assembling a protein cage from at least one polypeptide, preferably 24 peptides, wherein said polypeptide comprises an amino acid sequence I consisting of: MX 13 QAIGILELX1SIAAGMELGDAMLKSAX 14 VX 15 LLVSKTISX2GKFLLMLGGDIX8AIX9X 12 AIX 10 TGTX 11 QAGX3LLVDSLVLAX 16 IHPSVLPAIX 17 GX 18 NX 19 VX 20 X7X 21 QAVGIVETX4SVAACISAADX 22 AVX 23 GSX 24 VTLVRVHMAX5GIGGKCYMVVAGDVSDVALAVTVASSSAGAYGX6LVYASLIPX 25 PHX 26 AMWX 27 QMVX 28 GX 29 E (SEQ ID NO: 1), where X1 to X 29are each independently an amino acid, provided that at least three of X1 to X6 are each independently a positively charged amino acid, and 29 wherein up to five amino acids at positions other than those indicated by may be replaced by any amino acid; and encapsulating the surfactant composition of the present invention in a protein cage without disassembling the protein cage.

[0236] In a preferred embodiment, the amino acid sequence I is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, and 20 to 22. In a preferred embodiment, the amino acid sequence I is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, 20, and 21. In a preferred embodiment, the at least one polypeptide is defined as 24 polypeptides.

[0237] In a preferred embodiment, the method for producing a lipoprotein cage of the invention comprises the further step of producing a polypeptide of the invention, preferably by recombinant expression (e.g., in a bacterial cell, preferably in an E. coli cell). Upon expression, the polypeptide of the invention self-assembles into a well-defined protein cage that can be isolated.

[0238] In a further aspect, the present invention provides a method for producing a complex of the present invention, which comprises the step of mixing a lipoprotein cage of the present invention with one or more cargo molecules, wherein the cargo is encapsulated in the lipoprotein cage of the present invention without degrading the lipoprotein cage.

[0239] In a further aspect, the present invention provides a method of treating a cell with a complex of the present invention, the method comprising contacting the cell with the complex of the present invention. Preferably, the method for treating a cell is an in vitro method. In a preferred embodiment, the cell is a eukaryotic cell. More preferably, the cell is an animal cell, and even more preferably, a mammalian cell. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 2] [Example]

[0240] Example 1 - Materials and Methods Materials. All chemicals were used as supplied without further purification. Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Fluorochem (UK). Lysozyme was purchased from PanReac Axon Lab AG (Switzerland). For His-tagged protein isolation, Ni-NTA Agarose from Qiagen (Germany) was used. DNase I was from Roche (Switzerland), and RNase A was from Merck (Germany). GelRed was purchased from Biotium, Inc. (USA). Sodium dodecyl sulfate and cholesterol sulfate were purchased from Sigma-Aldrich (Merck, Germany).

[0241] Instrumentation. Protein quantification was performed using a NanoDrop 2000c spectrophotometer manufactured by ThermoFisher Scientific Inc. (USA). All size-exclusion chromatography was performed using an NGC™ Medium-Pressure Chromatography System manufactured by Bio-Rad Laboratories, Inc. (USA). Agarose gel electrophoresis (AGE) was performed using a Mini-Sub™ cell GT manufactured by Bio-Rad Laboratories, Inc. (USA). Gel images were taken using an EOS 1100D manufactured by Canon (Japan). Transmission electron microscope (TEM) images were obtained using a Morgagni 268 manufactured by FEI (USA). Fluorescence quantification was performed using a QuantaMaster™ 50 fluorometer manufactured by Photon Technology International (USA). Confocal fluorescence microscope images were obtained using an SP8-AOBS manufactured by Leica (Germany). Flow cytometry was performed on an LSRFortessa from BD Biosciences (USA).

[0242] Protein production. Proteins were expressed in E. coli strain BL21-Gold(DE3). Cells were cultured at OD in LB medium containing kanamycin sulfate (86 μM). 600 The cells were grown at 37 °C until an RI of 0.6-0.8 was reached, and protein overexpression was induced with IPTG (0.1 mM). After approximately 18 h of incubation at 25 °C, the cells were harvested by centrifugation (5,000 × g) at 4 °C for 15 min. The cell pellet was stored at -20 °C until purification. OP cages were isolated from the E. coli cell pellet and purified by Ni affinity and size exclusion chromatography as previously reported (Edwardson et al., 2018, op.cit.).

[0243] Preparation of OP:SDS and OP:SDS:CS complexes. Protein cage-micelle complexes were formed directly from aqueous solutions of purified empty OP cages and anionic surfactants. Unless otherwise specified, the molar ratio of total surfactant molecules to OP cages was 800:1 in all experiments. The buffers used were PBS (9.5 mM NaHPO, 1.4 mM KHPO, 136 mM NaCl, 2.7 mM KCl, pH 7.4) and TSEC (25 mM Tris-HCl, 200 mM NaCl, 5 mM EDTA, pH 7.4). To form the complexes, an appropriate volume of concentrated SDS solution (1–100 mM in PBS) or CS solution (8–16 mM, DMSO) was first diluted with PBS buffer to a concentration of less than 1 mM to avoid protein denaturation. The required volume of OP solution (2-20 μM capsid, PBS or TSEC buffer) was then added, and the mixture was incubated at room temperature for 1 h to allow complete complex formation. For small molecule encapsulation, a concentrated solution of the fluorescent probe / drug in acetone or DMSO was added to the preformed OP:SDS:CS complex and incubated for an additional 15 min at room temperature. In both cases, the total fraction of organic solvent was kept below 10% v / v.

