Serum-protein coated fusogenic lipid particles for targeted drug delivery

Abstract

The invention is based on the finding that fusogenic lipid particles, when coated with serum proteins such as apolipoproteins, retain their fusogenic properties. By coating fusogenic particles with serum proteins such as apolipoproteins, the inventors provide protein-coated fusogenic lipid particles that can be used for targeted drug delivery. The invention pertains to said protein-coated fusogenic lipid particles, their method of manufacturing and therapeutic use, und to methods for targeted delivery of drugs and of the fusogenic lipid particles that rely on the coating of the fusogenic lipid particles with serum proteins. In particular embodiments, the protein-coated fusogenic particles of the invention comprise a polyphenol.

Description

[0001] SERUM-PROTEIN COATED FUSOGENIC LIPID PARTICLES FOR TARGETED DRUG DELIVERY

[0002] FIELD OF THE INVENTION

[0003] [1] The invention is based on the finding that fusogenic lipid particles, when coated with serum proteins such as apolipoproteins, retain their fusogenic properties. By coating fusogenic particles with serum proteins such as apolipoproteins, the inventors provide protein-coated fusogenic lipid particles that can be used for targeted drug delivery. The invention pertains to said protein-coated fusogenic lipid particles, their method of manufacturing and therapeutic use, und to methods for targeted delivery of drugs and of fusogenic lipid particles that rely on the coating of the fusogenic lipid particles with serum proteins. In particular embodiments, the protein-coated fusogenic particles of the invention comprise a polyphenol.

[0004] DESCRIPTION

[0005] [2] Conventional phospholipid-based liposomes are spherically closed lipid bilayers enclosing an aqueous core, which can be used for the encapsulation of lipophilic drugs into the lipid bilayers and for hydrophilic drugs into the aqueous core. Such conventional liposome systems are known as drug delivery vehicles with a strong preference for clathrin-dependent or clathrin-independent endocytotic uptake. However, the efficiency of this endocytotic uptake pathway is limited, often below 1%. Increasing efficiencies is therefore of major interest and approached by enhancing the uptake probability of whole liposomes. For example, fusogenic lipid particles with extraordinarily high fusion ability have been developed that act on a different protein-independent fusion mechanism.

[0006] [3] Fusogenic lipid particles generally comprise of a membrane of positively charged lipids and aromatic molecules. It is believed that the positively charged lipids of the fusogenic lipid particles polarize the delocalized TT-electrons of highly aromatic molecules, inducing temporal dipoles within the lipid membrane of the particles. These dipoles are thought to promote local instabilities in molecular lipid arrangements, reducing the fusion barrier and allowing intermediate fusion formation. This interaction allows a direct drug delivery to the cell-membrane and to the cytoplasm without the cargo undergoing lysosomal cargo degradation. Nevertheless, this drug delivery using such fusogenic lipid particles is associated with a systemic drug-uptake and fails to accumulate high drug concentrations in specific tissues and to avoid systemic side-effects.

[0007] [4] Polyphenols such as resveratrol, as radical scavengers, are frequently used antioxidants. Polyphenols are of particular interest as therapeutics in age-related neuronal diseases, as they have been shown to influence the survival and differentiation of neuronal cells and thus improve neuronal function and regeneration. They are explored for application in medicaments that halt the progression of neurodegenerative diseases. However, one of the main drawbacks of these aromatic compounds is their low bioavailability. Polyphenols are generally poorly soluble and are poorly absorbed by the human body. To improve the bioavailability of such compounds, fusogenic liposomes, a type of fusogenic lipid particle in the shape of a liposome, have been explored for resveratrol delivery. However, due to lacking a specific targeting mechanism, only a small fraction of fusogenic lipids can successfully reach target tissues such as the brain after intravenous delivery.

[0008] [5] Nanoparticle-based therapeutics can improve the therapeutic index of drugs by increasing their localization to specific tissues, organs, or cells and decreasing potential side effects simultaneously. Such tissue-targeted delivery can be achieved by attaching a distinct molecule to the nanoparticle surface with a high binding affinity to the target tissue. For example, Apolipoprotein E (ApoE) is a serum protein that binds to the LRP receptor that is highly expressed in the cerebral endothelium of blood vessels.

[0009] [6] Topal et al. demonstrate a protein coating in a system consisting of negatively charged solid lipid nanoparticles (SLN) that are coated with apolipoprotein E via a biotin-avidin coupling. Compared to the uncoated particles, the ApoE-coated SLNs of Topal exhibit increased uptake in endothelial and neuronal brain cells and increased blood brain barrier (BBB)-permeation. Similarly, Kuo et al. have shown the delivery of ApoE-coated negatively charged liposomes to endothelial cells in the brain. In Kuo, the ApoE was grafted onto the carboxylic acid groups on the liposomes’ surface. Kuo also describes that their ApoE-coated particles would show improved blood brain barrier penetration. Notably, neither Kuo and Topal use a full-length ApoE protein for their coating. They only used the receptor binding part of the ApoE, linked with a chemically reactive group making the coating process complex and expensive. Both the particle-types of Kuo and Topal exhibit a negative zeta-potential before and after loading with ApoE and the absolute zeta-potential of the particles in these publications was decreased by the coverage with the ApoEcoating.

[0010] [7] As indicated above, fusogenic lipid particles merge with cells by a mechanism that relies on the interaction of a positively charged lipid particle surface containing polarized aromatic dipoles with the cell membrane that the particles are merging with. However, this mechanism lacks a targeting of specific cells. Prior to the present invention, a targeting as shown in Kuo et al. or Topal et al. was believed to be not compatible with the fusion mechanism of fusogenic lipid particles. This has numerous reasons: a protein coating introduces an additional “layer” between the fusogenic surface of the particle and the lipids of the cell membrane. The cell membrane therefore interacts with this protein layer instead of the lipids of the particle surface directly. Introducing a surface coating also increases the distance between surface of the particle and the cell membrane, therefore decreasing the strength of the charge and / or dipole-based interaction of the fusogenic lipid particles. Furthermore, a protein coating masks to a certain degree the surface properties of the drug delivery particles that lie underneath the coating. For example, this can be seen in reduced absolute zeta-potentials of coated lipid particles.

[0011] [8] The inventors found that fusion liposomes, when incubated in human serum samples, form a protein corona that comprises serum proteins. Surprisingly fusogenic lipid particles retain their fusogenic properties despite being coated with serum proteins. Based on this, they developed a new method for drug delivery and for targeted delivery of fusogenic lipid particles such as fusogenic liposomes, in which fusogenic lipid particles are pre-coated with serum proteins.

[0012] [9] The inventors further determined that serum proteins on the surface of the fusion proteins are enriched with apolipoproteins compared to their concentration in plasma, indicating a strong high binding affinity to the fusogenic lipid particles. The inventors use this binding affinity to coat fusogenic lipid particles enriched with apolipoproteins. In contrast to previously shown lipid-based particles, no covalent binding of the apolipoproteins to the surface of the fusogenic particles is required.

[0013]

[0010] Furthermore, the inventors found that ApoE-coated fusogenic particles of the invention can be used for the direct delivery of drugs to the membrane and the cytoplasm of endothelial cells of BBB. Amongst others, this delivery can rely on the binding interaction of the ApoE with interaction the LRP1 receptor. While previous studies on negatively charged lipid particles emphasize that an ApoE-coating increases the particles capabilities to traverse the blood brain barrier, the present invention demonstrates that ApoE-coated fusogenic lipid particles can be used for a targeted treatment of the microvascular system in the brain, specifically the endothelial cells.

[0014]

[0011] Binding ApoE to the nanoparticle surface can be used to guide the particles to the brain, where they accumulate. At the current stage of knowledge, none of the drug delivery systems known in the literature is able to accumulate in the cerebral vascular endothelium and at the same time, fuse with its plasma membrane, directly modifying its properties. The inventors make use of the protein-coated fusogenic lipid particles of the invention to deliver a wide range of aromatic compounds, such as naturally occurring polyphenols. These aromatic compounds are directly included in the lipid membrane and contribute to the fusogenic mechanism of the positively charged protein-coated fusogenic particles with the cellular plasma membrane.

[0015] BRIEF DESCRIPTION OF THE INVENTION

[0016]

[0012] Generally, and by way of brief description, the main aspects of the present invention can be described as follows:

[0017]

[0013] In a first aspect, the invention pertains to a protein-coated fusogenic lipid particle comprising (i) a compound A that is a positively charged lipid molecule; and

[0018] (ii) a compound C that is an aromatic molecule wherein the protein-coated fusogenic lipid particle comprises a protein coating that comprises an apolipoprotein.

[0019]

[0014] In a second aspect, the invention pertains to a protein-coated fusogenic lipid particle comprising

[0020] (i) a compound A that is a positively charged lipid molecule; and

[0021] (ii) a compound C that is an aromatic molecule; wherein the protein-coated fusogenic lipid particle comprises a protein coating that comprises ApoE; wherein the ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an Apolipoprotein E.

[0022]

[0015] In a third aspect, the invention pertains to a method of manufacturing a protein-coated fusogenic lipid particle according to any other aspect of the invention, the method comprising:

[0023] (i) Mixing Compound A and Compound C as defined herein in an organic or aqueous solvent;

[0024] (ii) Drying the mixture of step (i);

[0025] (iii) Redispersing the dried mixture obtained in step (ii) in a polar solvent such as ethanol and subsequently quickly adding, more preferably injecting, an aqueous buffer to obtain a dispersion containing a fusogenic lipid particle;

[0026] (iv) Mixing the dispersion containing the fusogenic lipid particle obtained in step (iii) with a dispersion comprising an apolipoprotein as defined herein.

[0027]

[0016] In a fourth aspect, the invention pertains to a therapeutic composition comprising a protein-coated fusogenic lipid particle of any other aspect of the invention and a pharmaceutically acceptable carrier or excipient.

[0028]

[0017] In a fifth aspect, the invention pertains to a therapeutic composition use in the treatment of a condition or disease.

[0029]

[0018] In a sixth aspect, the invention pertains to a method for the targeted delivery of a fusogenic lipid particle, wherein the method comprises an in-vitro step of pre-coating a non-coated fusogenic lipid particle with a serum protein, wherein the non-coated fusogenic lipid particle comprises (i) a Compound A that is a positively charged lipid molecule according to any aspect herein;

[0030] (ii) a Compound C that is an aromatic molecule according to any aspect herein.

[0031]

[0019] In a seventh aspect, the invention pertains to a method for targeted drug delivery, wherein the method comprises using a protein-coated fusogenic lipid particle as a drug-delivery vehicle, wherein the non-coated fusogenic lipid particle comprises

[0032] (i) a Compound A that is a positively charged lipid molecule according to any aspect herein;

[0033] (ii) a Compound C that is an aromatic molecule according to any aspect herein wherein the protein-coated fusogenic lipid particle comprises a protein coating that comprises a serum protein and wherein the protein- coated fusogenic lipid particle comprises a drug that is to be delivered, preferably wherein the protein-coated fusogenic lipid particle is a protein coated fusogenic lipid particle according to any other aspect of the invention.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035]

[0020] In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0036]

[0021] In a first aspect, the invention pertains to a protein-coated fusogenic lipid particle comprising

[0037] (i) a compound A that is a positively charged lipid molecule; and

[0038] (ii) a compound C that is an aromatic molecule wherein the protein-coated fusogenic lipid particle comprises a protein coating that comprises an apolipoprotein.

[0039]

[0022] In an alternative of this first aspect, the invention pertains to a protein-coated fusogenic lipid particle comprising

[0040] (i) a compound A that is a positively charged lipid molecule; and

[0041] (ii) a compound C that is an aromatic molecule; wherein the protein-coated fusogenic lipid particle comprises a protein coating that comprises an apolipoprotein; wherein the apolipoprotein is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an apolipoprotein.

