WO2014063757A1 - Procédés d'administration - Google Patents

Procédés d'administration Download PDF

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Publication number
WO2014063757A1
WO2014063757A1 PCT/EP2012/071320 EP2012071320W WO2014063757A1 WO 2014063757 A1 WO2014063757 A1 WO 2014063757A1 EP 2012071320 W EP2012071320 W EP 2012071320W WO 2014063757 A1 WO2014063757 A1 WO 2014063757A1
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WIPO (PCT)
Prior art keywords
peptide
lipid
cell
molecule
complementary
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PCT/EP2012/071320
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English (en)
Inventor
Alexander Kros
Hana Robson Marsden
Frank VERSLUIS
Harshal Raghunath ZOPE
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Universiteit Leiden
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Priority to PCT/EP2012/071320 priority Critical patent/WO2014063757A1/fr
Publication of WO2014063757A1 publication Critical patent/WO2014063757A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome

Definitions

  • This invention relates to methods of delivering agents to cells, and to molecules and cells useful in such methods.
  • Membrane fusion is a key process in all living cells as it facilitates the transport of molecules between and within cells.
  • a primary mechanism by which molecules are conveyed to the appropriate location is to encapsulate them in liposomes that deliver the cargo by fusing with the lipid membrane of the target cell or compartment.
  • In vivo membrane fusion is triggered by the specific interaction of SNARE (soluble N- ethylmaleimide-sensitive factor attachment protein receptors) fusion proteins that brings two membranes into close proximity followed by local disruption of the lipids and merging of the membranes (T.
  • SNARE soluble N- ethylmaleimide-sensitive factor attachment protein receptors
  • a further complicating factor concerning traditional cellular delivery systems is that the mechanism through which active agents (e.g. biomolecules/macromolecules) are delivered into the cytoplasm of cells is most likely by endocytosis (Alberts et al; Molecular Biology of the Cell 4 th Edition).
  • active agents e.g. biomolecules/macromolecules
  • endocytosis Alberts et al; Molecular Biology of the Cell 4 th Edition.
  • the present invention addresses the foregoing deficiencies by providing molecules that may be used in cellular delivery methods, modified cells that are receptive to receiving active agents, methods of modifying such cells, and methods for delivering active agents to cells.
  • a first aspect of the invention provides a molecule comprising a peptide capable of forming a coiled coil motif with a complementary peptide, and a steroid moiety.
  • a peptide capable of forming a coiled coil motif with a complementary peptide we include the meaning of any peptide that is capable of interacting with a complementary peptide by means of a coiled-coil interaction.
  • a coiled-coil interaction consists of two or more peptides able to adopt an alpha-helical conformation which interact with each other in a superhelical fashion, with their contacts mediated by hydrophobic interactions of amino acid side chains to produce a helical bundle.
  • the peptide must be one whose alpha helical secondary structure is able to orientate with the alpha helical secondary structure of another peptide such that the two peptides interact.
  • the peptide capable of forming a coiled coil motif with a complementary peptide is one that interacts specifically with a complementary peptide that is different from the peptide so as to form a hetero coiled coil, rather than a homo coiled coil.
  • the peptide contains an amino acid sequence such that it is able to form a hetero coiled coil motif as is described further below. This allows the peptide to interact with another peptide in a specific manner. The specificity of coiled-coil binding is determined by the primary amino acid sequence.
  • Suitable sequences of amino acid residues for the peptide capable of forming a coiled coil with a complementary peptide are described, for example, in Woolfson (The design of coiled-coil structures and assemblies, Fibrous Proteins: Coiled-Coils, Collagen And Elastomers, Elsevier Academic Press Inc: San Diego, 2005; Vol. 70, pp 79-112) and in Mason et al, 2004 (ChemBioChem 5: 170-176), both of which are incorporated herein by reference.
  • the peptide may comprise the sequence of one of a heterodimeric peptide pair derived from the leucine zipper (Moll et al, 2001, Prot Sci 10: 649-655) such as the fos/jun system (O'Sheaei al, 1989, Science 245: 646-648).
  • a heterodimeric peptide pair derived from the leucine zipper (Moll et al, 2001, Prot Sci 10: 649-655) such as the fos/jun system (O'Sheaei al, 1989, Science 245: 646-648).
  • the peptide may be naturally occurring or non-naturally occurring (i.e. synthetic).
  • the peptide is synthetic since such peptides have a lower tendency to interact with naturally occurring peptides or proteins at cellular membranes.
  • Naturally occurring coiled coil motifs are found in a variety of proteins including keratin, myosin, tropomyosin or laminin, or proteins such as those which contain a leucine zipper motif such as GCN4 or haemagglutinin, or other proteins such as IF, ATPase inhibitor protein. It will be understood that it is not necessary to use the entire naturally occurring protein in which coiled-coil regions are found. In the case of large coiled-coil proteins which have multiple heptad repeats capable of forming coiled-coil structures, it is also not a requisite to use the entire coiled-coil region. A portion of that region may be used subject to the size and functional limitations discussed herein. Preferably, the portion of the region is one that is capable of forming a hetero coiled coil.
  • Synthetic peptides may be synthetic variants of naturally occurring proteins known to have coiled coil motifs, or regions thereof, provided that they retain the ability to form coiled-coils. Generally, such variants differ from the naturally-occurring proteins by substitution, particularly conservative substitutions, or by insertion of one or more complete heptad repeats. Where one or more heptad repeats are to be inserted into the structure of a naturally occurring coiled-coil protein, the insertion will be such as to maintain the a-g residue structure mentioned below. Conveniently, the insertion will be of one or more complete ag repeats between the g and a of successive repeats of the naturally-occurring protein. However insertions may also be made between other residues of the heptad repeat so long as the insertion maintains the heptad structure.
  • synthetic peptides may be a peptide whose amino acid sequence has been designed to form a coiled coil motif.
  • the amino acid sequence is one that allows the peptide to form a hetero coiled coil motif with a complementary peptide.
  • the peptide comprises from 2 to about 200 (e.g. about 3 to about 100, such as from about 3 to about 10, 20, 30, 40, 50, 60, 70, 80 or 90) heptad repeats, enabling the peptide to form a coiled coil motif, and preferably a hetero coiled coil motif, with a complementary peptide.
  • the peptide comprises between 2 and 7 heptad repeats (eg 3-6 heptad repeats).
  • heptad repeats eg 3-6 heptad repeats.
  • the peptide When the peptide is prepared by solid phase peptide synthesis (SPPS) or solution phase synthesis, it may comprise from about 2 to about 10 heptad repeats, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 heptad repeats.
  • SPPS solid phase peptide synthesis
  • solution phase synthesis it may comprise from about 2 to about 10 heptad repeats, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 heptad repeats.
  • a heptad repeat in the peptide may be denoted (a-b-c-d-e-f-g) n , and, in the complementary peptide to which the peptide is capable of forming a coiled coil with, (a'- b'-c'-d'-e'-f'-g')n, using the helical wheel representation.
  • positions a and d are non-polar core amino acid residues found at the interface of the complementary peptide helices, and positions e and g are solvent exposed, occupied by polar amino acid residues.
  • each heptad may start with any of a, b, c, d, e, f or g (or a', b', c', d', e', f or g'), not necessarily a or a'.
  • the heptad repeat may be denoted (g-a-b-c-d-e-f) n -
  • each heptad in the peptide may contain the same repeating sequence of seven amino acids.
  • each heptad in the peptide may be the same or each may be different.
  • each heptad unit in the peptide is (E I A A L E K) (SEQ ID No: 1).
  • the peptide may comprise (E I A A L E K) n , preferably wherein n is from about 3 to about 10.
  • the peptide may be (E I A A L E K) 3 (SEQ ID No: 2), also known as peptide ⁇ " (Marsden et al J Am Chem Soc 2008, 130(29): 9386-9393, incorporated herein by reference).
  • each heptad unit in the peptide is (K I A A L K E) (SEQ ID No: 3).
  • the peptide may comprise (K I A A L K E) thread, preferably wherein n is from about 3 to about 10.
  • n 3
  • the peptide may be (K I A A L K E) 3 (SEQ ID No: 4), also known as peptide U K".
  • the complementary peptide to which the peptide portion of the molecule is capable of forming a coiled coil motif with may have a structure as defined above in relation to that peptide portion, for example in terms of heptad repeats.
  • Peptides comprising the heptad unit (E I A A L E K) are capable of forming a hetero coiled coil with a complementary peptide comprising the heptad unit (K I A A L K E).