[0244] Native agarose gel electrophoresis. All native gel electrophoresis runs were performed using 2% (w / v) agarose gels in Tris-acetate-EDTA buffer (40 mM Tris-HCl, 19 mM acetic acid, 1 mM EDTA, pH 8.3). After visualization of fluorescent molecules by UV transillumination, the gels were stained with Coomassie blue for protein visualization. In a typical experiment, approximately 100 pmol of capsid (relative to monomer) was loaded in 10 μL of buffer plus 2 μL of 70% (v / v) aqueous glycerol per lane.

[0245] Size exclusion chromatography. Analytical SEC was performed on a Superose6 Increase 10 / 300 GL column (GE Healthcare, USA). The sample was 800 μL of 10–50 μM protein monomer, and the mobile phase was 0.75× TSEC buffer. Peaks were detected by absorbance at 280 nm.

[0246] Dynamic light scattering. DLS measurements were performed on a Zetasizer Nano (Malvern Instruments, UK) at 25 °C using samples prepared from 0.22 μm filtered solutions of protein and surfactant. Sample concentrations ranged from 30 to 100 μM protein monomer.

[0247] Transmission electron microscopy. Negative-stain transmission electron microscopy (TEM) was performed as previously reported (Beck, T., Tetter, S., Kunzle, M. & Hilvert, D. Construction of Matryoshka-Type Structures from Supercharged Protein Nanocages. Angew. Chem. Int. Ed. 54, 937-940 (2015)). For all TEM experiments, samples were 2–4 μM OP monomer in PBS buffer.

[0248] Nile Red fluorescence. For a typical fluorimetry experiment, 800 μL of sample in PBS buffer was used. For surfactant loading kinetics, the ionic strength was adjusted by dissolving an appropriate amount of NaCl in PBS buffer. Stock solutions of Nile Red in acetone:water (1:1 v / v) or DMSO at concentrations ranging from 50 to 500 μM were used. The excitation wavelength was set at 535 nm for all experiments.

[0249] Effective concentration calculation. The volume of the OP lumen (255,528 Å) was estimated as a sphere with a radius of 39.4 Å. This radius was determined by averaging the distances between lumen-exposed residues from a reported crystal structure (Edwardson et al., 2018, op.cit.) using UCSF Chimera software (Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)). The effective concentration of SDS was simply estimated as the number of moles of SDS per lumen volume. The expected number of SDS molecules was estimated by first determining the volume occupied by an individual SDS molecule in the micelle from the reported average values ​​of the SDS micelle radius (17.5 Å) and aggregation number (n = 64). Dividing the OP cavity volume by the average volume of a single SDS molecule packed into the micellar aggregates yields an estimate of 729 molecules per OP cage, which is within the error range of the volume estimates and experimental results and is close to 800 molecules.

[0250] Cell culture. HeLa cells were maintained in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 2 mM GlutaMAX, and 1 μg / mL gentamicin. Cells were cultured at 37°C in 5% CO2 and typically split at a 1:4 ratio every 3 days. Only passages 7–20 were used for all experiments.

[0251] Flow cytometry. HeLa cells were seeded at a density of 30,000 cells per well in 24-well plates in 500 μL of culture medium and allowed to recover for 24 h at 37°C and 5% CO2 until they reached 60–80% confluence. Both the OP protein solution and the detergent solution were sterilized by filtration through a 0.22 μm membrane, and stocks were prepared in sterile PBS. Nile red solution (50 μM) in 1:1 EtOH:HO was used without sterilization. OP capsid-micelle complexes were prepared as described above. For each well, 20 μL of sample in PBS was added to 200 μL of culture medium to obtain a final concentration of 200 nM. Cells were incubated at 37°C and 5% CO2 for 16–20 h, then washed with PBS and trypsinized (0.05% Trypsin-EDTA (Thermo Fisher Scientific, USA), 4 min at 37°C). Cells were collected in cold culture medium, washed twice with cold PBS, and then resuspended in flow cytometry buffer (PBS containing 5% FBS). A representative flow cytometry analysis using all gating is shown in Figure 9.

[0252] Fluorescent labeling of the OP cage. To provide a specific handle for fluorophore conjugation, a single serine-to-cysteine ​​mutation was introduced at residue 38 of the OP protein by "QuikChange" (Agilent) site-directed mutagenesis. This lumen-resident residue was chosen to avoid interference with the outer surface of the OP cage, which could disrupt cellular uptake profiles. Successful molecular cloning was confirmed by Sanger sequencing (Microsynth AG, Switzerland) of the pET29b(+)_OPS38C plasmid used for protein expression, and the protein was produced as previously reported. 19Labeling of OP cages with Atto425-maleimide (Sigma-Aldrich) was performed by simply mixing purified protein in TSEC buffer with the dye solution (10 mM, DMSO) and incubating overnight at room temperature in the dark. To stop the reaction, two equivalents (wrt maleimide) of β-mercaptoethanol were added, and after 30 min of incubation, the protein was purified using a PD Minitrap G-10 column (GE Healthcare, USA). Labeling efficiency was assessed by UV-Vis absorbance measurements and the ε of Atto425 and OP proteins. 280 and ε 439 The experiment shown in Figure 4c used a sample with an average labeling of 1.9 dyes per OP capsid.