[0042]

[0023] In preferred embodiments of the invention, the protein-coated fusogenic lipid particle of the invention comprises a coating with a molar apolipoprotein-content of at least 30%, more preferably of at least 50%, more preferably of at least 70%, more preferably of at least 90%. Preferably, the protein-coated fusogenic lipid particle of the invention comprises a protein coating that consists of an apolipoprotein. In particularly preferred embodiments, the protein-coated fusogenic lipid particle of the invention comprises a coating with a molar apolipoprotein-content of at least 50%. With a higher amount of apolipoprotein in the protein coating of the lipid particles of the invention, the stability of the protein corona and the targeting of the protein-coated fusogenic lipid particles is increased.

[0043]

[0024] Therefore, in preferred embodiments, the invention pertains to a protein-coated fusogenic lipid particle comprising

[0044] (i) a compound A that is a positively charged lipid molecule; and

[0045] (ii) a compound C that is an aromatic molecule; wherein the protein-coated fusogenic lipid particle comprises a protein coating that consists of an apolipoprotein.

[0046]

[0025] The term “apolipoprotein” (also called “Apo”) preferably refers to the protein component of a lipoprotein. In a lipoprotein, the apolipoprotein component enables the transport of waterinsoluble lipids in blood. Instead of the complete designation of e.g. “apolipoprotein E”, it has become customary to simply speak of “ApoE”.

[0047]

[0026] In preferred embodiments, the protein-coated fusogenic lipid particle further comprises a Compound B that is a neutrally charged lipid molecule. Preferably, Compound A, Compound B and Compound C are defined as in any other aspect herein.

[0048]

[0027] In preferred embodiments of the invention, the Compound A, ,the optional Compound B, and the Compound C and the proteins of the protein corona form the protein-coated fusogenic lipid particles of the invention. The protein- coated fusogenic lipid particles of the invention comprise a lipid membrane that comprises at least a fraction (up to all) of the Compound A and of the Compound C. Without wishing to be bound by theory, it is believed that the positively charged Compound A polarizes the delocalized TT-electrons of Compound C inducing temporal dipoles within the lipid membrane of the particles. These dipoles promote local instabilities in molecular lipid arrangements, reducing the fusion barrier, resulting in the fusion mechanism of the protein-coated fusogenic particles of the invention.

[0049]

[0028] The inventors found that non-coated fusogenic lipid particles, when subjected to human blood serum, are coated with a protein corona. The ratio apolipoproteins in this corona is higher compared to the ratio of apoproteins in the serum indicating that the binding interaction of apolipoproteins to the surface of the fusogenic lipid particles is stronger than for other serum protein. Nevertheless, the protein corona of such non-coated apolipoproteins (that have not been coated prior to subjecting into serum) is comprised of many different proteins, including many different apolipoproteins. This results in particles that retain their fusogenic properties, but are without specificity to any tissue. By further enriching the fusogenic particles with apolipoproteins, the inventors have created protein-coated fusogenic lipid particles with an increased tissue specificity that depends on the amount and type of apolipoproteins in the protein corona of these protein-coated fusogenic lipid particles. The apolipoproteins inhibit the adhesion of other proteins to the surface of the lipid particles. Therefore such apolipoprotein-coated particles retain their target specificity, when administered to a subject. Despite the coating, the protein-coated fusogenic lipid particles retain their fusogenic properties.

[0050]

[0029] In preferred embodiments of the invention, the protein-coated fusogenic lipid particle comprises a protein coating with a molar apolipoprotein E (ApoE)-content of at least 10%, more preferably of at least at least 30%, more preferably of at least at least 50%, more preferably of at least at least 70%, more preferably of at least at least 90%. Preferably, the protein-coated fusogenic lipid particle of the invention comprises a protein coating that consists of ApoE. In particularly preferred embodiments, the protein-coated fusogenic lipid particle comprises a protein coating with a molar ApoE-content of at least at least 30%. In binding affinity experiments, the inventors found that the apolipoprotein E comprises a particularly high affinity for the fusogenic lipid particles of the invention (see Example 2). Therefore, a coating with ApoE provides the fusogenic lipid particles of the invention with a high stability and is protecting the surface of the fusogenic liposomes against subsequent uncontrolled protein corona formation. Furthermore, it provides the fusogenic lipid particles of the invention with a targeting of cells that express a receptor for ApoE, such as LRP-1. With a higher ApoE content in the protein-corona, the stability of the protein corona is further increased and / or targeting of cells is increased.

[0051]

[0030] In a second aspect, the invention pertains to a protein-coated fusogenic lipid particle comprising

[0052] (i) a compound A that is a positively charged lipid molecule; and (ii) a compound C that is an aromatic molecule; wherein the protein-coated fusogenic lipid particle comprises a protein coating that comprises ApoE; wherein the ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an Apolipoprotein E.

[0053]

[0031] In preferred embodiments of the invention, the protein-coated fusogenic lipid particle comprises a protein coating with a molar apolipoprotein E (ApoE)-content of at least 10%, more preferably of at least at least 30%, more preferably of at least at least 50%, more preferably of at least at least 70%, more preferably of at least at least 90%. Preferably, the protein-coated fusogenic lipid particle of the invention comprises a protein coating that consists of ApoE. In particularly preferred embodiments, the protein-coated fusogenic lipid particle comprises a protein coating with a molar ApoE-content of at least at least 30%. In binding affinity experiments, the inventors found that the apolipoprotein E comprises a particularly high affinity for the fusogenic lipid particles of the invention (see Example 2). Therefore, a coating with ApoE provides the fusogenic lipid particles of the invention with a high stability and is protecting the surface of the fusogenic liposomes against subsequent uncontrolled protein corona formation. Furthermore, it provides the fusogenic proteins of the invention with a targeting of cells that express a receptor for ApoE, such as LRP-1. With a higher ApoE content in the protein-corona, the stability of the protein corona is further increased and / or targeting of cells is increased.

[0054]

[0032] In particularly preferred embodiments of the invention, the protein-coated fusogenic lipid particle comprises

[0055] (i) a compound A that is a positively charged lipid molecule; and

[0056] (ii) a compound C that is an aromatic molecule; wherein the protein-coated fusogenic lipid particle comprises a protein coating that consists of ApoE; wherein the ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an Apolipoprotein E.

[0057]

[0033] In preferred embodiments of the invention, the ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an ApoE. In preferred embodiments, the term “ApoE” refers to a protein that comprises an amino acid sequence with at least 80% sequence identity to any one of SEQ ID NO: 1 to 4. Preferably, the term ApoE refers to a protein that comprises an amino acid sequence with at least 80% sequence identity to the ApoE protein that is described by any one of the UNIPROT entries (https: / / www.uniprot.org / , dated 30thAugust 2024) P02649 (SEQ ID NO: 1), E7ERP7 (SEQ ID NO: 2), H0Y7L5 (SEQ ID NO: 3), E9PEV4 (SEQ ID NO: 4). More preferably, the ApoE refers to a protein that comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 1. In particularly preferred embodiments, the term ApoE refers to a protein that comprises an amino acid sequence with at least 80% sequence identity to the ApoE protein that is described by the UNIPROT entry P02649 (date 30thAugust 2024).

[0058]

[0034] Preferably, the term “LRP1”, refers to the protein that is called “Prolow-density lipoprotein receptor-related protein 1” and / or a variant thereof. Preferably, the term “LRP1” refers to a protein that comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 5. Preferably it refers to the receptor that is described by the UNIPROT entry Q07954 (date 30thAugust 2024) or an isoform or variant thereof. LRP1 is also called apolipoprotein E receptor (APOER). Said receptor is known to bind to ApoE. Amongst others, the inventors use the binding interaction between the LRP1 receptor and ApoE for the targeted delivery of protein-coated fusogenic lipid particles.

[0059]

[0035] The terms “identical” or percent “identity”, as used herein in the context of two or more nucleic acid or protein / polypeptide sequences, refer to two or more sequences or subsequences that are the same or have (or have at least) a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, and more preferably at, or at least, about 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region - preferably over their full length sequences - , when compared and aligned for maximum correspondence over the comparison window or designated region), as measured using a sequence comparison algorithm, or by manual alignment and visual inspection (see, e.g., NCBI web site). In a particular embodiment, the percentage identity can be determined by the Blast searches; in particular for amino acid identity, those using BLASTP 2.2.28+ with the following parameters: Matrix: BLOSUM62; Gap Penalties: Existence: 11 , Extension: 1; Neighboring words threshold: 11 ; Window for multiple hits: 40.

[0060] Therefore, throughout this application, whenever the expression “ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an Apolipoprotein E”, “wherein the apolipoprotein is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an apolipoprotein” and such is used, this preferably include identities of at least 80%, 85%, 90%, 91 %, 92%, 93% or 94%, identity, and more preferably at, or at least, about 95%, 96%, 97%, 98%, 99% or higher identity such as 100% as defined above.

[0061]

[0036] In preferred embodiments, the protein-coated fusogenic lipid particles further comprises a Compound B as defined herein that is a neutrally charged lipid molecule.

[0062]

[0037] In preferred embodiments of the invention, the protein-coated fusogenic lipid particle comprises

[0063] (i) a Compound A that is a positively charged lipid molecule;

[0064] (ii) a Compound B that is a neutrally charged lipid molecule

[0065] (iii) a Compound C that is an aromatic molecule; wherein the protein- coated fusogenic lipid particle comprises a protein coating with a molar ApoEcontent of at least 30%; wherein the ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an Apolipoprotein E.

[0066]

[0038] Compound A is a positively charged lipid molecule. As a lipid, it is an amphipathic compound indicating that the molecule has both a polar (hydrophilic) and non-polar (hydrophobic section). Therefore, Compound A comprises a positively charged hydrophilic section and a hydrophobic section. The term “positively charged” in this context refers to a net positive charge at pH 7.4.. In hydrophilic solvents, such as aqueous systems, Compound A preferably forms colloidal particles, in which the hydrophilic section of compound A faces the surrounding solvent. As the positively charged hydrophilic section of the Compound A preferably faces the outside of the fusogenic particles of the invention, the fusogenic lipid particles of the invention are able to interact with the negatively charged cell membranes. The hydrophobic section of the amphipathic Compound A preferably faces in the interior of the fusogenic particle of the invention.

[0067]

[0039] In preferred embodiments of the invention, the hydrophobic section of Compound A comprises a C10-30 alkyl, a C10-30 alkenyl, and / or a C10-30 alkynyl group. Preferably, compound A comprises a C12-20 alkyl, a C12-20 alkenyl, and / or a C12-20 alkynyl group.

[0068]

[0040] The hydrophobic section of the Compound A preferably creates a hydrophobic environment in the protein-coated fusogenic lipid particle. This hydrophobic environment is advantageous, when encapsulating lipophilic drug molecules. Such drug molecules can be enclosed in this lipophilic environment to increase their availability in aqueous solvent systems and for transport by the protein-coated fusogenic lipid particle of the invention into target cells by the fusion mechanism of the protein-coated fusogenic lipid particle.

[0069]

[0041] Preferably the hydrophobic section of the compound A comprises one or more double bonds, such as in a C10-30 alkenyl group. Without wishing to be bound by theory, the presence of a double bond can increase a phase transition of the protein coated fusogenic lipid particles from a 2D bilayer phase into a 3D lipid phase under biologically relevant conditions (for example pH 7.4, 37°C and isosmotic conditions). During such a phase transition, lipid disorder significantly increases resulting in local membrane instabilities and fusion induction. Therefore, the presence of a double bond positively contributes to the fusion probability of the protein- coated fusogenic lipid particle of the invention with the cell membrane.

[0070]

[0042] In preferred embodiments of the invention, Compound A is selected from the group consisting of 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1 ,2-di-O-octadecenyl-3- trimethylammonium (DOTMA), Didodecyldimethylammonium (DDAB), and 1-[2-(oleoyloxy)ethyl]- 2-oleyl-3-(2-hydroxyethyl)imidazolinium (DOTIM). In particular preferred embodiments, the compound A is DOTAP.