  • the peptide comprises (E I A A L E K) (eg Peptide ⁇ ")
  • it is capable of forming a coiled coil with a complementary peptide that comprises (K I A A L K E) (eg Peptide "K”
  • the peptide comprises (K I A A L K E) (eg Peptide "K”
  • EIQALEEENAQLEQENAALEEEIAQLEY (Peptide P1; SEQ ID No: 5) which is capable of forming a coiled coil with KIAQLKEKNAALKEKNQQLKEKIQALKY (Peptide P2; SEQ ID No: 6); EIQQLEEEIAQLEQKNAALKEKNQALKY (Peptide P3; SEQ ID No: 7) which is capable of forming a coiled coil with KIAQLKQKIQALKQENQQLEEANAALEY (Peptide P4; SEQ ID No: 8); ENAALEEKIAQLKQKNAALKEEIQALEY (Peptide P5; SEQ ID No: 9) which is capable of forming a coiled coil with KNAALKEEIQALEEENQALEEKIAQLKY (Peptide P6; SEQ ID No: 10); and EIQALEEK
  • the peptide may be any of Peptides P1-P8 that is capable of forming a coiled coil with its respective complementary peptide.
  • the peptide may comprise any amino acid sequence enabling it to form a coiled coil motif, and preferably a hetero coiled coil motif, with a complementary peptide.
  • Such peptides can be readily found by the skilled person, for example based on the scientific literature on coiled coil motifs as mentioned herein.
  • coiled-coil assembly is determined by the amino-acid sequence, including the oligomerization state (two or more peptides), size ( ⁇ 2 nm - 200 nm long), helix orientation (parallel or antiparallel), homo- or heterotopic complexes, stability of the individual helix as well as the complex, rigidity, and solubility and interaction with lipid membranes.
  • the non-covalent association of these peptides is also sensitive to changes in the environment, for example pH, temperature, ionic strength and metal ions, which affect the electrostatic and/or hydrophobic interactions.
  • the amino acid sequence is one that promotes heterotopic complexes (eg heterodimers).
  • the peptide of the molecule is between about 14 amino acids and 100 amino acids in length.
  • the peptide may have less than 90 amino acids, such as less than 80, 70, 60 or 50 amino acids.
  • the peptide has between 14 and 49 amino acids, such as between 14 amino acids and any of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 amino acids.
  • any suitable method for preparing the peptide capable of forming a coiled coil motif with a complementary polypeptide may be used.
  • the peptide may be synthesised by solution phase or by SPPS (both can be done manually or by automation) or by using recombinant DNA techniques in order to genetically modify an organism to express it.
  • an amino acid sequence to form coiled-coils may be tested by routine methods known in the art.
  • the sequence of any putative coiled-coil region may be examined by the algorithms of Lupas et al or Berger et al.
  • the sequences may also be synthesised, for example by recombinant DNA techniques or solution phase synthesis or solid phase synthesis, and solutions of the sequence examined for circular dichroism spectra at different concentrations. Where coiled-coil formation occurs (as opposed to non-specific aggregation) the spectra will remain diagnostic of coiled coils at a range of dilutions (whereas non-specific aggregation will be diluted out).
  • the peptide portion of the molecule may also contain one or more non coiled-coil amino acids.
  • the peptide may comprise sequences flanking the heptad repeats that are not involved in formation of the coiled coil. These one or more amino acids may be necessary to maintain the overall structure of the coiled coil portion, thereby allowing the alpha-helical coil structure of the heptad repeats to be formed, and/or when the one or more amino acids are fluorescent (eg tryptophan), they may be used to make the molecule detectable.
  • the non coiled-coil forming amino acids may be located at either or both of the N-and/or C-terminals of the coiled coil region of the peptide (i.e. the region containing those amino acid sequences capable of forming a coiled coil with a complementary peptide).
  • one or more non-coiled coil amino acids may link the coiled-coil amino acids either to the steroid moiety or to the spacer moiety discussed below.
  • one or more non-coiled amino acids may be attached to the end of the peptide portion which end is not attached to the steroid moiety (or spacer moiety discussed below). Any such amino acids may be used provided that they do not affect the peptide's capability to form a coiled coil with a complementary peptide.
  • a steroid moiety we include the meaning of any molecular moiety which comprises a polycyclic steroid backbone comprising a nucleus of three six-carbon rings and a five carbon ring (eg a cyclophenanthrene nucleus).
  • the steroid moiety can contain varying degrees of unsaturation and may contain a variety of functional groups.
  • steroid moieties have a hydroxyl group or a keto group at the C-3 position.
  • an alkyl side chain or fatty acid chain may be present at the C-17 position, and such chains may be branched or unbranched, and such chains may be saturated or unsaturated.
  • Steroid moieties in which one or more rings are aromatic are also contemplated.
  • the steroid moiety is a sterol moiety which comprises the four cycloalkane rings but wherein a hydroxyl group is present at C-3.
  • the steroid moiety may be attached to the peptide portion of the molecule either directly or indirectly (eg via a linker molecule).
  • the steroid moiety is attached to the peptide portion of the molecule via a functional group that is attached to the cycloalkane ring structure.
  • the sterol moiety may be attached (either directly or indirectly) to the peptide portion of the molecule via the oxygen atom of the hydroxy group at position C-3.
  • the peptide portion of the molecule may be attached (either directly or indirectly) to the sterol moiety via a fatty acid present at the C-17 position.
  • the steroid moiety comprises cholesterol or a derivative thereof. Included in derivatives of cholesterol are steroid acids including bile acids such as cholic acid.
  • the molecule of the first aspect of the invention may be used to modify cell membranes such that the cell can be targeted by delivery vehicles that subsequently fuse with the cellular membrane.
  • the steroid moiety of the molecule eg cholesterol or derivatives thereof
  • the steroid moiety of the molecule is one that is capable of inserting into a lipid membrane.
  • lipid membrane we include the meaning of any of a lipid membrane of a cell, an organelle, a liposome, a proteoliposome, a lipid bilayer, a lipid monolayer, or bead particles coated with a lipid bilayer.
  • the lipid membrane may comprise any natural and/or synthetic lipids as described further herein.
  • the membrane is a bilayer of amphiphilic molecules, most preferably one or more phospholipids, eg phosphatidylcholines and/or phosphatidylethanolamines.
  • phospholipids eg phosphatidylcholines and/or phosphatidylethanolamines.
  • the membrane may comprise any one or more other phospholipids.
  • the peptide portion of the molecule may be directly attached to the steroid moiety, for example by linking of a steroid moiety's hydroxyl group to the C- or N- terminus of the peptide, preferably it is indirectly attached to the steroid moiety by means of a spacer moiety.
  • the inventors believe that the presence of a spacer reduces steric hindrance and/or enables a greater degree of rotation such that formation of a coiled coil is facilitated.
  • the spacer moiety may also suppress homocoiling and/or increase the specificity of coiled coil formation in the presence of competing molecules.
  • the steroid moiety may be attached to a spacer moiety which is, in turn, attached to the peptide portion of the molecule.
  • the spacer moiety is a water-soluble polymer, i.e. any polymer that is more soluble in water or other polar solvents (e.g. protic solvents such as alcohols) than in oil or other hydrophobic solvents (e.g. hydrocarbons).
  • polar solvents e.g. protic solvents such as alcohols
  • hydrophobic solvents e.g. hydrocarbons
  • the spacer moiety is a poly(alkylene oxide) such as any of polyethylene glycol (PEG), a polypropylene glycol, a polyethylene imine and a polyethylene oxide.
  • suitable polymers include any of alpha-substituted polyalkylene oxide derivatives such as methoxypolyethylene glycols; alkyl-substituted derivatives such as those containing C n -C 4 alkyl groups; monomethyl-substituted PEG homopolymers; polyethylene glycol (PEG) homopolymers and derivatives thereof; polypropylene glycol homopolymers and derivatives thereof; alkyl-capped polyethylene oxides; bis-polyethylene oxides; copolymers of poly(alkylene oxides); block copolymers of poly(alkylene oxides) or activated derivatives thereof; and branched and star PEGs such as those commercially available from Shearwater Polymers, Inc.
  • the spacer moiety comprises PEG.
  • the spacer moiety comprises from about 1 to about 48 ethylene glycol units, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 ethylene glycol units, and it is particularly preferred if the spacer moiety comprises between 2 and 4 ethylene glycol units.
  • the spacer moiety may be a water soluble polymer that is a hydrophilic polypeptide comprising one or more hydrophilic amino acids.
  • Hydrophilic amino acid residues are generally considered to be Arg (A), Asn (N), Asp (D), Gin (Q), Glu (E), Lys (K), Ser (S) and Thr (T). Hydrophobic residues are generally considered to be Ala (A), He (I), Leu (L), Met (M), Phe (F), Trp (W), Tyr (Y) and Val (V). Any sequence of amino acid residues may be used in the spacer moiety, provided that the spacer moiety is, overall, hydrophilic in nature.
  • the spacer moiety may also include any non-natural or modified amino acid having the general structure and/or R 2 may, for example, independently represent a fluorinated side chain (e.g. a fluorinated alkyl group) or a urea derived side chain.
  • One of Ri or R 2 may be a side chain found in natural amino acids, ⁇ amino acids may also be used.
  • hydrophilic polypeptides examples include any of KRKYW KNLK (SEQ ID No: 13) (synaptobravin), KYQSKARRKK (SEQ ID No: 14) (syntaxin) and RKYWWKNLK (SEQ ID No: 15) (VAMP) (see Meyenberg et al Chem Commun (Camb) 2011, 47, 9405- 9407; Lygina et al Angew Chem Int Edit 2011 , 50, 8597-8601), or the same sequences in C to N terminus direction (ie reversed sequences).