[0253] Confocal microscopy. Cells were seeded at a density of 15–20,000 cells per well onto μ-Slide 8-well chamber overslips with an ibiTreat surface (ibidi GmbH, Germany). Cells were incubated in 200 μL of culture medium at 37°C and 5% CO2 for 24 h before adding the samples. Sterile samples were prepared in PBS, and 10 μL of sample solution was added to 100 μL of culture medium per well to obtain the desired final concentrations of protein and fluorophore. After incubation with the samples for 24 h at 37°C and 5% CO2, the cells were washed with PBS and nuclear stained with 100 μL of Hoechst 33342 solution (5 μg / mL in PBS) for 10–15 min at 37°C. Cells were then washed twice with PBS and microscopically observed in PBS containing 10% FBS at 37°C.

[0254] Cell viability assay. Cytotoxicity was assessed using Sigma's WST-8-based Cell Counting Kit-8 according to the manufacturer's instructions. HeLa cells were seeded at a density of 5,000 cells per well in 100 μL of culture medium in a 96-well plate and allowed to recover for 24 h at 37°C and 5% CO2. Protein, surfactant, and drug samples were prepared by serial dilution in sterile PBS. A total volume of 25 μL of sample was added to each well to obtain the final concentrations shown in Figures 4e and 4f. As a negative control, 10 μL of 10% Triton X-100 in PBS was used per well. The positive control was PBS alone. After incubating the cells at 37°C and 5% CO2 for 24 h, 10 μL of CKK-8 reagent was added to each well. The plates were then incubated at 37°C and 5% CO2 for 2–4 h, after which the absorbance was measured at 450 nm. The absorbance of CKK-8 in cell-free culture medium was used for background subtraction, and samples were normalized to untreated cells to obtain the values ​​shown in Figures 4e and 4f. Samples were measured in sextuplicate using biological replicates of different protein batches.

[0255] Example 2 - Preparation and analysis of assembled lipoprotein cages Self-Assembly of Lipoprotein-Mimetic Cages To prepare exemplary lipoprotein cages according to the present invention, the artificial assembly OP was selected as a protein scaffold: a small, porous capsid with a positively charged internal cavity (Edwardson et al., 2018, op.cit.). After expression in E. coli, the OP protein is isolated as a complete octahedral assembly containing 24 monomers with approximately 3.5 nm pores on each of its six faces (Figure 1b). The outer diameter of the cage is approximately 13 nm, and the diameter of the spherical internal cavity is approximately 8 nm. These dimensions were assumed to be suitable for scaffolding small micellar aggregates with internal cavities.

[0256] Starting with a positively charged OP cage, we encapsulated a negatively charged amphiphile, which then phase-separated to create a hydrophobic core within the protein cage. The resulting protein-scaffolded lipid droplet then acts as a hydrophobic compartment capable of sequestering small molecules.

[0257] Cage-Templated Micelle Formation. Sodium dodecyl sulfate (SDS) was chosen as the anionic surfactant (Figure 2a) due to its aqueous solubility and the potential for favorable hydrogen bond formation between the sulfate headgroup and the many arginine residues on the inner surface of the OP capsid. Because SDS is typically used as a protein denaturant, we were interested in whether the charge and shape complementarity of the OP cage could be exploited to promote the formation of scaffolded micelles, rather than allowing hydrophobic interactions with the protein core to dominate the pathway leading to disruption of protein structure. As such, OPs were subjected to increasing molar equivalents of SDS, up to 2,000 molecules per cage, and analyzed by native gel electrophoresis (Figure 2b). The data show no change in OP quaternary structure up to approximately 800 equivalents of SDS per protein cage, but above 1,000 equivalents, bands of increased mobility emerged, potentially due to disruption or denaturation of the cage structure.

[0258] To better understand its resistance to SDS, we further investigated OP in the presence of 800 equivalents of SDS per cage through a combination of biophysical techniques (Figures 5 and 6). As illustrated by size-exclusion chromatography and transmission electron microscopy (Figures 2c, 2d, and 2e), there was no discernible change to the protein cage structure, even though the effective SDS concentration (approximately 5.2 M in lumen volume) was orders of magnitude higher than concentrations typically used to denature proteins (mM range).

[0259] To determine whether SDS molecules were drawn into the internal cavity of the OP cage as expected, we used a fluorescently labeled oligonucleotide probe. If SDS molecules were localized within the cavity via the intended sulfate-guanidinium interaction, other potential negatively charged guests would be prevented from entering. This blockage is due to both the cancellation of the high positive charge, which is the driving force for encapsulation, and the occlusion of the entrance pore. Because oligonucleotides are rapidly internalized by OP cages with high affinity (Edwardson et al., 2018, op.cit.), they provided an ideal probe to test this hypothesis. Atto488-labeled 21-nt DNA was added to either empty OP cages or those preincubated with SDS, and the complexes were analyzed by native gel electrophoresis (Figure 2f). While empty OP cages quantitatively encapsulated the probe, OP:SDS complexes failed to encapsulate the DNA strand, consistent with SDS molecules occupying the cavity.