[0071]

[0043] In preferred embodiments, the protein-coated fusogenic lipid particles further comprises a Compound B that is a neutrally charged lipid molecule. Individual atoms in the neutrally charged molecule can exhibit a formal charge however the term “neutrally charged” in this context indicates that the lipid molecule overall has a net neutral charge. As a lipid, Compound B is an amphipathic molecule indicating in the context of compound B that it has a both polar (hydrophilic) and non-polar (hydrophobic section). Preferably, Compound B comprises a hydrophilic section with no formal charge and a hydrophobic section.

[0072]

[0044] Compound B preferably functions as an optional helper lipid. Protein-coated fusogenic lipid particles according to the invention that comprise a Compound B have a greater stability compared to protein-coated fusogenic lipid particles without Compound B. The optional Compound B can increase the stability of the protein-coated fusogenic lipid particle of the invention by being integrated in the membrane of the protein-coated fusogenic lipid particle and counteracting the repelling forces between the molecules of the positively charged Compound A.

[0073]

[0045] In preferred embodiment, the Compound B is an inverted conical lipid. Without wishing to be bound by theory, using an inverted conical lipid increases lipid-bilayer instabilities preferably forming 3D lipid phases such as hexagonal or cubic. Such phases tend to release their energy excess through membrane fusion while the high-curvature 3D structures relax in 2D lipid bilayers. Therefore, when compound B is an inverted conical lipid, this increases the fusion probability of the protein-coated fusogenic-lipid particles of the invention.

[0074]

[0046] In preferred embodiments of the invention, the Compound B is a phospholipid. The advantages of Compound B being a phospholipid are based on a greater similarity of the protein- coated fusogenic lipid particle with the membrane of animal target cells, as described in the context of Compound A.

[0075]

[0047] In preferred embodiments of the invention, Compound B comprises a C10-30 alkyl, a C10-30 alkenyl, and / or a Cio-3oalkynyl group. More preferably compound B comprises a C12-20 alkyl, a C12- 20 alkenyl, and / or a C12-20 alkynyl group.

[0076]

[0048] Preferably the hydrophobic section of Compound B, comprises one or more double bonds, such as in a C10-30 alkenyl group. This induces an inverted conical molecular shape even more and due to insertion into the protein-coated fusogenic lipid particles of the invention, positively contributes to the fusion probability of the liposome with the cell membrane.

[0077]

[0049] In preferred embodiments of the invention, Compound B is a phosphatidylethanolamine.

[0078] In preferred embodiments of the invention, compound B is selected from the group consisting of phosphatidylethanolamine, phosphatidylcholine, 1 ,2-Dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 ,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine (DM PE), 1 ,2-dielaidoyl-sn-Glycero-3- phosphoethanolamin (DEPE), 1 ,2-diphytanol-sn-Glycero-3-phosphoethanolamine, 1 ,2- dilinoleoyl-sn-Glycero-3-phosphoethanolamine and 1 ,2-dioleoyl-sn-glycero-3-phosphatidylcholin (DOPC). In most preferred embodiments, Compound B is DOPE.

[0079]

[0050] The hydrophobic section of the Compound B preferably creates a hydrophobic environment within the protein-coated fusogenic lipid particle, which can be used to encapsulate lipophilic drugs. This enhances the bioavailability of these drugs that are usually not soluble in hydrophilic solvents.

[0080]

[0051] In preferred embodiments of the invention, Compound C is an aromatic molecule that comprises an aryl, heteroaryl, substituted aryl and / or substituted heteroaryl group.

[0081]

[0052] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, the aryl comprises one or more aromatic rings that contain 3 to 14 carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. The term “aryl” does not encompass fullerenes. Preferably, Compound C comprises a substituted aryl. The term "heteroaryl" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms (such as O, S, or N, preferably O and N). For example, a 3- to 14-membered heteroaryl encompasses a monocyclic heteroaryl (e.g., 5- or 6-membered), a bicyclic heteroaryl (e.g., 9- or 10-membered), and a tricyclic heteroaryl (e.g., 13- or 14- membered). Preferably, the term “heteroaryl” refers to an aromatic bicyclic ring system wherein one or two carbon atoms are replaced with the same or different heteroatoms of O, N, or S, preferably O or N. Preferably, the aryl, heteroaryl, substituted aryl and / or substituted heteroaryl group comprises two aromatic rings that are connected via a bridging Ci- alkylene, C2- alkenylene, or a bridging C2-3oalkynylene group. Preferably, the aryl, heteroaryl, substituted aryl and / or substituted heteroaryl group comprises two aromatic rings that are connected via a bridging Ci-yalkylene, C2-yalkenylene, or a bridging C2-?alkynylene group, more preferably a C2- yalkenylene group. Preferably, the aryl, heteroaryl, substituted aryl and / or substituted heteroaryl group comprises two aromatic rings that are connected via a bridging Ci-salkylene, C2- salkenylene, or a bridging C2-3 alkynylene group, most preferably a C2-3alkenylene group.

[0053] In this context "substituted aryl” or “substituted heteroaryl” means that one or more hydrogen atoms of the aryl group or heteroaryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen in the context of the "substituted aryl” or “substituted heteroaryl” is independently selected from the group consisting of halogen, -CN, - OR1, -(CH2)O-3COOR1, -SR1, -(CH2)O-3N(R1)2, alkyl, alkenyl, and alkynyl, wherein R1is independently selected from the group of -H, halogen, Ci-ealkyl, C2-6alkenyl, and C2-6alkynyl. Preferably, the substituent other than hydrogen in the context of the "substituted aryl” or “substituted heteroaryl” is selected form the group consisting of C1-30 alkyl, C2-3o alkenyl and or C2. 30 alkynyl.

[0082]

[0054] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 30, preferably 1 to 20 carbon atoms, more preferably 1 to 13 carbon atoms, such as 1 to 6 or 1 to 3 carbon atoms.

[0083]

[0055] The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Preferably, the alkenyl group comprises from 2 to 30 (e.g., 2 to 20, more preferably 2 to 13) carbon atoms. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration.

[0084]

[0056] The term "alkynyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. Preferably, the alkynyl group comprises from 2 to 30 (preferably 2 to 18, more preferably 2 to 12) carbon atoms.

[0085]

[0057] In preferred embodiments of the invention, the compound C comprises a delocalized it- electron system, preferably wherein the Compound C comprise a dye molecule, a vitamine and / or a polyphenol. In preferred embodiments of the invention, Compound C comprises a dye molecule and / or a polyphenol. Preferably the dye molecule is a fluorescent dye molecule. In particular preferred embodiments, the dye molecule is DiO (also known as DiOC18(3) and 3,3'- dioctadecyloxacarbocyanine perchlorate), DiD (also known as 1 ,1'-dioctadecyl-3,3,3',3'- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) or DiR (also known as DilCis(7) (1 ,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanin-lodid). Preferably the dye is DiO. By incorporating a dye molecule, preferably a fluorescent dye molecule, into the protein-coated fusogenic lipid particle of the invention, the protein-coated fusogenic lipid particle can monitored after administration to a subject, for example with light microscopy. This allows the quantification of intact protein-coated fusogenic lipid particles, therefore protein- coated fusogenic lipid particles that that have not yet been uptaken by target cells or otherwise been cleared from the subject. Furthermore, during the fusion process, aromatic dye molecules can be incorporated into the target cell membrane. By this, the uptake of the fusogenic particles into target cells, the location of target cells, and information on the state that the target cells are in can be acquired.

[0058] In particular preferred embodiments, the Compound C is a polyphenol and / or a derivative thereof. Preferably, the term “polyphenol” indicates an aromatic molecule that comprises one or more aromatic rings, wherein overall at least two hydrogen atoms of one or more aromatic rings are substituted with a hydroxy group. Preferably, the aromatic ring is a substituted aryl as defined above. Preferably, the polyphenol of the invention is selected from a polyphenol that is of plant origin, produced by a microorganism, or chemically synthesized.

[0086]

[0059] In preferred embodiments of the invention, the polyphenol is a stilbenoid, a flavonoid, a phenolic acid, a lignan or a derivative thereof, in particular wherein the derivative is an ester, ether (such as an alkylether), or a carboxylic acid derivative thereof. In particularly preferred embodiments of the invention, the polyphenol is a stilbenoid such as resveratrol. Preferably, the resveratrol is a trans-isomer of resveratrol (also known as trans-3, 5, 4'-Trihydroxystilben).

[0087]

[0060] In preferred embodiments of the invention, the Compound C is selected from the group consisting of resveratrol, curcumin, quercetin, dodecylgallat, xantohumol, 6-6-dihydroxyflavon and genistein. Preferably, the Compound C is selected from the group consisting of resveratrol, curcumin, quercetin, dodecylgallat and xantohumol.

[0088]

[0061] Polyphenols are known for their antioxidative, antihypertensive, immunomodulatory, antimicrobial, and antiviral and anticancer properties. There is a high interest for the usage of polyphenols in the treatment and prevention against many diseases, including diabetes, cancer, inflammation and degenerative diseases. However, a major hindrance is their general low aqueous solubility, which makes their clinical application difficult. In embodiments of the invention, in which Compound C comprises a polyphenol, this disadvantage is moot. In these embodiments, the aromatic polyphenol is encompassed by the protein-coated fusogenic lipid particle of the invention.

[0089]

[0062] In preferred embodiments of the invention, the polyphenol is comprised in the lipid membrane of the protein- coated fusogenic lipid particle. In preferred embodiments of the invention, the polyphenol is comprised in the hydrophilic center of the protein-coated fusogenic lipid particle. As mentioned above, polyphenols generally comprise a poor solubility in polar solvents such as aqueous solvents.

[0090]

[0063] The protein-coated fusogenic lipid particles of the invention can include a polyphenol, for example in the form of the aromatic Compound C. The polyphenol may be comprised in a lipid bilayer of the fusogenic lipid particles of the invention.

[0091]

[0064] In preferred embodiments of the invention the protein-coated fusogenic lipid particle, preferably a protein-coated fusogenic liposome, comprises a further therapeutically active compound D.

[0065] In these embodiments, the protein-coated fusogenic lipid particle of the invention can used for targeted drug delivery. Incorporating drug molecules in the protein-coated fusogenic lipid particles of the invention provides them with protection of degradation, a fusion-based uptake mechanism, and the concentration of further drug molecules, that would otherwise feature a poor solubility in aqueous solvent systems or blood can be increased.

[0092]

[0066] In preferred embodiments of the invention, the protein-coated fusogenic lipid particle comprises a lipid bilayer, preferably wherein particle is a liposome with a hydrophilic center or a nanodisc. In preferred embodiments of the invention, wherein the liposome is a unilamellar or a multilamellar liposome.

[0093]

[0067] In polar solvents, the protein- coated fusogenic lipid particles of the invention are in a dispersion state, preferably wherein the dispersion comprises the particles of the invention, wherein the particles of the invention in the form of a liposome or a nanodisc.

[0094]

[0068] In preferred embodiments of the invention, the protein-coated fusogenic lipid particle is in the shape of a nanodisc. A nanodisc in the context of the invention is a discoidal lipid bilayers. In this state, the Compound A (and optionally the Compound B) a forms a discoidal lipid bilayer wherein the hydrophilic section of the Compound A (and optionally of the Compound B) in the two layers faces away from the center of the bilayer, and wherein the hydrophobic section of the Compound A (and optionally of the Compound B) in the two layers faces towards each other. When the protein-coated fusogenic lipid particles are in the shape of a nanodisc, they preferably do not enclose a hydrophilic center. The amount of molecules (such as Compound A, Compound C, the optional Compound B) that are contained in the lipid bilayer section of the protein-coated fusogenic lipid particle is maximized. Furthermore, the amount of surface area of the protein- coated fusogenic lipid bilayer particle of the invention is increased compared to other shapes. Therefore, the amount of protein coating per protein-coated fusogenic lipid particle can be increased compared to other shapes.