  • Other appropriate polypeptides include those sequences found in natural occurring SNARE proteins (Jahn and Scheller, Nat. Rev. Mol. Cell Biol., 2006, 7: 631).
  • the spacer moiety is a hydrophilic polypeptide
  • the spacer moiety may be covalently linked to the N-terminus or the C-terminus of the peptide portion of the molecule.
  • the peptide portion of the molecule comprises Peptide ⁇ ", or Peptide "K", or any of Peptides P1-P8, the steroid moiety comprises cholesterol, and the spacer moiety comprises PEG.
  • the peptide portion of the molecule comprises the modified Peptide ⁇ ", G(EIAALEK) 3 -NH2, or the modified Peptide "K", (KIAALKE) 3 GW-NH2.
  • Amino acid residues described herein are generally in the natural "L” isomeric form. However, residues in the "D” isomeric form can be substituted for L-amino acid residues in certain situations, provided that the amino acid sequence still retains its function. Thus, in the context of the peptide capable of forming a coiled coil with a complementary peptide, the peptide must still be able to form a coiled coil, and in the context of the spacer moiety, the peptide must not interfere with formation of a coiled coil by the peptide portion of the molecule.
  • the definition also includes, unless otherwise specifically indicated, chemically-modified amino acids, including amino acid analogues (such as penicillamine, 3-mercapto-D-valine), naturally-occurring non-proteogenic amino acids (such as norleucine), beta-amino acids, azapeptides, N-methylated amino acids and chemically-synthesised compounds that have properties known in the art to be characteristic of an amino acid.
  • amino acid analogues such as penicillamine, 3-mercapto-D-valine
  • non-proteogenic amino acids such as norleucine
  • beta-amino acids such as norleucine
  • beta-amino acids such as norleucine
  • Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatised to form O-acyl or O-alkyI derivatives. Also included as derivatives are those peptide portions that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.
  • the peptide parts of the molecule of the invention can be peptide "mimetics", i.e. peptidomimetics which mimic the structural features of peptides comprising or consisting of the amino acid sequence as described herein.
  • Peptidomimetics can be even more advantageous in therapeutic use, in the resistance to degradation, in permeability or in possible oral administration.
  • the peptide portion of the molecule is made using solid phase peptide synthesis (for example using either Fmoc or Boc chemistry) methods or by solution phase synthesis.
  • solid phase peptide synthesis for example using either Fmoc or Boc chemistry
  • solution phase synthesis for example using either Fmoc or Boc chemistry
  • the general Fmoc and Boc methods are widely used and are described in the following references: errifield, J. A. Chem. Soc, 88:2149 (1963); Bodanszky and Bodanszky, The Practice of Peptide Synthesis, Springer-Verlag Berlin, 7-161(1994); Stewart, Solid Phase Peptide Synthesis, Pierce Chemical Rockford, (1984).
  • a second aspect of the invention provides a composition comprising a molecule according to the first aspect of the invention, wherein the steroid moiety of the molecule is inserted into a lipid membrane.
  • the composition may comprise a molecule according to the first aspect of the invention, the steroid moiety of which molecule is inserted into a lipid membrane of a cell, an organelle, a liposome, a proteoliposome, a lipid bilayer or a lipid monolayer.
  • composition of the second aspect of the invention may be any of a cell, an organelle, a liposome, a proteoliposome, a lipid bilayer or a lipid monolayer, wherein the molecule of the first aspect of the invention is inserted in the lipid membrane of that respective cell, an organelle, a liposome, a proteoliposome, a lipid bilayer or a lipid monolayer.
  • Methods for inserting lipidated peptides into a lipid membrane are standard practice in the art and may involve insertion of the lipidated peptide into preformed lipid membranes (eg by exposing the preformed membrane to the lipidated peptide) particularly when the lipid membrane is part of a naturally occurring structure such as a cell or organelle. In relation to cellular membranes, further details are provided below in relation to the fourth aspect of the invention.
  • the lipidated peptide may become inserted into the lipid membrane of that synthetic structure by adding the lipidated peptide to the lipid constituents of the synthetic structure when it is originally prepared.
  • the lipidated peptide may be mixed to the lipid constituents in an organic solvent (eg methanol/chloroform), the mixture is dried, and upon reconstitution of the dried mixture in an appropriate buffer, the synthetic structure is formed with the lipidated peptide inserted into its lipid membrane.
  • the method may further comprise using standard techniques such as sonication or extrusion to modify the diameter of the liposome to an appropriate size. Further detail on how lipidated peptides may be inserted into lipid membranes is provided in Marsden et al (Angew Chem. Int. Ed. , 2009, 48, 2330-2333 and in Liposomes 2 nd ed by V.P. Torchilin and V. Weissig (Oxford University Press 2003).
  • the molecule of the first aspect of the invention is integrated into the lipid membrane of a lipid delivery vehicle, and so the composition of the second aspect of the invention may be a lipid delivery vehicle.
  • lipid delivery vehicle we include any lipid based delivery vehicle that may be used to deliver one or more agents to a cell.
  • lipid delivery vehicles are any of a liposome (eg a proteoliposome), a vesicle, a polymersome, a nanoparticle (eg inorganic nanoparticle such as silica nanoparticle), a nanoemulsion, a nanogel, a viral particle, a cross linked micelle, or any self-assembled system (eg any amphiphilic molecule (eg macromolecule or biomolecule) which upon dispersion in an aqueous solution spontaneously forms a larger assembly held together by non-covalent interactions).
  • a liposome eg a proteoliposome
  • a vesicle e.g a vesicle
  • a polymersome eg inorganic nanoparticle such as silica nanoparticle
  • nanoemulsion emulsion
  • nanogel emulsion
  • viral particle emulsion
  • a cross linked micelle e.g any self-assembled system (eg any amphiphilic molecule (eg
  • the lipid delivery vehicle (eg liposome) further comprises a detectable marker such as a fluorescent marker, for example texas red, Lissamine rhodamine, fluorescein or NBD labelled lipids.
  • the lipid delivery vehicle is a liposome.
  • liposome we include both unilamellar and multilamellar vesicles, although unilamellar vesicles are preferred.
  • Liposomes typically range from 30 to 1000 nm in diameter.
  • the liposomes are between 30-300 nm in diameter, such as 70 - 150 nm, and most preferably the liposomes have a diameter of around 100 nm.
  • Liposomes 2 nd ed by V.P. Torchilin and V. Weissig (Oxford university press 2003 Isbn 978-0-19-963654-9), and Liposomes, Methods and Materials (Volume 1: Pharmaceutical Nanocarriers Edited by V. Weissig Part of Springer protocols; methods in molecular biology 605 Isbn 978-1-60327-360-2).
  • the lipid delivery vehicle comprises an active agent such as any of a small molecule, a drug, a polymer, a protein, a peptide, an antibody, a nucleic acid (e.g. DNA or RNA such as siRNA), a lipid or a carbohydrate.
  • the active agent may be hydrophilic (ie water soluble) or hydrophobic.
  • the active agent is a therapeutic agent that is to be delivered to a cell to adjust one or more cellular activities for therapeutic purposes.
  • the invention provides an alternative to polycationic transfecting agents such as lipofectamine.
  • These transfecting agents can be effective for the delivery of polyanionic compounds (such as plasmids) but are often not able to transfer other (bio)molecules into cells which are not charged.
  • the active agent is an imaging agent that is to be delivered to a cell to identify that cell or a cellular compartment thereof more easily.
  • the active agent may comprise a fluorescent label, or it may be any of a molecular beacon (S. Tyagi, 1996 Nat Biotechnol, 14: 303-308; S. Tyagi et al, 1998 Nat Biotechnol 16: 49-53; and Mhlanga and Tyagi, 2006 Nat Protoc 1: 1392-1398), a quantum dot (Michalet et al, 2005 Science 307: 538) or a gold nanorod (Huang et al 2009 Adv Mater 2 ⁇ : 4880-4910).
  • This embodiment is of particular use in a diagnostic setting by allowing the entry of detectable molecules into cells that would otherwise be unable to gain access.
  • the lipid membrane comprises a collection of amphipathic molecules that contains both a hydrophilic and a hydrophobic moiety.
  • the lipid membrane comprises one or more phospholipids such as one or more glycerophospholipids and/or sphingomyelins.
  • Phospholipids are amphiphilic molecules with nonpolar aliphatic tails and polar phosphoryl based head groups.
  • Glycerophospholipids are the major lipid component of biological membranes and comprise sn-glycerol-3-phosphate joined at their C1 and C2 positions to fatty acids and at their phosphoryl group to a simple organic molecule such as choline.
  • the glycerophospholipids are classified according to the identity of the organic molecule. Thus, when the organic molecule is choline, the glycerophospholipid is phosphatidylcholine, and so on.
  • glycerophospholipids include phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol, and so the lipid membrane may comprise one or more of these glycerophospholipids.