[0260] Cryo-electron microscopy provides a means to directly probe both protein cage structure and the presence of internalized guests. Thus, we analyzed both empty OP cages and surfactant (SDS)-filled OP cages (Figures 2g-j, 7 and 8). Particles imaged with a 200 kV microscope were classified multiple times in 2D, and the best class was ultimately used to refine a single-class three-dimensional model. First, note that the protein structure changes only slightly in the presence of SDS (Figure 2g, 2 h). Second, comparison of OP and OP:SDS complexes reveals electron density differences corresponding to internalized surfactant, revealing the formation of a distinct protein-scaffolded micellar core (Figure 2j). The structural fidelity of the OP:SDS complex demonstrates the potential for well-defined protein structures to act as templates for the self-assembly of non-protein components, providing an alternative means for creating hybrid assemblies.

[0261] Hydrophobic Core Formation, Cargo Capacity, and Kinetics. To gain a deeper understanding of the internal structure of OP-templated micelles, we used the solvatochromic dye Nile Red. This small-molecule fluorophore is nearly non-emissive in aqueous media but exhibits fluorescence in nonpolar environments (Greenspan, P., Mayer, E.P., & Fowler, S.D. Nile Red: A Selective Fluorescent Stain for Intracellular Lipid Droplets. J. Cell Biol. 1985, vol. 100, pp. 965–973). To identify whether the OP cages chaperoned SDS molecules into micelle-like aggregates with hydrophobic cores, we measured Nile Red fluorescence in the presence of each component of the system.

[0262] Below the critical micellar concentration (4-5 mM in PBS buffer) (De Paula, R., da Hora Machado, A.E. & de Miranda, J.A., 3-Benzoxazol-2-yl-7-(N,N-diethylamino)-chromen-2-one as a fluorescence probe for the investigation of micellar microenvironments. J. Photochem. Photobiol. A: Chem. 2004, vol. 165, pp. 109-114), SDS had only a slight effect on the fluorescence of a 500 nM aqueous solution of Nile Red (Figure 3a). Similarly, the OP cage did not enhance the fluorescence of the fluorescent probe. However, in the presence of both OP and SDS (800 equiv.), a large increase in Nile Red fluorescence was observed. This signal increase was accompanied by a blue shift in the emission maximum, indicating the localization of the probe within a nonpolar environment (Greenspan et al., 1985, op. cit.).

[0263] Having established the suitability and utility of Nile Red, we conducted further experiments to determine the optimal number of SDS molecules per capsid and the number of Nile Red guests that could be encapsulated.

[0264] Fluorescence monitoring of two equivalents of Nile Red in the presence of OP:SDS complexes with increasing SDS content revealed a plateau at approximately 800 surfactant molecules per cage (Figure 3b, 3c). This number corresponds to the volumetric capacity of the OP cavity (approximately 256 nm 3 ) and the average volume occupied by an SDS molecule in a micelle (Methods section), this corresponds to a value of approximately 730. This confirmation suggests that the molecules are arranged with some structural similarity to their typical oblate ellipsoidal micellar morphology (Hammouda, B. Temperature Effect on the Nanostructure of SDS Micelles in Water. J Res Natl Inst Stand Technol 2013, vol. 118, pp. 151-167).

[0265] Because cargo loading is a key feature of protein-micelle complexes, we also used fluorescence titration to determine the number of Nile Red molecules that could be accommodated per cage. One equivalent of Nile Red was added stepwise to a solution of OP:SDS complexes, and the fluorescence spectrum was measured for each addition (Figure 3d). Between 1 and 5 molecules of Nile Red per cage, a logarithmic increase in fluorescence emission was observed with each equivalent. From 5 to 20 molecules of Nile Red, a steady decrease in fluorescence was observed, and above 20, a steady increase in fluorescence was observed. We attribute these three distinct phases to i) Nile Red encapsulation, ii) self-quenching, and iii) excess free dye. In the first phase, the addition of each Nile Red molecule gave an increasing decrease in emission due to self-quenching between the dyes; this process began to dominate in the second phase due to the high effective molar concentration of the fluorophore (30-130 mM). In the third step, the emission increase per equivalent is very close to that observed for Nile Red in bulk media, associated with a red shift and suggesting localization in aqueous environments. From these data, we can conclude that there is a clear loading capacity of the OP:SDS complex, corresponding to an effective concentration (approximately 130 mM) of approximately 20 molecules, far exceeding the solubility limit of Nile Red in organic solvents (<5 mM).

[0266] Measurement of Biological Activity. OP assemblies can deliver small interfering RNA to the cytosol of mammalian cells and induce efficient gene knockdown. Therefore, we were interested in whether micelles containing OP cages could improve the cellular uptake of poorly soluble compounds. Human cancer cells (HeLa) were treated with OP:SDS complexes carrying Nile Red or free fluorophores themselves. Analysis by flow cytometry (Figure 4a) showed that the OP:SDS complexes enhanced the cellular uptake of small molecule fluorophores. This finding was confirmed using confocal fluorescence microscopy (Figure 4b), which also revealed that Nile Red was distributed throughout the cell. Based on the reported intracellular trafficking of OP cages, most of which are localized in endosomes (Edwardson et al., 2018, op.cit.), this result suggests that the Nile Red cargo can escape from the cage after cellular uptake. To test this hypothesis, we performed the same experiment using Atto425-labeled OP cages (Figure 4c) and confirmed that while the majority of OP cages were transported through the endolysosomal system for degradation, the small molecule cargo was released and escaped into the cytosol.