[0095]

[0069] In preferred embodiments of the invention, the protein-coated fusogenic lipid particle is in the shape of a liposome. In a liposome, Compound A (and optionally Compound B) forms a closed spherical shape that comprises at least one lipid bilayer. Preferably, the lipid bilayer comprises two layers that contain Compound A (and optionally the Compound B), wherein the hydrophilic section of the Compound A (and optionally of the Compound B) in the two layers faces away from the center of the bilayer, and wherein the hydrophobic section of the Compound A (and optionally of the Compound B) in the two layers faces towards each other. Preferably, the liposome encloses a hydrophilic center. The hydrophilic center may be comprised by the hydrophilic sections of the compound A (and the optional compound B) that are face to the interior of the liposome and / or of by polar solvent molecules that are enclosed in the interior of the liposome. Embodiments, in which a protein-coated fusogenic lipid particle of the invention is in the shape of a liposome feature a great flexibility on their drug loading properties. Therapeutically active molecules, for example Compound A or other hydrophobic drugs, can be incorporated into the lipid-bilayer membrane of the protein-coated fusogenic lipid particle and / or hydrophilic drugs can be enclosed by the liposome. For example, hydrophilic drug molecules can dissolved the hydrophilic center of the lipid bilayer particles.

[0096]

[0070] In preferred embodiments of the invention, the protein-coated fusogenic liposome comprises a mean positive Zeta-potential, preferably a mean positive Zeta-potential of at least 20 mV, more preferably a mean positive Zeta-potential of at least 30 mV, most preferably a mean positive Zeta-potential of at least 40 mV. In preferred embodiments of the invention, the protein- coated fusogenic lipid particle comprises a mean positive Zeta-potential of 20 mV to 70 mV, preferably a mean positive Zeta- potential of 20 mV to 60 mV, most preferably a mean positive Zeta-potential of 20 mV to 50 mV. Preferably, these zeta- potential values are obtained, when the protein-coated fusogenic lipid particles are measured in an Zetasizer Pro (Malvern Panalytical Ltd, UK) in an aqueous 20 mM HEPES buffer at pH = 7.4 with an assumed refractive index of the particle of 1.43. The protein-coated fusogenic lipid particles of the invention comprise positively amphipathic molecules A. The positive charge of the amphipathic molecules is related to a positive zeta potential, that the particles exhibit under the abovementioned conditions. Particles with a higher absolute zeta- potential are less prone to aggregation under fluctuating pH conditions and can consequently easier be stored and formulated. Furthermore, a positive zeta- potential is an indicator that the particles of the invention are positively charged and retain their fusogenic properties.

[0097]

[0071] In preferred embodiments of the invention, the protein-coated fusogenic lipid particle comprises a mean hydrodynamic diameter of 50 to 600 nm, more preferably of 50 to 350 nm, more preferably of 50 to 250 nm. In particularly preferred embodiments, the protein-coated fusogenic lipid particles comprise a mean hydrodynamic diameter 75 to 150 nm. Preferably, these hydrodynamic particle diameters refer to intensity-based particle size distributions obtained by dynamic light scattering, when the protein-coated fusogenic lipid particles are measured in an Zetasizer Pro (Malvern Panalytical Ltd, UK) in an aqueous 20 mM HEPES buffer at pH = 7.4, with an assumed refractive index of the particle of 1.43. The inventors found that the particles of the invention are better to inject with decreasing hydrodynamic diameter.

[0098]

[0072] In preferred embodiments of the invention, the molar ratio RAB of the compounds A:B in the protein-coated fusogenic lipid particle is between 1 :0 to 1 :1 , preferably between 1 :0.5 and 1 :0.8, most preferably around 1 : 1.

[0099]

[0073] In preferred embodiments of the invention, the wherein compound C comprises a fraction of 2 mol% to 30 mol%, more preferably of 4% to 20 mol%, more preferably of 4% to 10 mol% of the protein-coated fusogenic lipid particle, most preferably of 4% to 7% of the protein-coated fusogenic lipid particle.

[0100]

[0074] In preferred embodiments of the invention, the ratio RABC of the compounds A:B:C in the protein-coated fusogenic lipid particle is 2(±0.5):0:(0.05 to 5), preferably 2(±0.2):0:(0.05 to 2.5), preferably 2(±0.2):0:(0.1 to 1), most preferably 2(±0.2):0:(0.2 to 0.6).

[0101]

[0075] In preferred embodiments of the invention, the ratio RABC of the compounds A:B:C in the protein-coated fusogenic lipid particle 1(±0.5):1(±0.5):(0.05 to 5), preferably 1 (±0.2):1 (±0.2):(0.05 to 2.5), preferably 1 (±0.2):1 (±0.2):(0.1 to 1), most preferably 1(±0.2):1 (±0.2):(0.2 to 0.6).

[0102]

[0076] In the context of the ratio RABC described above, the amount of Compound C refers to the overall amount of Compound C. For example, in this context, if a fusogenic lipid particle of the invention comprises a dye molecule and a polyphenol, the amount of Compound C is the sum of the dye molecule and the polyphenol.

[0103]

[0077] In preferred embodiments of the invention, the lipid / ApoE molar ratio of the protein-coated fusogenic lipid particle is 50,000 / 1 to 500 / 1 , preferably 20000 / 1 to 500 / 1 , preferably 10000 / 1 to 1000 / 1 , preferably around 6000:1 to 4000:1 , most preferably about 5000 / 1.

[0104]

[0078] In preferred embodiments of the invention, the lipid / ApoE molar ratio of the protein-coated fusogenic lipid particle is 20000 / 1 to 500 / 1 , preferably 10000 / 1 to 1000 / 1 , preferably around 6000:1 to 4000:1 , most preferably about 5000 / 1. In preferred embodiments of the invention, the molar lipid / ApoE ratio of the protein coated protein-coated fusogenic lipid particle is at least 4000:1 , preferably around 6000:1 to 4000:1. The inventors found that this ratio leads to protein- coated lipid particles with increased homogeneity compared to lower lipid / ApoE ratios.

[0105]

[0079] In a third aspect, the invention pertains to a method of manufacturing a protein-coated fusogenic lipid particle according to any other aspect of the invention, the method comprising:

[0106] (i) Mixing Compound A and Compound C as defined herein in an aqueous or organic solvent;

[0107] (ii) Drying the mixture of step (i);

[0108] (iii) Redispersing the dried mixture obtained in step (ii) in a polar solvent such as ethanol and subsequently quickly adding, more preferably injecting in, an aqueous buffer to obtain a dispersion containing a fusogenic lipid particle;

[0109] (iv) Mixing the dispersion containing the fusogenic lipid particle obtained in step (iii) with a dispersion comprising an apolipoprotein as defined herein.

[0110]

[0080] In preferred embodiments of the third aspect, in step (i) the lipid compounds are mixed in an organic solvent, such as chloroform, methanol, ethanol or a mixture thereof. This allows to homogeneously dissolve the lipid components in step (i).

[0081] In preferred embodiments of the third aspect, in step (iii), an aqueous HEPES buffer (preferably 10-100 mM) is injected to the polar solvent to obtain the dispersion containing the lipid particle.

[0111]

[0082] In preferred embodiments of the third aspect, in step (iv), the dispersion comprising the apolipoprotein is an aqueous buffer containing sodium-bicarbonate and / or HEPES. More preferably, the in step (iv), the dispersion comprising the apolipoprotein is an aqueous buffer containing 0,5-50 mM sodium-bicarbonate and 15-25 mM HEPES.

[0112]

[0083] In a preferred embodiment, the method of manufacturing a protein-coated fusogenic lipid particle comprises:

[0113] (i) Mixing Compound A, and Compound C as defined herein in an organic solvent selected from chloroform, methanol, ethanol or a mixture of thereof;

[0114] (ii) Drying the mixture of step (i);

[0115] (iii) Redispersing the dried mixture obtained in step (ii) in a polar solvent (such as ethanol) and subsequently quickly adding, more preferably injecting in, an aqueous HEPES buffer to obtain a dispersion containing a fusogenic lipid particle;

[0116] (iv) Mixing the dispersion containing the fusogenic lipid particle obtained in step (iii) with a dispersion comprising an apolipoprotein as defined herein, wherein the dispersion comprising the apolipoprotein is an aqueous buffer containing sodium-bicarbonate and HEPES.

[0117]

[0084] In preferred embodiments of the third aspect, step (i) includes mixing the optional Compound B as defined according to any other aspect of the invention with Compound A and Compound C.

[0118]

[0085] In preferred embodiments, the method comprises a step (v) wherein the fusogenic lipid particles are incubated with apolipoprotein between 4°C and 25°C for 10-60 min under gentle mixing.

[0119]

[0086] In preferred embodiments of the third aspect, in step (ii) a film of the fusogenic lipid particles is obtained. The film obtained by step (ii) comprises fusogenic lipid particle without a protein coating.

[0120]

[0087] In preferred embodiments of the third aspect, the apolipoprotein is ApoE as defined in any of the other aspects herein. Preferably, in step (iv) the lipid / ApoE ratio during mixing is 20000 / 1 to 500 / 1 , preferably 10000 / 1 to 1000 / 1 , preferably around 6000:1 to 4000:1 , most preferably about 5000 / 1. In preferred embodiments of the invention, the molar lipid / ApoE ratio during mixing is at least 4000:1 , preferably around 6000:1 to 4000:1. The inventors found that this ratio leads to protein-coated lipid particles with increased homogeneity compared to lower lipid / ApoE ratios.

[0088] In a fourth aspect, the invention pertains to a therapeutic composition comprising a protein-coated fusogenic lipid particle of any other aspect of the invention and a pharmaceutically acceptable carrier or excipient.

[0121]

[0089] Preferably, the therapeutic composition is formulated in a liquid form, for example in the form of a dispersion. Preferably, the therapeutic composition comprises the protein- coated fusogenic lipid particles of the invention, wherein compound C is a polyphenol, such as resveratrol. Preferably, the pharmaceutically acceptable carrier or excipient is selected from of the group consisting of a) a diluent, e.g., purified water or an aqueous solution such as saline, an aqueous dextrose solution, and / or a solution containing glycerol. Preferably, the pharmaceutical composition is formulated for injection.

[0122]

[0090] In a fifth aspect, the invention pertains to a therapeutic composition use in the treatment of a condition or disease.

[0123]

[0091] The term “treatment” or “treating” with regard to a subject, refers to improving at least one symptom of the subject’s condition disorder. Treating includes curing, improving, or at least partially ameliorating the condition or disorder.

[0124]

[0092] Administration of the fusogenic particles of the invention, in particular wherein compound C is a polyphenol such as resveratrol induces angiogenesis, resulting in increased blood supply, cellular recovery and reduced cellular stress. Preferably, Compound C is resveratrol.

[0125]

[0093] In preferred embodiments of the invention, the disease is associated with the cardiovascular system, preferably its structure and / or function. In preferred embodiments of the invention, the disease is a neurological disorder of the central nervous system, preferably wherein the disease associated with the brain. The protein-coated fusogenic lipid particles of the invention, particular the fusogenic lipid particles of the invention that comprise ApoE in their protein corona, may be used to target a certain organ, tissue or cell-type such as the brain, liver, lung and / or kidney. In particular the protein-coated fusogenic lipid particles of the invention that comprise ApoE in their protein corona can be used to target endothelial cells such as endothelial cells of the brain.

[0126]

[0094] In preferred embodiments of the invention, the disease is a neurodegenerative disease, preferably wherein the disease is an age-related neurodegenerative disease.