  • the lipid membrane comprises phosphatidylethanolamine and phosphatidylcholine, which are commonly found in cellular membranes. Also included are phosphatidylglycerol lipids such as cardiolipins.
  • Cardiolipins (lUPAC name "1,3-bis(sn-3'-phosphatidyl)-sn-glycero ) are important components of the inner mitochondrial membrane, constituting about 20% of total lipid composition where they are important for the optimal function of numerous enzymes that are involved in mitochondrial energy metabolism.
  • Sphingomyelins are also major membrane components that are similar in structure to glycerophospholipids. However, rather than being derived from glycerol, they are derivatives of the C 8 amino alcohols sphingosine, dihydrosphingosine, and their C 16 , C 17 , C 9 and C 20 homologs (possible chains include 16:0, 18:0, 18:1, 20:0, 22:0, 22:1, 23:0, 23:1, 24:0, 24:1), i.e. they are sphingolipids. Sphingomyelin (or ceramide phosphoryl choline) consists of a ceramide unit with a phosphorylcholine moiety attached to position 1.
  • lipid components of the lipid membrane include the sphingolipids cerebrosides (eg galactocerebrosides or glucocerebrosides) and gangliosides.
  • sphingolipids cerebrosides eg galactocerebrosides or glucocerebrosides
  • gangliosides eg galactocerebrosides or glucocerebrosides
  • these components like sphingomyelins, are derivatives of the C 18 amino alcohols sphingosine, dihydrosphingosine, and their C 6 , C 7 , C 9 and C 20 homologs, but unlike sphingomyelins, the hydrophilic unit is one or more sugar residues.
  • the phospholipids and sphingolipids described above may contain any unsaturated or saturated fatty acid chain of any length.
  • the lipid membrane will be one that contains phospholipids and/or sphingolipids that comprise aliphatic (i.e. unsaturated or saturated) fatty acid chains having 8 to 24 carbon atoms, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 carbon atoms. Most typically, the fatty acid chains have between 12 and 20 carbon atoms, and yet more typically between 14 and 18 carbon atoms.
  • the two fatty acid chains that make up a phospholipid or sphingolipid may be the same or different.
  • saturated C 6 and Ci 8 fatty acids usually occur at the C1 position of glycerophospholipids and the C2 position is often occupied at an unsaturated Ci 6 to C 20 fatty acid.
  • the lipid membrane is one that comprises a phosphatidylethanolamine such as 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).
  • DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • POPE 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
  • the lipid membrane is one that comprises a phosphatidylcholine such as 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1- Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).
  • DOPC 1,2-Dioleoyl-sn-glycero-3-phosphocholine
  • POPC 1- Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • the lipid membrane is one that comprises a phosphatidylglycerol lipid such as cardiolipin.
  • the lipid membrane of the composition of the second aspect of the invention may contain a sterol such as cholesterol.
  • the lipid membrane comprises a sterol (eg cholesterol) and one or more phospholipids such as phosphatidylethanolamine and phosphatidylcholine, and optionally a cardiolipin.
  • a third aspect of the invention provides a kit of parts comprising (a) a molecule according to the first aspect of the invention or a composition according to the second aspect of the invention; and (b) the complementary peptide.
  • the complementary peptide will be any peptide that is capable of forming a coiled coil motif with the peptide portion of the molecule of the first aspect of the invention, and so the same preferences as described above apply.
  • the complementary peptide is capable of forming a hetero coiled coil motif with the peptide portion of the molecule of the first aspect of the invention.
  • the complementary peptide comprises from 2 to about 200 (e.g. about 3 to about 100, such as from about 3 to about 10, 20, 30, 40, 50, 60, 70, 80 or 90) heptad repeats, enabling the complementary peptide to form a coiled coil motif with the peptide portion of the molecule of the first aspect of the invention.
  • the complementary peptide When the complementary peptide is prepared by solid phase peptide synthesis (SPPS) or solution phase synthesis, it may comprise from about 2 to about 10 heptad repeats, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 heptad repeats. Most preferably, the complementary peptide comprises 2-7 (eg 3-6) heptad repeats.
  • SPPS solid phase peptide synthesis
  • solution phase synthesis e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 heptad repeats.
  • the complementary peptide comprises 2-7 (eg 3-6) heptad repeats.
  • each heptad unit in the complementary peptide is (E I A A L E K).
  • the complementary peptide may comprise (E I A A L E K) n , preferably wherein n is from about 3 to about 10.
  • the complementary peptide may be (E I A A L E K) 3 , also known as peptide ⁇ " (Marsden et al J Am Chem Soc 2008, 130(29): 9386-9393, incorporated herein by reference).
  • each heptad unit in the complementary peptide is (K I A A L K E).
  • the complementary peptide may comprise (K I A A L K E) n , preferably wherein n is from about 3 to about 10.
  • n 3
  • the complementary peptide may be (K I A A L K E) 3 , also known as peptide "K”.
  • a peptide that comprises (E I A A L E K) (eg Peptide ⁇ "), is capable of forming a coiled coil with a peptide that comprises (K I A A L K E) (eg Peptide "K”), and so the complementary peptide may comprise (E I A A L E K) (eg Peptide "E") when the peptide portion of the molecule comprises (K I A A L K E) (eg Peptide "K”), and vice versa.
  • EIQALEEENAQLEQENAALEEEIAQLEY (Peptide P1) which is capable of forming a coiled coil with KIAQLKEKNAALKEKNQQLKEKIQALKY (Peptide P2); EIQQLEEEIAQLEQKNAALKEKNQALKY (Peptide P3) which is capable of forming a coiled coil with KIAQLKQKIQALKQENQQLEEANAALEY (Peptide P4); ENAALEEKIAQLKQKNAALKEEIQALEY (Peptide P5) which is capable of forming a coiled coil with KNAALKEEIQALEEENQALEEKIAQLKY (Peptide P6); and EIQALEEKNAQLKQEIAALEEKNQALKY (Peptide P7) which is capable of forming a coiled coil with KIAQLKEENQQLEQKIQALKEEN
  • the complementary peptide is between about 14 amino acids and 100 amino acids in length.
  • the complementary peptide may have less than 90 amino acids, such as less than 80, 70, 60 or 50 amino acids.
  • the complementary peptide has between 14 and 49 amino acids, such as between 14 amino acids and any of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 amino acids.
  • the composition of the second aspect of the invention may be useful in the delivery of active agents to cells, for example when the composition comprises the molecule of the first aspect of the invention inserted into the lipid membrane of a lipid delivery vehicle.
  • the complementary peptide to which the peptide portion of the molecule is capable of forming a coiled coil with
  • component (b) of the kit of parts is anchored to the cell membrane of the cell to which the active agent is to be delivered, either by means of an anchor moiety or by direct attachment to the cell membrane (eg via chemical functionalisation of a membrane component). This 'decorates' the cell surface with the complementary peptide.
  • lipid delivery vehicle i.e. an example of the composition of the second aspect of the invention that constitutes component (a) of the kit of parts.
  • the peptide portion of the molecule in the composition then forms a coiled coil with the complementary peptide exposed on the surface of the cell, ultimately leading to lipid delivery vehicle-cell membrane fusion and subsequent delivery of the active agent into the cell.
  • the complementary peptide in the kit of parts may be attached to an anchor moiety that can insert into a lipid membrane such that the complementary peptide can be anchored to the cell membrane of a cell.
  • component (b) of the kit of parts is used to decorate the surface of a cell with the complementary peptide and component (a) of the kit of parts corresponds to the lipid delivery vehicle that can deliver an active agent to that cell.
  • the complementary peptide in the kit of parts is attached to an anchor moiety.
  • anchor moiety we include the meaning of any moiety which is capable of inserting into a lipid membrane, for example by favourably interacting with the hydrophobic portions of the lipids that make up the lipid membrane.
  • the complementary peptide can be tethered to the cell membrane, and hence decorate its surface.
  • the anchor moiety may be attached directly or indirectly (eg via a linker moiety) to the complementary peptide.
  • the anchor moiety is a lipid moiety and so the complementary peptide is lipidated.
  • lipidated we include the meaning that the complementary peptide is attached to a lipid moiety that is capable of inserting into a lipid membrane (eg lipid bilayer). In this way, the complementary peptide is anchored to the membrane.
  • the lipid moiety may be any suitable lipid that is capable of integrating into a lipid membrane.
  • the lipid moiety may be any hydrophobic or amphiphilic molecule such as any of a free fatty acid, a di- or tri- acyl lipid, a steroid moiety, a phospholipid or a sphingolipid including any of the particular examples mentioned above.
  • Other suitable lipid moieties are depicted in Figure 4 and are described in Haipeng et al (Angen Chem Int Ed, 2011 , 50, 7052-7055).
  • An example of a diacyl lipid is depicted below, which would be attached to complementary peptide via the hydroxyl group.
  • the lipid moiety is a phospholipid such as one that comprises aliphatic (i.e. unsaturated or saturated) fatty acid chains having 8 to 24 carbon atoms (eg 12-20 or 14-18 carbon atoms).