[0267] To test the encapsulation, transport, and intracellular release of bioactive small molecules, we select lapatinib, a dual tyrosine kinase inhibitor, as a model compound (Moy, B. & Goss, P. E. Lapatinib: Current Status and Future Directions in Breast Cancer. The Oncologist 2006, vol. 11, pp. 1047-1057). Further suitable cargo molecules with a preferred size of less than 600 Da are listed in Table 3 below. Lapatinib has been used as a treatment for solid tumors and has been shown to benefit from nanoparticle-mediated delivery due to its low solubility and serum protein binding (Bonde, G. V. et al. Lapatinib nano-delivery systems: a promising future for breast cancer treatment. Expert Opin. Drug. Deliv. 2018, vol. 15, pp. 495-507). To reduce the thermodynamic cost of encapsulation and promote the formation of more stable complexes, we modified the surfactant composition with the endogenous steroid, cholesterol sulfate (CS), to facilitate loading of larger, planar guest molecules. We found that a surfactant composition of 75% SDS and 25% CS was well tolerated by the OP cage (Figures 11 and 12) and enabled stable loading of lapatinib (Figure 13a, b). Furthermore, flow cytometry analysis of Nile red-treated cells or Nile red encapsulated in the OP:SDS:CS complex revealed an even greater enhancement of intracellular delivery, 2- to 5-fold, compared to the OP:SDS complex (Figure 4d). This result may be due to the increased stability of the inclusion complex provided by the addition of CS, highlighting the inherent modularity of this strategy and the potential to tailor the surfactant composition to achieve the desired physical properties of different cargo molecules. [Table 3]

[0268] To determine how stably the OP:SDS:CS complexes retained their lapatinib cargo, they were dialyzed against medium containing 10% fetal bovine serum. After 72 h, fluorescence spectroscopy demonstrated lapatinib content in the OP:SDS:CS samples, revealing a signal within the range of the control sample (Figure 13c). Because lapatinib is known to be sequestered by serum albumin, this data confirms that the OP:SDS:CS complexes exhibit high affinity for this guest molecule.

[0269] Finally, we assayed the ability of the OP:SDS:CS complex to increase the effective cytotoxicity of lapatinib in human cancer cells. Cells were treated with either free lapatinib or lapatinib packaged in the OP:SDS:CS cage, and cell viability was monitored after 18 hours of incubation (Figure 4e). At a concentration of 2.5 μM, lapatinib had negligible effects on cell viability. However, packaging in the OP cage had a significant effect on potency, killing 60% of the cells. Importantly, the OP cage-containing surfactant itself exhibited negligible toxicity, confirming that the increased efficacy was due to the delivery of lapatinib to its intracellular target. A dose-response comparison of free and encapsulated drug (Figure 4f) reveals a 3-fold increase in potency provided by the OP:SDS:CS complex without the addition of specific targeting or uptake-enhancing ligands. In addition to solubilizing the drug molecules, encapsulation within the OP-surfactant complex prevents sequestration by serum proteins present in the medium, which may also reduce cellular uptake.

[0270] Example 3 - Preparation and analysis of assembled lipoprotein cages with fluorophore-conjugated surface cysteine ​​residues. Energy transfer between chemically conjugated fluorophores and Nile Red. To demonstrate the compatibility of lipoprotein cage formation and small molecule cargo loading with cysteine-containing proteins and their chemical conjugation, we examined complex formation with two protein variants, OP-K93C (SEQ ID NO: 4) and OP-S38C (SEQ ID NO: 22), which contain a single cysteine ​​mutation per monomer, displayed on either the external or internal surface, respectively. These proteins form the same cage structure as OP (SEQ ID NO: 2), as shown by transmission electron microscopy (Figure 14a, b). Purified proteins were site-specifically labeled with a cysteine-reactive Atto495-maleimide fluorophore using the manufacturer's recommended protocol. Lipoprotein cage formation with SDS was monitored by fluorimetry using Nile Red as a probe (Figure 14c). Due to the overlap between the excitation wavelength of Nile Red (535 nm) and the fluorescence emission of Atto495 (Emax = 527 nm), Förster resonance energy transfer (FRET) is expected when Nile Red is encapsulated in lipoprotein cage complexes. Indeed, this effect was observed with both OP-K93C and OP-S38C, consistent with the formation of SDS-containing lipoprotein cages. Furthermore, the significant difference in FRET efficiency is consistent with the location of Atto495 either inside the protein cage, closer to Nile Red, or on the outer surface, further away from the micelle core. These data demonstrate that chemically conjugated cysteine ​​mutants of OP proteins can be used to form lipoprotein cages that retain their functionality. Furthermore, the compatibility of OP-S38C with different detergent compositions (75% SDS and 25% CS) and its efficient delivery to cells were also demonstrated (Figures 4c, 10).

[0271] Example 4 - Preparation and analysis of assembled lipoprotein cages with C-terminal peptide tags. Formation of Lipoprotein Cages with C-Terminal Appendages. Attachment of peptides, such as degradation tags, targeting tags, cell-penetrating tags, and endosomal escape tags, to the C-terminus of proteins allows for tuning of the functionality of this encapsulation system. To demonstrate the compatibility of lipoprotein cage formation with this type of protein modification, we tested four protein variants with different C-terminal peptide tags created by gene fusion. The four proteins tested were OP-96 (SEQ ID NO: 24), OP-ZHER2 (SEQ ID NO: 25), OP-ZEGFR (SEQ ID NO: 26), and OP-SP94 (SEQ ID NO: 27). Each of these four variants formed protein cage structures, as demonstrated by size exclusion chromatography (Figure 15a) and transmission electron microscopy (Figures 15b-e). Lipoprotein cage formation with SDS was examined by fluorimetry using Nile Red as a probe (Figure 16f). The fluorescence enhancement and blue shift in emission wavelength in the presence of the protein cage and SDS is consistent with lipoprotein cage formation and encapsulation of the small molecule cargo, Nile Red.