[0127]

[0095] In preferred embodiments of the invention, the disease is associated with a decreased cognitive function. As demonstrated herein in Radial Arm Water Maze experiments on mice, a treatment with the protein-coated fusogenic lipid particles of the invention, in particular the protein- coated fusogenic lipid particles of the invention that comprise ApoE in their protein coating, restores cognitive function.

[0128]

[0096] In preferred embodiments of the invention, the neurodegenerative disease is associated with vascular dysfunction in the brain.

[0129]

[0097] In preferred embodiments of the invention, wherein the neurodegenerative disease is associated with a breakdown of the blood brain barrier (BBB) and / or impaired neurovascular coupling (NVC).

[0130]

[0098] In preferred embodiments of the invention, the disease is associated with neuroinflammation and / or oxidative stress.

[0131]

[0099] In preferred embodiments of the invention, the treatment is a protection of cells against oxidative stress, preferably a protection of endothelial cells against oxidative stress.

[0132]

[0100] In preferred embodiment of the invention, the treatment comprises one or more of:

[0133] (i) a restoration and / or an improvement of the cerebromicrovascular endothelial function and

[0134] (ii) an improvement of vascular barrier properties and / or a restoration of the integrity of the BBB;

[0135] (iii) an improvement of tissue capillarization; and

[0136] (iv) an improvement of vascular dilatation

[0137]

[0101] According to the ‘neurovascular hypothesis’ of AD contributing factors to the cognitive decline and dementia in AD are cerebrovascular dysfunction such as a blood-brain barrier breakdown, microhaemorrhages and cerebral blood flow deficits. With the protein-coated fusogenic lipid particles of the invention, these factors can be alleviated. In preferred embodiments of the invention, the disease is vascular dementia (preferably age-related), the effects of a stroke and brain hemorrhage and / or Alzheimer’s disease.

[0138]

[0102] In preferred embodiments of the invention, treatment comprises an intra-arterial, intraarticular, intracardial, intracutaneous, intralumbar, intramuscular, intraperitoneal, intrathecal, intravenous, retroorbital, subcutaneous and / or intravitreal administration. Preferably, the treatment comprises a retroorbital administration of the therapeutic composition.

[0139]

[0103] In preferred embodiment of the invention, wherein the subject is a mammal such as a mouse, preferably a human.

[0140]

[0104] In a sixth aspect, the invention pertains to a method for the targeted delivery of a fusogenic lipid particle, wherein the method comprises an in-vitro step of pre-coating a non-coated fusogenic lipid particle with a serum protein, wherein the non-coated fusogenic lipid particle comprises (i) a compound A that is a positively charged lipid molecule according to any aspect herein;

[0141] (ii) a compound C that is an aromatic molecule according to any aspect herein.

[0142]

[0105] In this context, the term “pre-coating” is to indicate that the coating takes place, before the protein-coated fusogenic lipid particle is administered to a subject.

[0143]

[0106] The term “non-coated” is meant to indicated that the fusogenic lipid particle does not yet comprise a protein coating.

[0144]

[0107] In preferred embodiments of the method for the targeted delivery of a fusogenic lipid particle, the non-fusogenic lipid particle further comprises a Compound B that is a neutrally charged lipid molecule according to any aspect herein.

[0145]

[0108] In preferred embodiments of the method for the targeted delivery of a fusogenic lipid particle, the serum protein is an apolipoprotein, preferably ApoE. As indicated above, the inventors found that the protein corona of fusogenic lipid particles subjected to plasma are coated with apolipoproteins. In particular, Apolipoprotein E has a strong affinity for fusogenic lipid particles. The inventors found that by coating a fusogenic lipid particle with a serum protein such as an apolipoprotein, they are able to provide the fusogenic lipid particle with a targeting mechanism that targets organs, tissues and / or cells depending on interaction (such as a binding) of the serum protein with the organs, tissues and / or cell.

[0146]

[0109] In preferred embodiments of the method for the targeted delivery of a fusogenic lipid particle, the in-vitro step of pre-coating comprises mixing a dispersion containing a fusogenic lipid particle with a dispersion comprising the serum protein, preferably wherein the dispersion comprising the serum protein is an aqueous buffer containing sodium-bicarbonate and / or HEPES. In this context, the aqueous buffer is not blood plasma or blood serum.

[0147]

[0110] Generally, the step of pre-coating the non-coated fusogenic lipid particle with a serum protein, does not include subjecting the non-coated fusogenic lipid particle to blood plasma or blood serum. Such as step would result in a PC formation that contains a selection of proteins that depend on the blood plasma or blood serum composition and would not result in a targeting as defined herein.

[0148]

[0111] In preferred embodiments of the method for the targeted delivery of a fusogenic lipid particle, the resulting protein- coated fusogenic lipid particle comprises a protein-coating with a molar Apo-content at least 30%, more preferably of at least 50%, even more preferably of at least 70%, even more preferably of at least 90%. Preferably, the protein- coated fusogenic lipid particle of the invention comprises a protein coating that consists of an apolipoprotein. In particularly preferred embodiments, the resulting protein-coated fusogenic lipid particle comprises a proteincoating with a molar Apo-content at least 50%. With a higher amount of apolipoprotein in the protein coating of the lipid particles of the invention, the stability of the protein corona and the targeting of the protein-coated fusogenic lipid particles is increased. Preferably, the resulting protein-coated fusogenic lipid particle is a protein-coated fusogenic lipid particle according to any other aspect herein, in particular the first aspect.

[0149]

[0112] In preferred embodiments of the method for the targeted delivery of a fusogenic lipid particle, the resulting protein- coated fusogenic lipid particle comprises a protein-coating with a molar ApoE-content of at least 10%, more preferably of at least 30%, more preferably of at least 50%, more preferably of at least at least 70%, more preferably of at least at least 90%. Preferably, the protein-coated fusogenic lipid particle of the invention comprises a protein coating that consists of ApoE. In particularly preferred embodiments, the resulting protein-coated fusogenic lipid particle comprises a protein-coating with a molar ApoE-content of at least 30%. With a higher ApoE content in the protein-coating, the stability of the protein corona is further increased and / or targeting of cells is increased. Preferably, the resulting protein-coated fusogenic lipid particle is a protein-coated fusogenic lipid particle according to any other aspect herein, in particular the second aspect.

[0150]

[0113] In preferred embodiments of the method for the targeted delivery of a fusogenic lipid particle, the method targets the brain, liver, lung and / or kidney, in particular an endothelial cell therein. Preferably, the method targets the brain, more preferably brain endothelial cells. In preferred embodiments of the method for the targeted delivery of a fusogenic lipid particle, the serum protein is an apoplipoprotein, preferably an ApoE as defined herein. By precoating the non-coated fusogenic lipid particles with an ApoE coating, a targeting of the brain, liver, lung and / or kidney (in particular of the brain endothelial cells) can be achieved.

[0151]

[0114] In preferred embodiments of the method for the targeted delivery of a fusogenic lipid particle, the targeted delivery targets a cell that comprises an LRP1-receptor on the cell-surface.

[0152]

[0115] In preferred embodiments of the method for the targeted delivery of a fusogenic lipid particle, the targeted delivery targets is a cell of the blood brain barrier and / or a cerebrovascular endothelial cell.

[0116] In a seventh aspect, the invention pertains to a method for targeted drug delivery, wherein the method comprises using a protein-coated fusogenic lipid particle as a drug-delivery vehicle, wherein the non-coated fusogenic lipid particle comprises

[0153] (i) a Compound A that is a positively charged lipid molecule according to any aspect herein;

[0154] (ii) a Compound C that is an aromatic molecule according to any aspect herein

[0155]

[0117] wherein the protein-coated fusogenic lipid particle comprises a protein coating that comprises a serum protein and wherein the protein-coated fusogenic lipid particle comprises a drug that is to be delivered, preferably wherein the protein-coated fusogenic lipid particle is a protein coated fusogenic lipid particle according to any other aspect of the invention.

[0156]

[0118] Preferably, the drug that is to be delivered is Compound C and / or Compound D, as defined in any other aspects herein. This drug delivery system is particularly advantageous, as Compound C, which is an aromatic molecule, is comprised by the lipid membrane of the protein-coated fusogenic lipid particle. In the case that Compound C is the drug to be delivered, for example a polyphenol, the method offers the advantage of being a targeted drug delivery system that does not require further loading of a drug molecule. In such a case, the protein-coated fusogenic lipid particles themselves are an be used as a therapeutic.

[0157]

[0119] In preferred embodiments of the method for targeted drug delivery, the protein-coated lipid particle comprises a protein-coating with a molar ApoE-content of at least 10%; preferably at least 30%, more preferably at least 50%, even more preferably at least 70%, most preferably at least 90%;

[0158]

[0120] wherein the ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an Apolipoprotein E. In particularly preferred embodiments of the method for targeted drug delivery, the protein-coated lipid particle comprises a proteincoating with a molar ApoE-content of at least 30%, more preferably at least 50%, even more preferably at least 70%, most preferably at least 90%;

[0159]

[0121] wherein the ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an Apolipoprotein E. Apolipoprotein E has a strong affinity for fusogenic lipid particles. For example, by using a protein-coated fusogenic lipid particle with such an ApoE-content, the method can be used for targeting organs, tissues and / or cells that show a preferential uptake of ApoE.

[0122] In particular preferred embodiments of the method for targeted drug delivery, the protein- coated lipid particle comprises a protein-coating than consists of ApoE

[0160]

[0123] wherein the ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an Apolipoprotein E.

[0161]

[0124] In preferred embodiments of the method for targeted drug delivery, the method targets the brain, liver, lung and / or kidney, in particular an endothelial cell therein. Preferably, the method targets the brain, more preferably brain endothelial cells.

[0162]

[0125] In preferred embodiments of the method for targeted drug delivery, the protein-coated lipid particle comprises a protein-coating with a molar Apo-content of at least 30%, more preferably of at least 50%, even more preferably of at least 70%, even more preferably of at least 90% In preferred embodiments of the method for targeted drug delivery, the protein-coated lipid particle comprises a protein-coating that consists of an apolipoprotein. In particularly preferred embodiments of the method for targeted drug delivery, the protein-coated lipid particle comprises a protein-coating with a molar Apo-content of at least 50%. By using such a protein-coated fusogenic lipid particle, the method can be used for targeting organs, tissues and / or cell depending on the interaction (such as a binding) of the Apo protein with the organs, tissues and / or cell.

[0163]

[0126] In preferred embodiments of the method for targeted drug delivery, the targeted delivery targets a cell that comprises an LRP1-receptor on the cell-surface.

[0164]

[0127] In preferred embodiments of the method for targeted drug delivery, the targeted delivery targets a cell of the blood brain barrier and / or a cerebrovascular endothelial cell.

[0165]

[0128] In preferred embodiments of the method for targeted drug delivery, the drug is delivered to the target cell, preferably to the membrane and / or the cytoplasm of the target cell, more preferably, wherein the drug does not cross the BBB.

[0166]

[0129] The terms “identical” or percent “identity”, as used herein in the context of two or more nucleic acid or protein / polypeptide sequences, refer to two or more sequences or subsequences that are the same or have (or have at least) a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, and more preferably at, or at least, about 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region - preferably over their full length sequences - , when compared and aligned for maximum correspondence over the comparison window or designated region), as measured using a sequence comparison algorithm, or by manual alignment and visual inspection (see, e.g., NCBI web site). In a particular embodiment, the percentage identity can be determined by the Blast searches; in particular for amino acid identity, those using BLASTP 2.2.28+ with the following parameters: Matrix: BLOSUM62; Gap Penalties: Existence: 11 , Extension: 1; Neighboring words threshold: 11 ; Window for multiple hits: 40.

[0167] The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0168]

[0130] As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[0169]

[0131] It is to be understood that application of the teachings of the present invention to a specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.