  • the lipid moiety may be a phosphatidylethanolamine such as DOPE or a phosphatidylcholine such as DOPC.
  • the lipid moiety is a sterol moiety such as any of those described above in relation to the first aspect of the invention, including cholesterol.
  • the lipidated complementary peptide an example of component (b) of the kit of parts, may itself be a molecule of the first aspect of the invention that contains a peptide capable of forming a coiled coil with the peptide portion of the molecule that constitutes component (a) of the kit of parts.
  • the anchor moiety being a lipid moiety, it will be appreciated that it may be a transmembrane domain of a protein.
  • the transmembrane domain can be a region of a single pass type I or type II membrane protein or any one of the several transmembrane regions of a multispan membrane protein.
  • Examples include Synaptobrevin TMD MM I I L G V I C A I I L I I I I V Y F S T (SEQ ID No: 16) or Syntaxin TMD IMIIICCVILGIIIASTIGGIFG (SEQ ID No: 17) (see Meyenberg et al Chem Commun (Camb) 2011 , 47, 9405-9407; Lygina et al Angew Chem Int Edit 2011 , 50, 8597-8601 ).
  • the complementary peptide-anchor moiety may be expressed as one polypeptide in an appropriate prokaryotic or eukaryotic expression system using standard molecular biology approaches, wherein the anchor moiety is positioned at the N-terminus or C-terminus of the complementary peptide.
  • GPI anchors are glycolipids that can be attached to the C-terminus of a protein during post-translational modification.
  • the complementary peptide may conveniently be produced by using recombinant techniques to engineer a suitable signal sequence into the nucleic acid encoding the complementary peptide and expressing the engineered construct in a suitable expression system that has the capability to add GPI anchors.
  • the complementary peptide is indirectly attached to the anchor moiety (eg lipid moiety) by means of a spacer moiety.
  • Suitable spacer moieties include those described above.
  • the spacer moiety is a water soluble polymer such as a poly(alkylene oxide) (e.g. a polyethylene glycol (PEG), a polypropylene glycol, a polyethylene imine and a polyethylene oxide) or a hydrophilic polypeptide.
  • the spacer moiety is a hydrophilic polypeptide and the anchor moiety is a transmembrane domain
  • the complementary peptide - spacer moiety - anchor moiety construct may be expressed as a single polypeptide in a suitable prokaryotic or eukaryotic expression system.
  • the spacer moiety comprises from about 1 to about 48 ethylene glycol units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 ethylene glycol units.
  • the complementary peptide of the kit of parts may itself be anchored to a lipid membrane (either directly or by means of an anchor moiety), such as to any of a cell membrane, an organelle membrane, a liposome membrane, a proteoliposome membrane, a lipid bilayer or a lipid monolayer.
  • a lipid membrane either directly or by means of an anchor moiety
  • component (b) of the kit of parts may be a lipid delivery vehicle wherein the complementary peptide is anchored to the lipid membrane of the lipid delivery vehicle.
  • lipid delivery vehicles include a liposome, a vesicle, a polymersome, a nanoparticle (eg an inorganic nanoparticle such as silica nanoparticle), a nanoemulsion, a nanogel, a viral particle, a crosslinked micelle or any self-assembled system.
  • the lipid delivery vehicle is a liposome (eg a unilamellar liposome).
  • the lipid delivery vehicle may contain an active agent including any of those defined herein.
  • components (a) and (b) of the kit of parts may both be lipid delivery vehicles that respectively bear peptides that are capable of forming coiled coils with one another. Such kits may be useful in studying membrane fusion.
  • component (a) of the kit of parts may be used to decorate the surface of a cell with the complementary peptide (i.e. when component (a) simply constitutes the molecule of the first aspect of the invention) and component (b) of the kit of parts may be used as the lipid delivery vehicle that can deliver an active agent to the cell (i.e. when component (b) constitutes the lipid delivery vehicle wherein the complementary peptide is anchored into the lipid membrane of the lipid delivery vehicle).
  • component (b) of the kit of parts it is not essential for component (b) of the kit of parts to be a lipid delivery vehicle in order for it to deliver active agents to a cell that has been decorated by component (a) of the kit of parts.
  • the complementary peptide of component (b) may be directly or indirectly attached to the active agent without it being in the form of a lipid delivery vehicle.
  • the active agent will nevertheless be brought within the vicinity of the decorated cell by virtue of coiled coil formation between the peptides of components (a) and (b).
  • component (b) of the kit of parts may comprise the complementary peptide as part of a non-lipid based delivery vehicle (eg a cyclodextrin vesicle, inorganic particle or hydrogel).
  • a non-lipid based delivery vehicle eg a cyclodextrin vesicle, inorganic particle or hydrogel.
  • the complementary peptide is acting as a targeting moiety that brings the active agent to the decorated cell.
  • This may be useful, for example, to label a particular cell within a population of cells with a detectable moiety.
  • nanoparticles eg gold nanoparticles
  • nanogels eg. gold nanoparticles
  • polymersomes and silica nanoparticles may all be brought into the vicinity of cells using this technology. Preferences for the active agent are as described above in relation to the second aspect of the invention.
  • the molecule or composition may comprise a peptide comprising the heptad unit (E I A A L E K) (eg peptide ⁇ "), and the complementary peptide comprise the heptad unit (K I A A L K E) (eg peptide "K”), or vice versa.
  • the molecule or composition may comprise a peptide selected from any of Peptides P1-P8, and the complementary peptide comprise the respective complementary peptide selected from any of Peptides P1-P8, or vice versa.
  • the molecule is CPK and the complementary peptide is the lipidated complementary peptide CPE or the molecule is CPE and the complementary peptide is the lipidated complementary peptide CPK.
  • a fourth aspect of the invention provides a method of anchoring a lipidated peptide capable of forming a coiled coil motif with a complementary peptide, into a cell membrane, the method comprising contacting a cell with the lipidated peptide.
  • lipidated peptide By anchoring a lipidated peptide into a cell membrane, we include the meaning of inserting the lipid moiety of the lipidated peptide into a cell membrane wherein the lipid moiety favourably interacts with the hydrophobic portions of the lipids that make up the lipid membrane. In this way the peptide portion of the lipidated peptide is exposed at the cell surface and is available for forming coiled coil motifs with complementary peptides. In other words, the method allows one to decorate cell surfaces with peptides that are capable of forming coiled coils.
  • lipidated peptide capable of forming a coiled coil motif with a complementary peptide
  • a lipid membrane eg lipid bilayer
  • Suitable lipid moieties are defined above in relation to the first, second and third aspects of the invention, and include any suitable lipid that is capable of inserting into a lipid membrane.
  • the lipid moiety may be any amphiphilic or hydrophobic molecule such as any of a free fatty acid, a di- or tri-acyl lipid, steroid moiety, a phospholipid or a sphingolipid including any of the particular examples mentioned above.
  • the peptide may be any peptide that is capable of forming a coiled coil motif with a complementary peptide as defined in the first aspect of the invention, and so the same preferences described above apply. Most preferably, the peptide is capable of forming a hetero coiled coil motif with a complementary peptide.
  • each heptad unit in the peptide is (E I A A L E K).
  • the peptide may comprise (E I A A L E K) n , preferably wherein n is from about 3 to about 10.
  • the peptide may be (E I A A L E K) 3 , also known as peptide ⁇ ".
  • each heptad unit in the peptide is (K I A A L K E).
  • the peptide may comprise (K I A A L K E) n , preferably wherein n is from about 3 to about 10.
  • the peptide may be (K I A A L K E) 3 , also known as peptide "K”.
  • the peptide may be any of Peptides P1-P8 as defined above.
  • the lipidated peptide contains a spacer moiety between the lipid moiety and the peptide portion.
  • Suitable spacer moieties include those described above.
  • the spacer moiety is a water soluble polymer such as a poly(alkylene oxide) (e.g. a polyethylene glycol (PEG), a polypropylene glycol, a polyethylene imine and a polyethylene oxide) or a hydrophilic polypeptide.
  • the spacer moiety comprises from about 1 to about 48 ethylene glycol units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 ethylene glycol units.
  • lipidated peptide in this fourth aspect of the invention may be the same as the molecule of the first aspect of the invention or it may be the same as the lipidated complementary peptide defined as an embodiment in the third aspect of the invention, and so the same preferences described above for those aspects equally apply to this aspect also.
  • the lipidated peptide is CPK or CPE as defined above.
  • the method involves contacting the cell with a micellar solution of the lipidated peptide.
  • micellar solution we include the meaning of a solution that consists of a dispersion of the lipidated peptide in the form of micelles in a solvent such as Hanks Buffered Saline Solution or a cell growth medium such as Dulbecco's modified eagle medium (DMEM).
  • DMEM Dulbecco's modified eagle medium
  • a micelle is an aggregate form of surfactant molecules dispersed in a liquid colloid.
  • micelles in aqueous solution form an aggregate with the hydrophilic 'head' regions in contact with the surrounding solvent, sequestering the hydrophobic 'tail' regions in the micelle centre.