[0272] Example 5 - Preparation and analysis of assembled lipoprotein cages with N-terminal peptide tags. Formation of Lipoprotein Cages with N-Terminal Appendages. In addition to the C-terminus of proteins, the attachment of peptides such as degradation tags, targeting tags, cell-penetrating tags, and endosomal escape tags to the N-terminus of proteins also allows for tuning of the functionality of this encapsulation system. To demonstrate the compatibility of lipoprotein cage formation with this type of protein modification, we tested a protein variant, OP-93 (SEQ ID NO: 23), with an N-terminal peptide tag created via gene fusion. This protein also formed a protein cage structure, as demonstrated by size-exclusion chromatography (Figure 16a). Lipoprotein cage formation with SDS was investigated by fluorimetry using Nile Red as a probe (Figure 16b). The fluorescence enhancement and blue shift in emission wavelength in the presence of the protein cage and SDS are consistent with lipoprotein cage formation and encapsulation of the small molecule cargo, Nile Red.

[0273] Example 6 - Preparation and analysis of assembled lipoprotein cages using alternative surfactant compositions. Formation of lipoprotein cages with sodium dodecylbenzenesulfonate. The protein cages are highly stable, acting as a template for the formation of lipid / micelle cores in their internal cavities, meaning that a prior surfactant formulation step is not required. Therefore, the amphiphile does not need to form stable particles by itself before the addition of the protein, allowing for the use of a variety of different amphiphiles as long as the mixture possesses sufficient negative charge. To further demonstrate the system's generality to different surfactants, sodium dodecylbenzenesulfonate (SDBS) was used in a 1:1 mixture with SDS to form lipoprotein cages with the OP protein (SEQ ID NO: 2). Native agarose gel analysis revealed the formation of stable lipoprotein complexes capable of encapsulating three different types of small molecule cargo (Figure 17a). Furthermore, flow cytometry analysis of HeLa cells treated with the OP:SDS:SDBS:curcumin (1:400:400:8) complex showed an 8-fold increase in cellular uptake compared to the free drug (Figure 17b). These data highlight the modular nature of lipoprotein cage technology, as varying the lipid composition provides a facile means to tune the encapsulation properties of different cargo molecules.