[0170]

[0132] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0171]

[0133] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

[0172] BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES

[0134] The figures show:

[0173]

[0135] Figure 1 : Size and zeta potential distributions. (A) Size and (B) zeta potential distributions of FL / RSV with and without ApoE targeting motive directly after preparation. Zetapotential and hydrodynamic radius values are obtained, from coated and non-coated fusogenic liposomes when measured in an Zetasizer Nano ZS (Malvern Panalytical Ltd, UK) in an aqueous 20 mM HEPES buffer at pH = 7.4. (C) Percentage of ApoE retained by filtration at different lipid / ApoE ratios between 10000 / 1 and 1000 / 1 (mol / mol). Protein samples with identical protein content but without lipid nanoparticles were used as a reference. The protein retention in the presence of lipids indicates complete ApoE binding in FL / ApoE particles for all three lipid / ApoE ratios. (C) Size and (D) zeta potential distributions of FL / RSV particles coated with ApoE. (E) Encapsulation efficiency of resveratrol of fusogenic lipid-particles prior to the coating with Apolipoproteins. (F) Encapsulation efficiency of resveratrol during the synthesis of the Apo-coated fusogenic lipid particles of the invention. (G) Cryo-TEM micrographs of the fusogenic particles of the invention and particle diameters as determined by Cryo-TEM. Scalebars correspond to 100 nm.

[0174]

[0136] Figure 2: Lipid-bound ApoE proportion determined during ApoE-coating of FL via ELISA- assay. Over the whole tested range, no residual ApoE could be found in the binding solution. The ratio of lipid bound ApoE compared of the weighed-in ApoE was 100%.

[0175]

[0137] Figure 3. Proof of the ApoE presence on the cellular membrane surface of hCMEC. Cells have been treated with either FL / RSV without ApoE or with different amount of ApoE-coating on the surface (L / ApoE=5000 / 1 and 1000 / 1 mol / mol). As control samples cells without any treatment (control) and cells incubated with ApoE solution at 1 pg / ml concentration (ApoE) have been used. Particle cellular uptake has been identified in all cases as membrane fusion based on the homogenously distributed DiR signal (red) in the cellular plasma membrane. The presence of ApoE protein was proven by CoraLite Plus 488-conjugated ApoE antibody (green dotted signal). Scale bars, 50 pm, except of the lower right, 10 pm.

[0176]

[0138] Figure 4: Proof of membrane fusion as cellular uptake process. hCMEC cells were treated with RSV-loaded FLs complexes with different amount of ApoE. Lipid / protein molar ratio was varied between 10,000 / 1 and 1000 / 1 mol / mol. FL contained the fluorescent dye DiO for liposomal visualization during uptake processes. In all cases, upon incubation, homogenous (green) fluorescent signal appeared in the cellular membrane of hCMEC cells. Scale bar, 50 pm, applied to all.

[0177]

[0139] Figure 5: Proof of RSV protection upon incubation with ApoE / FL / RSV. hCMEC cells were incubated with 50 pM of cumine hydroperoxide solution for 1 h to induce cellular oxidative stress reactions. After stress application, cells were treated either with and without RSV containing ApoE / FL solution. However, all particles were internalized via membrane fusion by hCMECs, only the RSV-containing liposomes were able to reduce cells stress

[0178]

[0140] Figure 6: Monitoring of resveratrol-loaded liposomal uptake in the rodent cerebral microvasculature. Cationic liposomes (EL), fusogenic liposomes (FL) and ApoE-modified FL (APOE) were retro-orbitally injected in mice and liposomal uptake was monitored by two-photon microscopy (A). The total fluorescent intensity of DiO, incorporated into the liposomes was plotted vs. time as a significant characteristic for liposomal accumulation in the cerebral blood capillaries.

[0179]

[0141] Figure 7: Fluorescence signal distribution of ApoE-FL in different organs of C57BI6 mice. ApoE-FL particle (intense green) and endomucin (red) endothelial staining frequently overlap (yellow) indicating an overall vascular staining. Cumulative DiO signal analysis in different organs shows that ApoE-FL could be detected in the brain, kidney, liver and lung. Overall, ApoE-FL is primarily directed to the brain when compared to other organs e.g. heart, kidney, liver, lung, or skeletal muscles.

[0180]

[0142] Figure 8: FL / RSV and ApoE-FL / RSV significantly improves BBB integrity in aged mouse brain. Two-photon microscopy-based measurement of microvascular permeability to fluorescent tracers in brains of young control and aged BL6 mice that received liposomal treatment of FL / RSV or ApoE-FL / RSV, or orally administrated RSV (RSV per OS) (for 1 month). A) Representative intensity maps of the mouse brain during microvascular permeability measurement. Treatment with FL-RSV and ApoE-FL / RSV notably reduces extravascular tracer intensities in maximum projection images, indicating a decrease in vascular permeability. Scale bar represents 100 pm. B-C) Quantification of relative permeability changes in aged mice treated with FL / RSV, and ApoE-FL / RSV for 4 days (B) and 1 month (C).

[0181]

[0143] Figure 9: ApoE-FL / RSV treatments effect on learning and memory functions of mice. Radial Arm Water Maze (RAWM) study was used to characterize the repeated treatment of ApoE- FL / RSV compared to orally administrated RSV, as well as young and aged controls. The combined error rate was computed by adding one error for each incorrect arm entry plus an error for every 15 seconds of inactivity. Data are presented as mean±SEM (n = 10-15 per group). Statistical significance indicated by *p<0.05, **p<0.01 , ***p<0.001 using repeated measure AN OVA.

[0182]

[0144] The sequences show:

[0183] EXAMPLES

[0184]

[0145] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0185]

[0146] The examples show:

[0186]

[0147] Example 1 : Preparation of resveratrol-loaded fusogenic lipid nanoparticle with human ApoE targeting.

[0187]

[0148] The phospholipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, AL, USA).

[0188]

[0149] The fluorescent dyes T1-dioctadecyl-3,3’,3,3’-tetramethylindotricarbocyanine iodide (DiR) and 3,3' octadecyloxacarbocyanine perchlorate (DiO) were purchased from ThermoFisher (Waltham, MA, USA). The polyphenol resveratrol (RSV) was acquired from Merck (Darmstadt, Germany). Phospholipids (10 mg / mL in chloroform) and fluorescent dye (1 mg / mL in chloroform) were mixed in a DOPE / DOTAP / dye ratio of 1 / 1 / 0.1 (mol / mol).

[0189]

[0150] For polyphenol loading, the mixture was supplemented with RSV (5 mg / mL in ethanol), and the dye amount was reduced by half. The corresponding DOPE / DOTAP / dye / RSV ratio was 1 / 1 / 0.05 / 0.4 (mol / mol).

[0190]

[0151] Generation of fusogenic liposomes: All components were mixed vigorously and dried under reduced pressure. The corresponding lipid film was redispersed first in low amount of ethanol (10-20 pL) and injected subsequently in 20 mM 4-(2-hydroxyethyl)-1 -piperazineethane- sulfonic acid (HEPES) buffer to obtain RSV-loaded fusogenic liposomes (FL / RSV) at a lipid concentration of 2 mg / mL.

[0152] Protein-coating with ApoE: Human recombinant apolipoprotein E (ThermoScientific Inc.) was redispersed in 10 mM sodium-bicarbonate buffer at 1 mg / ml concentration and diluted in 20 mM Hepes buffer to 0.1 mg / ml concentration. ApoE solution was added to the FL / RSV suspension reaching the lipid / protein molar ratios from 10,000 / 1 to 1000 / 1 mol / mol. Samples were homogenized by pipetting up and down, as well as careful vortexing.

[0191]

[0153] The formed particles had an average size of 105 nm (PDI 0.22) and a zeta potential of 52 mV without ApoE surface coupling. In comparison, ApoE-loaded liposomal size increased to 120 nm (PDI 0.20) while zeta potential decreased to 40 mV, proving successful protein coupling (Figure 1A and B). Particle are stable during storage at 4°C at least one week.

[0192]

[0154] Further experiments were carried out similar to previous FL preparation. In further experiments, fusogenic liposomes were manufactured according to protocol above with a lipid(DOPE, DOTAP) I RSV ratio variation between 2 / 0.2 and 2 / 0.8 mol / mol. Subsequently, the particles were coated with ApoE as described above with a lipid / ApoE ratio of 5000 / 1 mol / mol. The resulting ApoE-coated particles were characterized via dynamic light scattering. The determined hydrodynamic diameters and zeta potentials are shown in Figure 1C and D. Loading of RSV change significantly neither the hydrodynamic diameter nor the polydispersity (PDI) nor the zeta potential of the particles.

[0193]

[0155] The inventors further determined the encapsulation efficiency of resveratrol in non-coated fusogenic lipid particles that were manufactured according to protocol above with a lipid(DOPE, DOTAP) I RSV ratio variation between 2 / 0.1 and 2 / 0.2 mol / mol, before they are coated with ApoE. Encapsulation efficiency of RSV was determined from the initial weigh-in of resveratrol and the remaining resveratrol in the synthesis solution, after separation of the particles of the invention. Notably, with increasing RSV, the fraction of RSV included in the particle increased. (Figure 1E)

[0194]

[0156] Similarly, they determined the resveratrol content of protein-coated fusogenic lipid particles of the invention that were manufactured according to the protocol above with a lipid(DOPE, DOTAP) / RSV ratio of 2 / 0.4 mol / mol, and varying lipid / ApoE ratios. Encapsulation efficiency of RSV was determined from the initial weigh-in of resveratrol and the remaining resveratrol in the synthesis solution, after separation of the particles of the invention, the inventors determined an encapsulation efficiency of around 90%, regardless of protein content. (Figure 1 F)

[0195]

[0157] The inventors examined the shape of fusogenic lipid particles that were manufactured according to the protocol above with a lipid(DOPE, DOTAP) I RSV ratio of 2 / 0.4 mol / mol, and varying lipid / ApoE ratios (no ApoE, 10000 / 1 and 5000 / 1) by Cryo-TEM (see Figure 1G). These Cryo-TEM micrographs indicate a liposome shape of the particles.

[0196]

[0158] Protein binding quantification: Resveratrol loaded FLs

[0197] (DOTAP / DOPE / DiO / RSV=1 / 1 / 0.05 / 0.4 mol / mol) with varying lipid / ApoE ratios were prepared as described above. 25 pL of each sample stock (2 mg / mL) were diluted 1 :4 (v / v) with 20 mM HEPES buffer and transferred into Nanosep® centricon tubes with a MWCO of 100 kDa. The samples were centrifuged for 30 min at 5000 ref and 4 °C until the whole solution went through the filter unit. The resulting filtrate was diluted 1 :4 (v / v) and tested for unbound ApoE using a human ApoE ELISA kit from ThermoFisher (Waltham, MA, USA). The assay was performed according to the manufacturer's manual. Absorbance measurements at 450 nm were conducted on an Infinite M1000 Pro platereader from Tecan (Maennedorf, Switzerland). For all tested concentrations, a complete binding of the ApoE to the FLs was determined (Figure 2)

[0198]

[0159] Example 2: Determination of ApoE binding affinity to fusogenic liposomes; ApoE shows a strong binding affinity to fusogenic lipid particles.

[0199]

[0160] Fluorescence quenching measurements were carried out on a fluorescence spectrometer, Fluorolog-3 from HORIBA Jobin Yvon (Bensheim, Germany; Kyoto, Japan) and were evaluated using the FluorEssence software (Bensheim, Germany; Kyoto, Japan). Samples were excited at 280 nm and fluorescence spectra were recorded from 300 to 500 nm using a photo multiplier. The signals were measured directly with an integration time of 0.1 s. Excitation and emission slits were set to 1.6 and 1.6 nm, respectively.