  • the micelle is composed of lipidated peptides.
  • Micellar solutions form when the concentration of amphiphile exceeds the critical micellar concentration (CMC) or critical aggregation concentration (CAC), and persist until the amphiphile concentration becomes sufficiently high to form a lyotropic liquid crystal phase.
  • CMC critical micellar concentration
  • CAC critical aggregation concentration
  • Such solutions are routinely prepared in the art and involve mixing (eg by sonication) an appropriate amount of amphiphile (in this case lipidated peptide) to an aqueous solvent.
  • the lipidated peptide is added to the solvent in a dried form.
  • the formation of micelles can be assessed using any suitable technique known in the art, including measuring the hydrodynamic radius using dynamic light scattering (DLS).
  • DLS dynamic light
  • the micellar solution of the lipidated peptide is one that contains a buffer selected from any of PBS, HBSS, HEPES and TRIS buffers.
  • the cell is incubated with the lipidated peptide (eg micellar solution thereof) for up to 30 minutes such as up to 25, 20, 15, 10 or 5 minutes. It is preferred if the cell is incubated with the lipidated peptide (eg micellar solution thereof) for between 10 and 15 minutes.
  • the lipidated peptide eg micellar solution thereof
  • Incubation may take place at any appropriate temperature such as between 10 and 45°C, for example between 15°C and 40°C, 20° and 40°C, 25° and 40°C, 30°C and 40° or 35° and 40°C. Most preferably, incubation takes place at 37°C.
  • a fifth aspect of the invention provides a cell comprising a lipidated peptide capable of forming a coiled coil with a complementary peptide, wherein the lipid component of the lipidated peptide is integrated into the cell membrane.
  • the lipidated peptide is CPK or CPE as defined above.
  • the cell may be one that has been prepared by carrying out the method of the fourth aspect of the invention.
  • the cell may be any cell, for example any cell to which it is desirable to deliver an active agent to.
  • Prokaryotic and eukaryotic cells are included.
  • the cell is a mammalian cell such as a human or animal cell.
  • establish cell lines include any of a CHO cell, a HeLa cell, a Hepg2 cell, a HEK cell, or a zebrafish cell (eg zebrafish skin cell).
  • a sixth aspect of the invention provides a method of delivering an active agent to a cell, the method comprising contacting the cell with a vehicle comprising a peptide capable of forming a coiled coil motif with a complementary peptide and the active agent, wherein the complementary peptide is lipidated and its lipid component is inserted into the cell membrane of the cell.
  • the agent may be any of a small molecule, a drug, a polymer, a protein, a peptide, an antibody, a lipid, a carbohydrate or a nucleic acid (e.g. DNA or RNA such as siRNA).
  • the agent may be a therapeutic agent or an imaging agent such as any of a molecular beacon, a quantum dot or a gold nanorod.
  • the active agent is water soluble.
  • the method may also be used to deliver hydrophobic active agents, such as drugs, proteins or lipids to the membranes of cells.
  • a vehicle comprising a peptide capable of forming a coiled coil motif with a complementary peptide and an active agent
  • a vehicle that contains the active agent, as defined above in relation to the second or third aspects of the invention.
  • the vehicle may be one in which the active agent is encapsulated and the peptide is present at the surface of the vehicle.
  • the peptide capable of forming of a coiled coil with a complementary lipidated peptide may itself be lipidated, such that the lipid portion of the peptide is inserted into the lipid membrane of the lipid delivery vehicle and the peptide is at the surface of the lipid delivery vehicle.
  • the peptide capable of forming a coiled coil with a complementary peptide may be attached to some other anchor moiety as defined herein (eg TMD) which allows the peptide to be at the surface of the lipid delivery vehicle by virtue of the anchor moiety being inserted into the lipid membrane of the lipid delivery vehicle.
  • anchor moiety as defined herein
  • appropriate lipid delivery vehicles include any of a liposome (eg a proteoliposome), a vesicle, a polymersome, a nanoparticle, a nanoemulsion, a nanogel, a crosslinked micelle, or a self-assembled system.
  • the vehicle comprising a peptide capable of forming a coiled coil with a complementary lipidated peptide may correspond to the lipid delivery vehicle described in the context of the second or third aspects of the invention, and the same preferences for the peptide, vehicle, and lipid composition of the vehicle apply.
  • the peptide is one that is capable of forming a hetero coiled coil motif with a complementary peptide.
  • the vehicle is a liposome (eg a unilamellar liposome) comprising a phosphatidylethanolamine such as 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE); and/or a phosphatidylcholine such as 1,2-Dioleoyl-s/?-glycero-3-phosphocholine (DOPC) or 1- Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); and/or cholesterol.
  • DOPE 1,2-Dioleoyl-s/?-glycero-3-phosphocholine
  • POPC Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • cholesterol e.g a unilamellar liposome
  • the liposome has
  • the vehicle is a liposome (eg a unilamellar liposome) comprising a phosphatidylethanolamine such as 1 ,2-Dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or 1-Palmitoyt-2-oleoyl-s/7-glycero-3- phosphoethanolamine (POPE); a phosphatidylcholine such as 1 ,2-Dioleoyl-sn-glycero-3- phosphocholine (DOPC) or 1-Palmitoyl-2-oleoyl-so-glycero-3-phosphocholine (POPC); and cholesterol, and the vehicle comprises a lipidated form of any of peptides E, K and P1-P8 (e.g. CPK or CPE). It is preferred if the liposome has a diameter of between 70 and 150 nm, such as around 100 nm.
  • DOPE 1-Palmitoyt-2-
  • the vehicle comprises a lipidated form of any of peptides capable of forming coiled coils described above, such as peptides that comprise (E I A A L E K) n , preferably wherein n is from about 3 to about 10, (eg Peptide ⁇ "), or peptides that comprise (K I A A L K E) n, preferably wherein n is from about 3 to about 10 (eg Peptide "K”), or any of Peptides P1-P8 as defined above.
  • the vehicle comprises CPK or CPE.
  • the lipidated complementary peptide, and the lipidated peptide of the vehicle contain a spacer moiety between their lipid and peptide portions.
  • Suitable spacer moieties include those described above.
  • the spacer moiety is a water soluble polymer such as a poly(alkylene oxide) (e.g. a polyethylene glycol (PEG), a polypropylene glycol, a polyethylene imine and a polyethylene oxide) or a hydrophilic polypeptide.
  • the spacer moiety comprises from about 1 to about 48 ethylene glycol units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 ethylene glycol units.
  • the lipidated complementary peptide and the lipidated peptide of the vehicle comprise the same type of lipid moiety.
  • the lipidated complementary peptide contains a steroid moiety
  • the lipidated peptide of the vehicle contains a steroid moiety
  • the lipidated complementary peptide contains a phospholipid
  • the lipidated peptide of the vehicle contains a phospholipid, and so on.
  • the lipid moieties may be identical between the lipidated complementary peptide and the lipidated peptide of the vehicle (in the embodiment when it is lipidated).
  • the vehicle is a lipid delivery vehicle (eg a liposome) and the active agent is water- soluble
  • the inventors have found that formation of a coiled coil between the peptide of the vehicle and the complementary peptide on the cell surface results in fusion of the lipid membrane of the lipid delivery vehicle (eg liposome) with the cell membrane, such that the active agent is delivered directly into the cytoplasm of the cell.
  • delivery of the active agent does not occur by endocytosis. Rather, the active agent is delivered as a result of cell-lipid delivery vehicle fusion.
  • the method of the sixth aspect of the invention will be carried out under conditions in which the peptide and complementary peptide are able to form a stable coiled coil motif.
  • the conditions may be readily optimised for any given pair of peptides.
  • the optimum pH will depend upon the amino acid sequence of the peptides, and for Peptides ⁇ " and "K", a pH of 5 and higher is appropriate.
  • the optimum temperature depends upon the amino acid sequence and number of heptad repeat units of the peptides and should remain below the melting temperature of the coiled coil motif. It will be appreciated that the higher the temperature the higher the rate of fusion.
  • a vehicle comprising a peptide capable of forming a coiled coil with a complementary peptide and an active agent
  • the active agent will nevertheless be brought to the cell by virtue of coiled coil formation between the peptide of the vehicle and the complementary peptide that is inserted into the cell membrane.
  • the peptide capable of forming a coiled coil with a complementary peptide may be part of a non-lipid based delivery vehicle that encapsulates an active agent (eg cyclodextrin vesicle, inorganic particles or hydrogels).
  • an active agent eg cyclodextrin vesicle, inorganic particles or hydrogels.
  • the vehicle may be any molecular structure containing the active agent, in which the peptide capable of forming a coiled coil with a complementary peptide is accessible and available to form that coiled coil interaction.
  • the lipidated complementary peptide may correspond to the molecule of the first aspect of the invention or to the lipidated form of the complementary peptide described in the context of the third aspect of the invention, and so the same preferences outlined above with respect to those aspects of the invention equally apply to this aspect of the invention.
  • any suitable peptide can be used for the complementary peptide provided that it can form a coiled coil motif, and preferably a hetero coiled coil motif, with the peptide of the vehicle.