[0274] Example 7 - Preparation and analysis of assembled lipoprotein cages with alternative small molecule cargoes. Formation of Lipoprotein Cages with Alternative Small Molecule Cargoes. To further demonstrate that the bilayer encapsulation concept can be generalized to alternative cargoes by exploiting hydrophobic effects, we tested the encapsulation of five different small molecules: Nile Red, lapatinib, daunorubicin, curcumin, and laurdan. These molecules have diverse molecular structures and unique biological and photophysical properties. Nevertheless, each of these molecules could be encapsulated and efficiently delivered to cells. Native agarose gel analysis of the OP:SDS:SDBS (1:400:400) protein cage in the presence of curcumin, lapatinib, and daunorubicin reveals the encapsulation of these bioactive compounds within the lipoprotein cage (Figure 17a). The encapsulation efficiency of small molecules could be improved by varying the surfactant composition, as seen in the use of OP:SDS:CS (1:600:200) for curcumin (Figure 17c), Nile Red (Figures 4, 11), and lapatinib (Figure 13). Importantly, improved encapsulation efficiency also led to increased cellular delivery of these compounds. For example, using OP:SDS:CS, a 2.5-fold increase in cellular uptake of Nile Red (Figure 4d) was observed compared to OP:SDS (1:800) cages. In the case of curcumin, OP:SDS:CS (1:600:200) lipoprotein cages provided a 2-fold increase in cellular delivery compared to their OP:SDS:SDSBS (1:400:400) counterparts (Figures 17a, 17d). In the case of the fluorescent probe Laurdan, packaging in OP:SDS (1:800) lipoprotein cages provided a significant increase in cellular uptake compared to the free molecule, as observed by confocal fluorescence microscopy (Figure 17e). The present invention includes the following preferred embodiments. (1) 1. A lipoprotein cage for intracellular delivery of cargo, the lipoprotein cage comprising: (i) a protein cage comprising at least one polypeptide comprising an amino acid sequence I consisting of: MX 13 QAIGILELX 1 SIAAGMELGDAMLKSAX 14 VX 15 LLVSKTISX 2 GKFLLMLGGDIX 8 AIX 9 X 12 AIX 10 TGTX 11 QAGX 3 LLVDSLVLAX 16 IHPSVLPAIX 17 GX 18 NX 19 VX 20 X 7 X 21 QAVGIVETX 4 SVAACISAADX 22 AVX 23 GSX 24 VTLVRVHMAX 5 GIGGKCYMVVAGDVSDVALAVTVASSSAGAYGX 6 LVYASLIPX 25 PHX 26 AMWX 27 QMVX 28 GX 29 E (SEQ ID NO: 1) where X 1 ~X 29 are independently amino acids, provided that X 1 ~X 6 at least three of which are independently positively charged amino acids, and X in SEQ ID NO: 1 1 ~X 29 Up to five amino acids at positions other than those indicated may be replaced by any amino acid, the protein cage having a positively charged interior; (ii) a surfactant composition comprising one or more amphiphiles, the one or more amphiphiles being selected such that the net charge of the composition is negative, each of the amphiphiles comprising a hydrophilic group and a hydrophobic group, the hydrophobic group comprising at least one hydrocarbon moiety selected from the group consisting of C4-C30 alkyl, C4-C30 alkenyl, C4-C30 alkynyl, C4-C30 alkoxy, or C5-C30 cycloalkyl; a surfactant composition as described above, wherein the surfactant composition is encapsulated in an assembled protein cage; a lipoprotein cage for intracellular delivery of said cargo comprising: (2) X 1 ~X 6 The lipoprotein cage according to (1), wherein each is independently arginine or lysine. (3) X 1 ~X 6 at least four of X are positively charged amino acids, preferably 1 ~X 6 at least five of X are positively charged amino acids, more preferably 1 ~X 6 The lipoprotein cage according to (1) or (2), wherein each of is a positively charged amino acid. (4) X 8 is glycine or a conservative substitution thereof, X 9 and X 12 are, independently of each other, glutamine, asparagine, or a conservative substitution thereof; X 10 is glutamate, aspartate or a conservative substitution thereof; X 11 is serine or a conservative substitution thereof, X 13 、X 17 and X 19 are each independently serine or a conservative substitution thereof, X 14 、X 16 and X 24 are independently asparagine, glutamine, or a conservative substitution thereof; X 15 、X 20 、X 26 and X 28 are independently aspartate, glutamate or a conservative substitution thereof, X 18 and X 29 are, independently of each other, leucine or a conservative substitution thereof, and X 21 、X 22 、X 23 、X 25 and X 27 are, independently of each other, arginine, lysine, or conservative substitutions thereof; The lipoprotein cage according to any one of (1) to (3). (5) The lipoprotein cage according to any one of (1) to (4), wherein the amino acid sequence I is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5, 10 to 16, and 20 to 22. (6) The lipoprotein cage according to any one of (1) to (5), wherein 20 mol % of the compounds contained in the surfactant composition have at least one single negative charge. (7) The lipoprotein cage according to any one of (1) to (6), wherein the hydrophilic group is an anionic hydrophilic group. (8) 8. The lipoprotein cage according to (7), wherein the anionic hydrophilic group is selected from the group consisting of carboxylate, sulfate, sulfonate, phosphonate, boronate, phosphate and amino acid moieties. (9) The lipoprotein cage according to any one of (1) to (8), wherein the hydrocarbon moiety is selected from the group consisting of branched or straight-chain alkyl, branched or straight-chain alkenyl, or cycloalkyl of C5 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16, C17, or C18, respectively. (10) The lipoprotein cage according to (8) or (9), wherein the anionic hydrophilic group is a sulfate moiety, and the hydrocarbon moiety is a linear alkyl, linear alkyl ether, or cycloalkyl residue, and the linear alkyl, linear alkyl ether, or cycloalkyl residue is C5 to C30, preferably C8 to C20, more preferably C10 to C18, even more preferably C12 to C18, and most preferably C12, C14, C16, C17, or C18. (11) The lipoprotein cage according to any one of (1) to (10), wherein at least one of the one or more amphiphilic substances is an ammonium salt, alkali salt, or alkaline earth salt of dodecyl sulfate, preferably sodium dodecyl sulfate. (12) The lipoprotein cage according to any one of (1) to (11), wherein at least one of the one or more amphiphiles is an anionic steroid, and preferably, the anionic steroid is cholesterol sulfate. (13) A complex comprising the lipoprotein cage according to any one of (1) to (12) and one or more cargo molecules, preferably the cargo molecules having a size of 1000 Da or less. (14) The complex according to (13), wherein the cargo molecule is encapsulated in the lipoprotein cage without disassembling the lipoprotein cage. (15) A method for producing the lipoprotein cage according to any one of (1) to (12), self-assembling a protein cage from at least one polypeptide comprising amino acid sequence I, preferably 24 polypeptides each comprising amino acid sequence I; encapsulating the surfactant composition according to (1) in a protein cage without decomposing the protein cage; A method for producing the lipoprotein cage according to any one of (1) to (12), comprising: [Sequence List Free Text]

[0275] Sequence Listing 1 <223> Synthetic sequences Sequence Listing 1 <223> Any amino acid Sequence Listing 1 <223> At least three of the X amino acids at positions 11, 39, 66, 103, 130, and 163 are, independently of each other, positively charged amino acids. Sequence Listings 2-27 <223> Synthetic sequences

Claims

1. 1. A lipoprotein cage for intracellular delivery of cargo, the lipoprotein cage comprising: (i) a protein cage comprising at least one polypeptide comprising amino acid sequence I, the amino acid sequence I is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 12-16, and 20-27; the protein cage having a positively charged interior; (ii) a surfactant composition comprising one or more amphiphiles selected such that the net charge of the composition is negative, each of the amphiphiles comprising a hydrophilic group and a hydrophobic group, the hydrophobic group comprising at least one hydrocarbon moiety selected from the group consisting of C4 to C30 alkyl, C4 to C30 alkenyl, C4 to C30 alkynyl, C4 to C30 alkoxy, or C5 to C30 cycloalkyl; a surfactant composition as described above, wherein the surfactant composition is encapsulated in an assembled protein cage; a lipoprotein cage for intracellular delivery of said cargo comprising:

2. The lipoprotein cage of claim 1, wherein the amino acid sequence I is an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, and SEQ ID NO: 22 to 27.