[0200]

[0161] 400 pL of the ApoE solution at a concentration of 1 pM were transferred into the measurement cuvette and homogenized for 5 min at RT prior to the measurement. Subsequently, 10 pL of a 0.1 mg / mL non-protein coated FL stock (prepared as described in Example 1 , with a DOPE / DOTAP ratio of 1 :1 mol / mol) were added to the protein solution. Sample was homogenized for 5 minutes and measured. The process was repeated ten times. For baseline determination, the cuvette was filled with 400 pL of the respective medium. It was homogenised for 5 minutes using magnetic stirring at room temperature and the fluorescence spectrum was recorded. Subsequently, 10 pL of the corresponding liposome formulation were pipetted into the cuvette, homogenised for 5 min, and measured again. This step was repeated ten times. Fluorescence intensity data was plotted vs. liposomal concentration and the slope, the Stern- Volmer constant (Ksv), as a characteristic metric for the molecular binding affinity, was determined. ApoE exhibited a high binding affinity to the fusogenic lipid particles of Ksv = 29 x 10-31 / M ± 10 x 10-31 / M.

[0201]

[0162] Resveratrol loaded FLs (DOTAP / DOPE / DiO / RSV=1 / 1 / 0.05 / 0.4 mol / mol) with varying lipid / ApoE ratios were prepared. 25 pL of each sample stock (2 mg / mL) were diluted 1 :4 (v / v) with 20 mM HEPES buffer and transferred into Nanosep® centricon tubes with a MWCO of 100 kDa. The samples were centrifuged for 30 min at 5000 ref and 4 °C until the whole solution went through the filter unit. The resulting filtrate was diluted 1 :4 (v / v) and tested for unbound ApoE using a human ApoE ELISA kit from ThermoFisher (Waltham, MA, USA). The assay was performed according to the manufacturer's manual. Absorbance measurements at 450 nm were conducted on an Infinite M1000 Pro platereader from Tecan (Maennedorf, Switzerland). The device was controlled with version 3.9.1.0 of Tecan 's i-control software.

[0202]

[0163] Example 3: Proof of ApoE attachment on the cellular surface of hCMEC due to ApoE-FL / RSV treatment

[0203]

[0164] In these experiments, HCMEC / D3 cells were treated with resveratrol containing fusogenic lipid particles, apolipoprotein E without fusogenic lipid particles, and various ApoE-coated fusogenic lipid particles of the invention.

[0204]

[0165] HCMEC / D3 cells were cultivated for 72 h on 8-well p-slides from ibidi. Prior to the treatment FLs with a lipid / ApoE ratio of 5000 / 1 and 1000 / 1 (mol / mol) and a lipid concentration of 2 mg / mL were prepared freshly and diluted 1 :20 (v / v) with cold PBS. The culture medium was aspirated and each chamber was treated with 200 pL FL stock. After 1 h incubation at 37 °C, the FL solution was replaced by 250 pL of fresh ECGM2. All cells were fixed for 15 min at RT using 250 pL of 3.7% (w / w) para formaldehyde in PBS. Each chamber was washed three times with 250 pL PBS and then blocked for 1 h at RT with 5% (w / w) milk powder in PBS. The previous washing procedure was repeated and the antibody stock was prepared by diluting the CoraLite Plus 488-conjugated ApoE antibody from ThermoFisher (Waltham, MA, USA) 1 : 100 (v / v) with 1% (w / w) milk powder in PBS. 150 pL of the resulting AB stock were added to each chamber and cells were stained for 2 h at RT. Staining was followed by washing the cells three times with 250 pL PBS. Each washing step included 5 min of gentle shaking on an orbital shaker. Cell nuclei were visualized using 1 drop of NucBlue reagent for fixed cells (ThermoFischer (Waltham, MA, USA)). Cells were directly imaged using LSM710 confocal microscope (Carl Zeiss Microscopy, Cologne, Germany). The inventors found that in samples that were treated with ApoE, the protein attaches to the cellular surface of hCMEC, indicating a binding interaction of ApoE with the surface of these endothelial cells. Similarly, with the ApoE-coated fusogenic lipid particles of the invention, ApoE was detected on the surface of the cells, in addition to the staining of the cells’ membrane by the DiR dye delivered by the protein-coated fusogenic lipid particles. (Figure 3). This shows that the binding interaction of ApoE to the surface of the endothelial cells is retained, when the ApoE is comprised in the coating of the protein-coated fusogenic lipid particles of the invention.

[0205]

[0166] Example 4: Proof of cellular uptake of ApoE-coated FL by membrane fusion

[0206] In vitro studies were performed on human cerebral microvascular endothelial cells (hCMEC / D3) purchased from Merck (Darmstadt, Germany). Cells were cultivated at 37 °C in a humidified atmosphere containing 5% (v / v) carbon dioxide. Cell nutrition and avoidance of bacterial contamination were achieved using endothelial cell growth medium 2 (ECGM2) from PromoCell (Heidelberg, Germany) containing 2% (v / v) serum, supplemented with 1% (v / v) penicillin- streptomycin from ThermoFisher (Waltham, MA, USA).

[0207] HCMEC / D3 cells were seeded on 8-well p-slides from ibidi and cultivated for 72 h. After formation of a confluent cell layer, the cells were treated with FL / RSV-ApoE suspension at a total lipid concentration of 0.1 mg / ml for 20 min. After removal of the treatment solution, cells were washed with phosphate buffer and resuspended in culture medium. Microscopic analysis revealed homogenous plasma membrane staining due to membrane fusion with FL / RSV or FL / RSV-ApoE suspensions (Figure 4).

[0208] In further experiments, HCMEC / D3 cells were seeded on 8-well p-slides from ibidi and cultivated for 72 h. After formation of a confluent cell layer, the cells were first subjected to 80 pm of cumine hydroperoxide solution for 1 h to induce cellular oxidative stress reactions. Then, the cells were treated with FL / ApoE suspension or FL / ApoE / RSV suspension at a total lipid concentration of 0.1 mg / ml for 20 min. After removal of the treatment solution, cells were washed with phosphate buffer and resuspended in culture medium. Microscopic analysis revealed that all particles were internalized, however only the RSV-containing ApoE-coated liposomes were able to reduce the stress of the cells (Figure 5).

[0209]

[0167] Example 5: Animals and treatments with liposomes

[0210]

[0168] The inventors employed p16-3MR transgenic mice to investigate the influence of ApoE- FL / RSV on age-related neurovascular impairment, microvascular rarefaction and BBB dysfunction. To characterize the time course of age-related changes in NVC, BBB and cortical capillary density and progression, young (2 month old), middle aged (10 month old) and aged (22 month old) mice were used. The mice were housed under specific pathogen-free conditions within the Rodent Barrier Facility at the University of Oklahoma Health Sciences Center (OUHSC). Subsequently, for the duration of the experimental procedures, they were relocated to the standard rodent colony at OUHSC. The research shown herein strictly adhered to the ethical guidelines outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978).

[0211]

[0169] Example 5.1 : Real-time monitoring of fusogenic liposomes uptake with ApoE corona with intravital two-photon microscopy.

[0212]

[0170] To investigate whether the ApoE targeting motive improves cerebral retention of FL / RSV in vivo, the inventors retro-orbital ly injected mice with 120 pL of conventional cationic liposomes without high fusiogenity (EL / RSV), FL / RSV, and FL / RSV with ApoE motive. Liposomal uptake was monitored by using intravital two-photon microscopy.

[0213]

[0171] Prior to in vivo microscopy cranial window surgery was carried out as described in Tarantini et al. (Tarantini S, Valcarcel-Ares NM, YabluchanskiyA, Fulop GA, Hertelendy P, Gautam T, Farkas E, Perz A, Rabinovitch PS, Sonntag WE, et al. Treatment with the mitochondrial- targeted antioxidant peptide SS-31 rescues neurovascular coupling responses and cerebrovascular endothelial function and improves cognition in aged mice. Aging Cell. 2018; 17. doi: 10.1111 / acel.12731).

[0214]

[0172] Mice, previously equipped with cranial window have been anesthetized and fixed into an anatomical stereotaxic frame as previously described. A LEICA SP8 confocal TPM system was used for the detection of the liposome particles in the brain vasculature with a SpectraPhysics tunable high power two-photon laser equiped with 800-nm laser line for excitation. Emitted light was detected by PMT detectors using three filter sets (420-460, 495-540, and 575-630 nm. Brain vasculature has been recorded after injection of 100 pl of 10 mg / ml 500 kDa FITC-dextran tracer. After the measurement of the baseline fluorescent intensity, FL-DiD and ApoE-FL / DiD have been injected in the indicated concentrations and brain tissue has been recorded in the anatomical identical area at multiple locations in the indicated time-points (before injection, 5 minutes after injection, 30 minutes, 1 h, 24 h, 48, and 96 h) with a 300 pm deep 700x700x300 pm size z-stack with 2 pm intervals.

[0215]

[0173] Recorded z-stacks have been exported, then processed and analyzed in IMARIS 10.2 software. First green (FITC-Dextran) channel has been processed by multi-level filtering 3D segmentation, then red (Di D) channel has been used for the detection and 3D segmentation of the liposomes. Moving and non-moving liposomes have been separated by supervised machine learning (implemented in IMARIS) based on the liposomes orientation, size, and shape. All measured data have been determined by the qunatification of the 3D vascular network and the surrounding brain tissue.

[0216]

[0174] The inventors observed that the liposomes were quickly and efficiently incorporated within the first hour after injection. They monitored a slow decay in fluorescent signal intensity in the vasculature over the course of 96 hours, which was in the case of the FL with ApoE targeting motive significantly prolonged compared to cationic liposomes (EL) or FL without ApoE targeting motive. Their findings indicate that the retention of FL / RSV-ApoE is significantly better than EL / RSV and FL / RSV, as evidenced by its higher signal intensity over time (Figure 6).

[0217]

[0175] Example 5.2: Determination of ApoE-FL biodistribution in a mouse model by fluorescence imaging of organs at 4h post-injection.

[0218]

[0176] ApoE-FL particles containing DiO as fusion inducing molecule (30 pl of 2 mg / ml ApoE-FL) were administrated to 6-month-old C57BI6 mice (n=4) via retro-orbital injection for 4 consecutive days. The animals were sacrificed on the 4thday and the fluorescence intensity of DiO liposomal dye was monitored via Fluoview FV1000 two-photon microscope, equipped with a water immersion objective (XLPLN25XWMP, 25x, 1.05 NA; Olympus, Tokyo, Japan) and an 800-nm laser line for excitation. Emitted light was detected by PMT detectors using three filter sets (420- 460, 495-540, and 575-630 nm. The organs brain, hearth, kidney, liver, lung and quadriceps were isolated and imaged. (Figure 7). Additional vascular endothelia staining was carried out using endomucine (red fluorescent). To visualize the different tissues total fluorescent intensity of DiO was collected within region of interests (ROI) with the same size of 300 pm x 300 pm.

[0219]

[0177] The inventors recognized significantly higher DiO fluorescence signal in the mouse brain compared to other organs. The DiO signal was co-localized in all cases with the specific endothelial staining of endomucine.

[0220]

[0178] Example 5.3: In vivo blood-brain barrier permeability measurment with two-photon microscopy.

[0221]

[0179] In each group mice equipped with chronic cranial window underwent isoflurane anesthesia (3% induction, 2% maintenance, with a flow rate of 0.6-0.8 L / min) and the head was securely positioned with ear bars into a stereotaxic frame. After adding eye ointment, the setup was moved under a Fluoview FV1000 two-photon microscope, equipped with a water immersion objective (XLPLN25XWMP, 25* , 1.05 NA; Olympus, Tokyo, Japan) and an 800-nm laser line for excitation. Emitted light was detected by PMT detectors using three filter sets (420-460, 495-540, and 575- 630 nm). Subsequently, Alexa Fluor594-conjugated Wheat Germ Agglutin (WGA-AF594, 1 mg / mL, 4 pL / g body weight, ref: W11262, ThermoFisher Scientific, MA, USA) was retro-orbitally injected for blood vessel visualization and Z-stacks with 5 pm z-intervals were captured to establish a baseline. FITC-conjugated dextrans of decreasing molecular weights (70-, 40-, 10-, and 3 kDa FITC-dextrans, 4 pL per gram of body weight, 2 mg / mL; ref: D1823, D1845, D1821 , D3305, respectively, ThermoFisher Scientific, MA, US ) were then retro-orbitally injected sequentially, and 15-minute time-Z-stacks (one Z-stack per minute) were acquired after each injection, resulting in a hyperstack.