  • Suitable pairs of peptides that are capable of forming coiled coils are described above and include, for example, a peptide comprising the heptad unit (E I A A L E K) (eg peptide "E"), and the complementary peptide comprise the heptad unit (K I A A L K E) (eg peptide "K”); as well as Peptides P1 and P2, Peptides P3 and P4, Peptides P5 and P6 and Peptides P7 and P8.
  • the lipid component of the lipidated complementary peptide may be inserted into the cell membrane by carrying out the method of the fourth aspect of the invention.
  • the invention provides a method of delivering a water-soluble active agent to a cell, the method comprising: (a) anchoring a lipidated complementary peptide into the cell membrane of the cell by contacting the cell with the lipidated complementary peptide; and (b) contacting the cell with a vehicle comprising a peptide capable of forming a coiled coil with the lipidated complementary peptide and the active agent.
  • an active agent eg water soluble active agent
  • the active agents may simply be tethered to the surface of the cell or hydrophobic active agents may be inserted into the cellular membrane or water-soluble active agents may be delivered to the cytoplasm of the cell.
  • Assessing whether the active agent has been delivered to the cell surface can be done using any suitable method known in the art.
  • the agent may be detectably labelled and the location of the agent assessed using standard imaging techniques (eg confocal imaging). Assessing whether the active agent has been delivered into the cell (as opposed to on the cell surface) may also make use of standard imaging techniques and may involve assessing whether there has been lipid mixing or content mixing, for example when a lipid delivery vehicle is used, as is described in the Examples.
  • the method may be carried out in vitro, for example on cultured cells or on zebrafish embryos, or in vivo.
  • the method may be used to carry out basic research or to study membrane dynamics. For example, through coiled coil formation, a cell membrane may be decorated with a fluorescent molecule or object, and the movement, lifetime and/or fate of this molecule or object subsequently measured over time. In this way, dynamics occurring in or at the membrane may be assessed.
  • the method has utility in preclinical pharmacological analysis as outlined further below.
  • the products and methods of the invention have particular value in screening for compounds, for example in drug screening, and so are useful in drug discovery.
  • the technology of the invention allows one to modify the membranes of cells so that agents can be subsequently delivered to the cells, and so the toxicity or efficacy of potential drug molecules may be screened.
  • such uses are carried out in vitro, but may also be carried out in vivo for example when conducting clinical trials of a particular drug.
  • the products and methods of the invention may be useful in pharmacological analysis, such as in a preclinical setting using animal models or zebrafish embryos.
  • the invention provides a molecule according to the first aspect of the invention, a composition according to the second aspect of the invention, a kit of parts according to the third aspect of the invention or a cell according to the fifth aspect of the invention, for use in drug discovery.
  • the products and methods of the invention may also be used in a medical context, for example to deliver therapeutic or diagnostic agents to particular cells in vivo.
  • the cells of the subject to which it is desired to deliver a particular active agent to may be 'decorated' with a lipidated peptide that is capable of forming a coiled coil with a complementary peptide, as described herein.
  • the cells may be 'decorated' either in vivo (in which case the lipidated peptide will be first administered with an appropriate targeting moiety), or in vitro. In the latter case, the appropriate cells may have been removed from a subject so that they can be 'decorated' with the peptide in vitro.
  • the cells may then be exposed to a vehicle comprising a peptide capable of forming a coiled coil with the peptide that was used to 'decorate' the cells and the active agent.
  • vehicle may be a lipid delivery vehicle as described herein, or simply a vehicle in which the peptide is attached, either directly or indirectly to the active agent.
  • the cells may be exposed to the vehicle in vivo or in vitro. When the cells are exposed to the vehicle in vitro, conveniently this immediately follows decoration of the cells such that the cells, together with delivered active agent, can be reintroduced into a subject.
  • the invention provides a molecule according to the first aspect of the invention, a composition according to the second aspect of the invention, a kit of parts according to the third aspect of the invention or a cell according to the fifth aspect of the invention, for use in medicine.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a molecule according to the first aspect of the invention, a composition according to the second aspect of the invention, a kit of parts according to the third aspect of the invention or a cell according to the fifth aspect of the invention, and a pharmaceutically acceptable carrier.
  • the various products of the invention Whilst it is possible for the various products of the invention to be administered alone, it is preferable to present them as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the product (and optionally the therapeutic agent contained within it) and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association a product of the invention with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association a product of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of a product of the invention; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • a product of the invention may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine a product of the invention in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross- linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of a product of the invention therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of a product of the invention.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • the invention includes a molecule, or a composition, or a cell, or a kit of parts substantially as described herein, optionally with reference to the Examples.
  • the invention includes a method of delivering an active agent to a cell substantially as described herein, optionally with reference to the Examples.
  • the invention includes a method of anchoring a lipidated peptide capable of forming a coiled coil with a complementary peptide, into a cell membrane, substantially as described herein, optionally with reference to the Examples.
  • the invention will now be described with the aid of the following Figures and Examples.
  • FIG. 1 (a) Space filling model of the lipidated oligopeptides LPE and LPK, consisting of a DOPE tail, linked via a PEG12 spacer to the coiled-coil forming oligopeptides E and K.
  • Oligopeptide E is a modified form of peptide ⁇ ' having the amino acid sequence G(EIAALEK) 3 -NH2, where the G residue is attached to the PEG12 spacer.
  • Oligopeptide K is a modified form of peptide 'K' having the amino acid sequence (KIAALKE) 3 GW-NH2, where the W residue acts as a fluorescent probe,
  • (b) The spontaneous incorporation of the DOPE tail in lipid bilayers results in liposomes decorated with either E or K peptides at the surface.
  • a liposome population carrying LPE (1) is mixed with a liposome population carrying LPK (2), coiled-coil formation (E K) initiates liposome fusion (3).
  • E K coiled-coil formation
  • Figure 4 Cartoon depiction of molecules of the invention, showing that molecules may be composed of three parts: a peptide capable of forming a coiled coil motif with a complementary peptide; a flexible spacer and a lipid anchor to allow insertion into a lipid membrane (eg cell membrane).
  • a peptide capable of forming a coiled coil motif with a complementary peptide
  • a flexible spacer and a lipid anchor to allow insertion into a lipid membrane (eg cell membrane).
  • lipid membrane eg cell membrane
  • Lipid (A) and content mixing (B) graphs upon mixing two batches of liposomes decorated with the lipidated peptides, showing that the peptides coupled to the cholesterol derivative induces fusion more efficiently than the peptides coupled to DOPE (CPK/CPE vs LPE/LPK).
  • FIG. 6 Confocal images obtained from CHO cells.
  • A, B, C First, a 5 ⁇ solution of CPK in HBSS was added to the cells, 5 min incubation time. Subsequently, CPE liposomes with 1 mol% DHPE-TR were added to the cells. Images D, E, F were obtained from CHO cells in a similar experiment, however no CPK was added.
  • Figure 7. Confocal images of CHO cells. A solution of CPK was added with a 5 min incubation time. Subsequently, CPE decorated liposomes with A) 1 mol% DHPE-OG and 1.5 mol% DHPE-TR and B) 1 mol% DHPE-OG were added with a 5 min. incubation.
  • Figure 8. Confocal images of CHO cells. CPK was added, 5 min incubation time. Subsequently, CPE liposomes with 1 mol% DHPE-TR were added, 5 min incubation time.
  • Figure 9. Fluorescence stereomicroscope images of zebrafish embryos incubated with 5 ⁇ CPK in egg water for 30 mins at 34°C followed by 0.25mM Liposomes decorated with 1% CPE and 1% TR (Texas red) for 30 min at 34°C where A and B are the images taken while having focus on the yolk and C and D images were focused on the skin. Control group was treated with fluorescent liposomes only.
  • FIG 10 Quantification of the fluorescence using pixel counter software. Experimental shows 3-5 fold more interaction of fluorescent liposomes with surface of zebrafish. A is analysis performed on the images focused on the yolk while B is focused on the skin.
  • Figure 11. Fluorescence graph showing that the cholesterol anchor is better at inducing lipid mixing than the DOPE anchor. Measurements were started after mixing 0.1 mM K decorated liposomes (1% lipopeptide) and 0.5 mM E decorated liposomes (1% lipopeptide).
  • Figure 12 Fluorescence image showing enhanced lipid membrane insertion properties of cholesterol anchor.
  • Example 1 Cell surface engineering and direct drug delivery mediated by lipidated coiled-coil motifs.
  • a cholesterol derived "K” peptide (denoted CPK) is inserted into the cell membrane through spontaneous self assembly by the addition of a CPK solution to the cell culture medium.
  • CPK cholesterol derived "K” peptide
  • This specific binding motif enables a large variety of opportunities to engineer cell membranes (i.e. cell surface).
  • One can think of a seemingly limitless amount of entities e.g.
  • liposomes liposomes, vesicles, polymersomes, (in)organic nanoparticles, nanoemulsions, self-assembled systems, biomolecules like proteins, antibodies etc) which could be conjugated to the ⁇ " peptide and subsequently bound to the "K" peptide at the surface of the cell.