3. The lipoprotein cage described in claim 1, wherein the amino acid sequence I is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 22 and SEQ ID NOs: 24 to 27.

4. The lipoprotein cage according to any one of claims 1 to 3, wherein 20 mol % of the compounds contained in the surfactant composition have at least one single negative charge.

5. A lipoprotein cage described in any one of claims 1 to 4, wherein the hydrophilic group is an anionic hydrophilic group.

6. The lipoprotein cage of claim 5, wherein the anionic hydrophilic group is selected from the group consisting of carboxylate, sulfate, sulfonate, phosphonate, boronate, phosphate and amino acid moieties.

7. A lipoprotein cage described in any one of claims 1 to 6, wherein the hydrocarbon portion is selected from the group consisting of branched alkyl, straight-chain alkyl, branched alkenyl, straight-chain alkenyl and cycloalkyl, each of C5 to C30.

8. A lipoprotein cage described in any one of claims 1 to 6, wherein the hydrocarbon portion is selected from the group consisting of branched alkyl, straight-chain alkyl, branched alkenyl, straight-chain alkenyl and cycloalkyl, each of C8 to C20.

9. A lipoprotein cage described in any one of claims 1 to 6, wherein the hydrocarbon portion is selected from the group consisting of branched alkyl, straight chain alkyl, branched alkenyl, straight chain alkenyl and cycloalkyl, each of C10 to C18.

10. A lipoprotein cage described in any one of claims 1 to 6, wherein the hydrocarbon portion is selected from the group consisting of branched alkyl, straight chain alkyl, branched alkenyl, straight chain alkenyl and cycloalkyl, each of C12 to C18.

11. A lipoprotein cage according to any one of claims 1 to 6, wherein the hydrocarbon moiety is selected from the group consisting of branched alkyl, straight chain alkyl, branched alkenyl, straight chain alkenyl and cycloalkyl of C12, C14, C16, C17 or C18, respectively.

12. A lipoprotein cage as described in claim 6 or 7, wherein the anionic hydrophilic group is a sulfate moiety and the hydrocarbon moiety is a straight chain alkyl, straight chain alkyl ether or cycloalkyl residue, and the straight chain alkyl, straight chain alkyl ether or cycloalkyl residue is C5 to C30.

13. A lipoprotein cage as described in claim 6 or 7, wherein the anionic hydrophilic group is a sulfate moiety and the hydrocarbon moiety is a straight chain alkyl, straight chain alkyl ether or cycloalkyl residue, and the straight chain alkyl, straight chain alkyl ether or cycloalkyl residue is C8 to C20.

14. A lipoprotein cage as described in claim 6 or 7, wherein the anionic hydrophilic group is a sulfate moiety and the hydrocarbon moiety is a straight chain alkyl, straight chain alkyl ether or cycloalkyl residue, and the straight chain alkyl, straight chain alkyl ether or cycloalkyl residue is C10 to C18.

15. A lipoprotein cage as described in claim 6 or 7, wherein the anionic hydrophilic group is a sulfate moiety and the hydrocarbon moiety is a straight chain alkyl, straight chain alkyl ether or cycloalkyl residue, and the straight chain alkyl, straight chain alkyl ether or cycloalkyl residue is C12 to C18.

16. The lipoprotein cage of claim 6 or 7, wherein the anionic hydrophilic group is a sulfate moiety and the hydrocarbon moiety is a straight chain alkyl, straight chain alkyl ether or cycloalkyl residue, and the straight chain alkyl, straight chain alkyl ether or cycloalkyl residue is C12, C14, C16, C17 or C18.

17. A lipoprotein cage described in any one of claims 1 to 16, wherein at least one of the one or more amphiphilic substances is an ammonium salt, alkali salt or alkaline earth salt of dodecyl sulfate.

18. A lipoprotein cage described in any one of claims 1 to 16, wherein at least one of the one or more amphiphilic substances is sodium dodecyl sulfate.

19. A lipoprotein cage described in any one of claims 1 to 16, wherein at least one of the one or more amphiphilic substances is an anionic steroid.

20. A lipoprotein cage described in any one of claims 1 to 16, wherein at least one of the one or more amphiphilic substances is cholesterol sulfate.

21. A complex comprising a lipoprotein cage according to any one of claims 1 to 20 and one or more cargo molecules.

22. The complex described in claim 21, wherein the cargo molecule has a size of 1000 Da or less.

23. 23. The complex of claim 21 or 22, wherein the cargo molecule is encapsulated in the lipoprotein cage without disassembling the lipoprotein cage.

24. A method for producing a protein cage comprising the steps of: self-assembling a protein cage from at least one polypeptide comprising amino acid sequence I; encapsulating the surfactant composition of claim 1 in a protein cage without disintegrating the protein cage; 21. A method for producing a lipoprotein cage according to any one of claims 1 to 20, comprising:

25. The method described in claim 24, wherein the step of self-assembling a protein cage from at least one polypeptide containing the amino acid sequence I is a step of self-assembling a protein cage from 24 polypeptides each containing the amino acid sequence I.

Images