[0222]

[0180] To quantify BBB permeability, a “relative permeability change over-time” paradigm was used. For this, the concatenated image time-Z-stacks and subtracted the maximal intensity projection images was taken to obtain the extravascular fluorescent intensity changes over baseline, measured in the green channel (FITC-dextrans). The masks of the vasculature, created using the “Trainable Weka Segmentation” plugin, included the autofluorescent signal, which was subsequently subtracted from the brain parenchyma to refine the analysis. The cumulative changes in green fluorescence (I [a.u.]) compared to the baseline (I0 [a.u.]) for each tracer were measured for each experimental animal and plotted against time, giving a function, and the area under the curve (AUC) has been used as the relative permeability (I / I0) of the blood-brain barrier.

[0223]

[0181] The inventors found, that treatment with ApoE-FL / RSV notably reduces extravascular tracer intensities in maximum projection images, indicating a significant decrease in vascular permeability and a significant improve in BBB integrity in aged mouse brain (Figure 8).

[0182] Example 5.4: Cognitive behavior study using Radial Arm Water Maze (RAWM).

[0224]

[0183] To determine how ApoE-FL / RSV treatment affect cognitive function, spatial memory and long-term memory, the performance of the abovementioned mice were examined in a radial arms water maze on mice, following the published protocols published by Bauer et al. (Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS, Lopez-Lluch G, Lewis K, et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature. 2006;444:337-342).

[0225]

[0184] The maze comprises eight arms, each leading to a submerged escape platform located at the end of one arm. Opague water, achieved by adding food coloring, filled the maze. Surrounding the maze were privacy blinds, supplemented with extramaze visual cues, while intramaze visual cues were positioned at the arm ends. Video tracking directly above the maze, coupled with Ethovision software (Noldus Information Technology Inc., Leesbur, VA, USA), was employed for monitoring mice, with experimenters unaware of the experimental conditions. During each daily session, mice underwent two blocks, each encompassing four consecutive acguisition trials, to learn the submerged escape platform's location. Starting in an arm without the platform, mice were given up to one minute to locate the escape platform, spending 30 seconds on it after each trial. The platform remained in the same arm for every trial. Over three days of training, mice exhibited gradual performance improvement. Errors were charged when a mouse entered an incorrect arm, defined as having all four paws within the distal half of the arm. After the group acguired the task, mice were placed in their home cage for seven days, followed by a recall / probe trial on day 10. On day 11 (reversal / extinction), mice were tested for their ability to relearn the task, with the platform relocated to a different arm not adjacent or diametrically positioned to the previous location. The mice underwent testing for two session blocks, with the second block, comprising four trials, used for comparison.

[0226]

[0185] During the learning phase (days 2 to 6) and on probe (P), retrieval (R) and on relearn (L) days aged mice displayed higher combined error rates compared to young mice.

[0227]

[0186] First week, 30 pl of ApoE-FL / RSV at a concentration of 2 mg / ml was retroorbitally injected on 4 consecutive days. Subseguently, treatment was repeated once a week for additional 3 weeks. The same amount of RSV was either administrated orally or retro-orbital ly incejted as ApoE-FL / RSV-formulation. Untreated young and aged mice were used as control.

[0228]

[0187] Treatment with ApoE-FL / RSV significantly enhanced learning performance in aged mice relative to their untreated counterparts demonstrating the beneficial effects of ApoE-FL / RSV treatments on enhancing cognitive functions in aged mice, particularly in spatial learning. (Figure 9) The inventors were able to return vascular dementia processes (compare also Example 5.3), the cognitive performance of the aged mice was brought to the level of young animals.

Claims

CLAIMS1. A protein-coated fusogenic lipid particle comprising(i) a compound A that is a positively charged lipid molecule; and(ii) a compound C that is an aromatic molecule; wherein the protein- coated fusogenic lipid particle comprises a protein coating that comprises an apolipoprotein, preferably wherein the protein-coated fusogenic lipid particle comprises a protein-coating with a molar apolipoprotein-content of at least 50%.

2. The protein-coated fusogenic lipid particle of claim 1, comprising(i) a compound A that is a positively charged lipid molecule; and(ii) a compound C that is an aromatic molecule; wherein the protein- coated fusogenic lipid particle comprises a protein coating that comprises ApoE, preferably wherein the protein-coated fusogenic lipid particle comprises a protein coating with a molar ApoE-content of at least 30%; wherein the ApoE is a protein that comprises an amino acid sequence with at least 80% identity to the amino acid sequence of an Apolipoprotein E.

3. The protein-coated fusogenic lipid particle of claim 1 or 2, wherein the protein-coated fusogenic lipid particle further comprises a compound B that is a neutrally charged lipid molecule.

4. The protein-coated fusogenic lipid particle of any one of claims 1 to 3, wherein the protein-coated fusogenic lipid particle comprises a lipid bilayer, preferably wherein particle is a liposome with a hydrophilic center or a nanodisc.

5. The protein-coated fusogenic lipid particle of claim 1 to 4, wherein the molar ratio RAB of the compounds A:B in the protein-coated fusogenic lipid particle is between 1:0 to 1:1, preferably between 1:0.5 and 1:0.8, most preferably around 1:1.

6. The protein- coated fusogenic lipid particle of any one of claims 1 to 5, wherein the molar ratio RABC of the compounds A:B:C in the protein-coated fusogenic lipid particle is(i) 2(±0.5):0:(0.05 to 5), preferably 2(±0.2):0:(0.05 to 2.5), preferably 2(±0.2):0:(0.1 to 1), most preferably 2(±0.2):0:(0.2 to 0.6), or(ii) 1(±0.5):1(±0.5):(0.05 to 5), preferably 1 (±0.2):1(±0.2):(0.05 to 2.5), preferably 1(±0.2):1(±0.2):(0.1 to 1), most preferably 1 (±0.2):1(±0.2):(0.2 to 0.6).

7. The protein-coated fusogenic lipid particle of any one of claims 1 to 6, wherein the lipid / ApoE molar ratio of the protein-coated lipid particle is 20,000 / 1 to 500 / 1 , preferably 10,000 / 1 to 1000 / 1, preferably around 6000:1 to 4000:1 , most preferably about 5000 / 1.

8. The protein-coated fusogenic lipid particle of any one of claims 1 to 7, wherein compound A comprises a C10-30 alkyl, a C10-30 alkenyl, and / or a C10-30 alkynyl group and / or wherein Compound A is selected from the group consisting of 1 ,2-dioleoyl-3- trimethylammoniumpropane (DOTAP), 1 ,2-di-O-octadecenyl-3-trimethylammonium (DOTMA), Didodecyldimethylammonium (DDAB), and 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium (DOTIM), preferably wherein the compound A is DOTAP.

9. The protein-coated fusogenic lipid particle of any one of claims 1 to 8, wherein the compound B comprises a C10-30 alkyl, a C10-30 alkenyl, and / or a C10-30 alkynyl group and / or wherein the compound B is selected from the group consisting of phosphatidylethanolamine, phosphatidylcholine, 1 ,2-Dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1 ,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1 ,2-dielaidoyl-sn- Glycero-3-phosphoethanolamin (DEPE), 1 ,2-diphytanol-sn-Glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-Glycero-3-phosphoethanolamine and 1 ,2- dioleoyl-sn-glycero-3-phosphatidylcholin (DOPC), preferably wherein compound B is DOPE.

10. The protein-coated fusogenic lipid particle of any one of claims 1 to 9, wherein the compound C comprises a delocalized n-electron system, preferably wherein Compound C comprises a dye molecule, a vitamine and / or a polyphenol, preferably wherein the polyphenol is a stilbenoid, more preferably resveratrol.

11. The protein-coated fusogenic lipid particle of any one of claims 1 to 10, wherein the protein-coated fusogenic lipid particle comprises a further therapeutically active compound D.

12. A method of manufacturing a protein-coated fusogenic lipid particle according to any one of claims 1 to 11 , the method comprising:(i) Mixing Compound A, Compound C according to any one of claims 1 to 11 in an organic or aqueous solvent, preferably an organic solvent such as chloroform, methanol, ethanol or a mixture thereof;(ii) Drying the mixture of step (i);(iii) Redispersing the dried mixture obtained in step (ii) in a polar solvent such as ethanol and subsequently injecting an aqueous buffer, preferably an aqueous HEPES buffer to obtain a dispersion containing a fusogenic lipid particle;(iv) Mixing the dispersion containing the fusogenic lipid particle obtained in step (iii) with a dispersion comprising an apolipoprotein, preferably wherein the dispersion comprising the apolipoprotein is an aqueous buffer containing sodium-bicarbonate and / or HEPES.

13. A therapeutic composition comprising a protein-coated fusogenic lipid particle according to any one of claims 1 to 11.

14. A therapeutic composition according to claim 13 for use in the treatment of a condition or disease.

15. The therapeutic composition for use according to claim 14, wherein the condition or disease is associated with the cardiovascular system, preferably its structure and / or function and / or wherein the disease is a, optionally age-related, neurodegenerative disease.

16. The therapeutic condition for use according to claim 14 or 15, wherein the treatment comprises one or more of:(i) a restoration and / or an improvement of the cerebromicrovascular endothelial function;(ii) an improvement of vascular barrier properties and / or a restoration of the integrity of the BBB;(iii) an improvement of tissue capillarization and;(iv) an improvement of vascular dilatation17. A method for the targeted delivery of a fusogenic lipid particle, wherein the method comprises an in-vitro step of pre-coating a non-coated fusogenic lipid particle with a serum protein, wherein the non-coated fusogenic lipid particle comprises(i) a compound A that is a positively charged lipid molecule according to any one of claims 1 to 11 ;(ii) a compound C that is an aromatic molecule according to any one of claims 1 to 11.

18. The method for the targeted delivery of a fusogenic lipoid particle according to claim 17 wherein the serum protein is an apolipoprotein according to claim 1 , preferably ApoE such as according to any one of claims 2 to 11 .

19. The method of claim 17 or 18, wherein the resulting coated fusogenic lipid particle comprises a protein coating with a molar Apo-content of at least 50% and / or wherein the resulting coated fusogenic lipid particle comprises a protein coating with a molar ApoEcontent of at least 30%.

20. The method of any one of claims 17 to 19, wherein the targeted delivery targets the brain, liver, lung and / or kidney, in particular an endothelial cell therein.

21. A method for targeted drug delivery, wherein the method comprises using a protein- coated fusogenic lipid particle as a drug-delivery vehicle, wherein the non-coated fusogenic lipid particle comprises(i) a Compound A that is a positively charged lipid molecule according to any one of claims 1 to 11 ;(ii) a Compound C that is an aromatic molecule according to any one of claims 1 to 11 ; wherein the protein-coated fusogenic lipid particle comprises a protein coating that comprises a serum protein and wherein the protein-coated fusogenic lipid particle comprises a drug that is to be delivered, preferably wherein the protein-coated fusogenic lipid particle is a protein coated fusogenic lipid particle according to any one of claims 1 to 11.

22. The method for targeted drug delivery of claim 21 , wherein the drug that is to be delivered is Compound C and / or Compound D.

23. The method of claim 21 or 22, wherein the targeted delivery targets the brain, liver, lung and / or kidney, in particular an endothelial cell therein.

24. The method of any one of claims 21 to 23, wherein the drug is delivered to the target cell, preferably to the membrane and / or the cytoplasm of the target cell, more preferably, wherein the drug does not cross the BBB.

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