  • CPE lipidated ⁇ " peptide
  • Membrane fusion is a key process in all living cells as it facilitates the transport of molecules between and within cells.
  • a primary mechanism by which molecules are conveyed to the appropriate location is to encapsulate them in liposomes that deliver the cargo by fusing with the lipid membrane of the target cell or compartment.
  • In vivo membrane fusion is triggered by the specific interaction of SNARE (soluble N- ethylmaleimide-sensitive factor attachment protein receptors) fusion proteins that brings two membranes into close proximity followed by local disruption of the lipids and merging of the membranes.
  • SNARE soluble N- ethylmaleimide-sensitive factor attachment protein receptors
  • proteoliposomes fusion-related proteins
  • liposomes i.e. proteoliposomes
  • proteoliposomes reconstituted in liposomes
  • these reconstituted proteoliposomes resulted in contradicting results[5] and to date, none of these systems have shown to posses all the characteristics of in vivo systems.
  • This synthetic fusion system is the first generic method for the direct introduction of (bio)molecules into the cytoplasm of cells.
  • SNARE proteins Native membrane fusion is initiated by a set of proteins called SNARE proteins.
  • the natural SNARE protein complex can be dissected in three distinct regions: I) a protein recognition domain.[17, 18] II) a flexible spacer domain and III) a transmembrane domain (TMD) anchoring the proteins to the lipid membranes.
  • TMD transmembrane domain anchoring the proteins to the lipid membranes.
  • the protein recognition domain of SNARE proteins is an 8 heptad repeat segment that has a high coiled-coil forming propensity.
  • the tetrameric coiled-coil forming domain is mimicked by a complementary pair of coiled- coil forming peptides composed of three heptad repeat units (E and K, Figure 1 b). These oligopeptides are the shortest known coiled-coil pair which assembles specifically into a stable heterodimer. [21-23] For isolated peptides the mixture E/K forms stable heterodimers (dissociation constant ⁇ 10-7 M at 25 °C), whereas homodimer formation is prevented by unfavourable electrostatic repulsions. In this model system the LPE/LPK complex formation is the driving force to bring two different liposomes close together.
  • the role of the flexible spacer is fulfilled by a short poly(ethylene glycol) chain with 12 ethylene glycol units (PEG12, Figure 1b).
  • PEG12 poly(ethylene glycol) chain with 12 ethylene glycol units
  • This spacer allows for extension of the oligopeptide component from the surface of the liposomes.
  • the lipidated oligopeptides are spontaneously anchored in the membrane by means of a phospholipid tail, 1,2- dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), mimicking the function of the transmembrane domain of SNARE proteins ( Figure 1b).
  • DOPE 1,2- dioleoyl-sn-glycero-3-phosphatidylethanolamine
  • cells are in a first step pretreated with a solution containing the "K" lipopeptide (CPK, vide infra) and in a second step liposomes bearing the complementary peptide amphiphile E (CPE, vide infra) at its surface are added resulting in coiled-coil formation (i.e. liposome binding to the surface) and subsequently liposome-cell membrane fusion (i.e. lipid mixing and content mixing).
  • CPE complementary peptide amphiphile E
  • Experimental embryos shows uniformly glowing skin which is a result of a coiled-coil interaction of CPK present on the membrane and CPE present on the surface of the fluorescently labeled liposomes.
  • respective images were analysed using pixel counter and Figure 10 shows that experimental group has 3-5 fold more interaction of liposomes with the surface of zebrafish which confirms the surface engineering of the zebrafish embryos.
  • the peptide segments (E (EIAALEK)3 and K (KIAALKE)3) of the hybrids were synthesized on a 100 prnol scale using Fmoc chemistry on an automatic Syro peptide synthesizer. A sieber amide resin with a loading of 0.69 mmol/g was used. Amino acid couplings were performed with 4 eq. of the appropriate amino acid, 4 eq. of the activator HCTU and 8 eq. of the base DIPEA, for 1 hour. Fmoc-deprotection was performed with piperidine:NMP (4:6 v/v).
  • Fmoc-NH-PEG12-COOH was coupled to the peptide on the resin.
  • the resin was swollen in N P for 1 hour.
  • 2 eq. of Fmoc-NH-PEG12-COOH, 4 eq. of DIC and 4 eq. of HOBT were dissolved in NMP and left to preactivate for 2 minutes before it was added to the resin.
  • the coupling was performed overnight and the Fmoc group was removed.
  • 4 eq. of the hydrophobic anchor, 4 eq. of DIC and 4 eq. of HOBT was dissolved in NMP.
  • the mixture was left to preactivate for 2 minutes and coupling was performed overnight.
  • the peptide hybrids were cleaved from the resin by shaking the resin with a mixture of TFA/TIS/H20 (95:2.5:2.5 v/v) for 1 hour.
  • the cleavage mixture was collected and after co-evaporation with toluene, the crude product was obtained.
  • the purification of the hybrid peptides was performed by RP-HPLC with a Shimadzu system with two LC- 8A pumps and a SPD-10AVP UV-VIS detector and a Gemini C18 column. UV detection was performed at 214 nm.
  • the peptide hybrids were dissolved in a mixture of tert- butanol:acetonitril:water (1:1:1 v/v) and eluted with a flow rate of 20 mUmin.and with a linear gradient from A to B, where A was H20 with 0.1 vol% TFA and B was acetonitrile with 0.1 vol% TFA.. Samples were eluted with a linear gradient from 10% to 90% B over 3 column volumes. The purity was checked via LCMS and estimated to be greater than 95%.
  • CHO cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% of fetal calf serum (iron supplied), 2% of L- glutamine, 1% of penicillin and 1% of streptomycin. The cells were cultured with 5% of C02. The medium was refreshed every two days and the cells were passaged by trypsinization before reaching 100% confluence. For liposome tagging experiments, cells were grown in an 8 chamber Lab-Tek slide to a confluency of around 80% over 48 hours.
  • DMEM Dulbecco's Modified Eagle's Medium
  • CPE and CPK stock solutions were prepared with a 10 ⁇ concentration in a 1:1 (v:v) mixture of chloroform.methanol.
  • Lipid stock with a total concentration of 1 mM solutions were constituted by 50% DOPC, 25% DOPE and 25% CH.
  • CPK solutions (5 ⁇ ) that were incubated with the cells were prepared by taking the appropriate amount of CPK stock solution, drying the solvent under a stream of air, adding Hanks Buffered Saline Solution (HBSS) and sonication for 1 minute at ⁇ 50 °C.
  • Fluorescent liposomes (250 ⁇ ) that were decorated with 1 mol% CPE were prepared by adding the appropriate amounts of lipid, CPE and fluorescent probe (DHPE-TR and/or DHPE-OG, typically 1 mol%). After the solvent was dried, HBSS was added and the solution was sonicated at ⁇ 50 °C for 1 minute. The hydrodynamic diameter of the liposomes was shown to be ⁇ 100 nm as determined by dynamic light scattering.
  • Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (zfin.org). In this study albino strain was used. Family crossing was used to perform fertilization. Embryos were grown at 28°C in egg water (60 pg ml Instant Ocean salts). Treatment was performed on 2 day post fertilized (dpf) embryos. Albino zebrafish surface (Skin) were engineered using Coiled- coil peptide bearing liposomes. Albino zebrafish embryos after 2 days of fertilization were incubated with 5 ⁇ CPK in egg water for 30 mins at 34 0C.
  • the membrane fusion model system initially consisted of the lipopeptides LPE and LPK. Upon studying the influence of the anchor that confines the peptides at the surface of the liposomes, it was found that the cholesterol anchor has several advantages over the DOPE anchor. Multiple rounds of fusion as observed through lipid mixing
  • the final stage of full fusion is the mixing of the aqueous compartments of the liposomes. It was observed that the cholesterol anchor induced content mixing more significantly than DOPE.
  • Example 3 Enhanced lipid membrane insertion property of cholesterol anchor
  • CHO cells were incubated with 1 ⁇ CPE (cholesteryl-Peg12-E-cf, cf is carboxyfluorescein) and lipid membrane insertion monitored by fluorescence microscopy.
  • CPE cholesterol-Peg12-E-cf, cf is carboxyfluorescein
  • the fluorescence images show that within 3-5 min, the lipopeptide is anchored on the cell membrane at 37°C.

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Abstract

L'invention concerne une molécule qui comporte un peptide, capable de former une superhélice avec un peptide complémentaire, et une fraction stéroïdienne. L'invention concerne également un procédé permettant d'administrer un agent actif à une cellule, le procédé consistant à amener la cellule à entrer en contact avec un véhicule comportant un peptide capable de former une superhélice avec un peptide lipidé complémentaire et l'agent actif, le constituant lipidique du peptide lipidé complémentaire étant inséré dans la membrane de la cellule.
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US11576872B2 (en) 2017-05-08 2023-02-14 Flagship Pioneering Innovations V, Inc. Compositions for facilitating membrane fusion and uses thereof

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