WO2010020798A2

WO2010020798A2 - Nanoparticles

Info

Publication number: WO2010020798A2
Application number: PCT/GB2009/002061
Authority: WO
Inventors: John Fabre; Louise Collins; Alan L. Parker
Original assignee: King's College London
Priority date: 2008-08-21
Filing date: 2009-08-21
Publication date: 2010-02-25
Also published as: WO2010020798A3

Abstract

The invention relates to a system for delivering biologically active molecules to the cytosol of cells. In particular, the invention relates to substantially spherical nanoparticles comprising two polyvalent components of opposite charge.

Description

Nanoparticles

The invention relates to a system for delivering biologically active molecules to the cytosol of cells. In particular, the invention relates to substantially spherical nanoparticles comprising two polyvalent components of opposite charge, the nanoparticles being capable of entering the cells by an endocytic process, and thereafter either escaping from the endocytic vesicles into the cytosol or crossing cells via transcytosis. Either component in the nanoparticles may be conjugated to biologically active molecules or moieties to allow stable (in vitro and in vivo) delivery of those molecules to and into the target cells and to hide those molecules from opsonins, immune recognition, oxidation, degradation or other harmful process. Additionally, the components or particles may be conjugated to diagnostic moieties. All these moieties may be passively incorporated into the nanoparticulate complex.

Self-assembly of nanoparticles from monomelic components is being pursued for a variety of objectives in the physical and biological sciences. The properties of monomelic components which can self-assemble can vary widely.

The inventors have investigated ways of delivering membrane-impermeable substances to the cytosol. The delivery to the cytosol of such substances (such as biologically active peptides and proteins), could have wide ranging experimental and clinical applications, and represent an entirely novel approach to therapeutics. However, transversing the plasma membrane represents a considerable problem. "Cell-penetrating" peptides are known in the art. They were originally considered to translocate directly across the plasma membrane and have been extensively investigated. These are small, usually arginine-rich peptides, which occur either as natural sequences, for example in the TAT protein of human immunodeficiency virus type-1, or as synthetic polyarginines. Such peptides, and the cargo molecules to which they are attached, are now believed to be internalised by endocytosis. This raises potential problems for optimisation of efficiency, in addition to problems of in vivo stability (especially susceptibility to proteases) and targeting.

Fusion peptides, comprising polyarginine and a fusogenic peptide, such as a part of the influenza virus have also been used to transport therapeutic agents into the cell cytosol (Michine et al., J. Biol Chem 2005. 280, No. 9, 8285-8289). It is also known to use targeted polylysine derivatives mixed with fusogenic peptides for enhanced delivery of DNA plasmids.

BRIEF DESCRIPTION OF THE INVENTION

In order to improve the delivery of therapeutic agents to the cytosol or across cells, the inventors have produced nanoparticles comprising a polyvalent charged components that may be conjugated to biologically active or diagnostic molecules. Surprisingly, the inventors have been able to produce regular nanoparticles by mixing the two components.

According to the invention, there is provided a nanoparticle comprising a plurality of polyvalent charged rigid brick molecules connected by a plurality of polyvalent oppositely charged flexible strap molecules, the brick molecules being proteins or peptides or other rigid structures. The brick molecules are preferably proteins or peptides. The nanoparticle is preferably substantially spherical. When combined the strap and brick components produce nanoparticles which may be positively, neutrally or negatively charged.

The inventors have surprisingly found that they are able to create spherical nanoparticles by combining oppositely charged molecules, some rigid and some flexible, the molecules interlinking by way of their charges to form the particles.

The brick and strap molecules are polyvalent, so each brick can link to at least two straps and each strap to at least two bricks. Preferably each brick is able to link to three or more straps and each strap to three or more bricks.

The first component used by the inventors is termed herein a brick molecule. This is a rigid structure, often a peptide oligomer or a protein. The term rigid, when used herein, means the level of rigidity normally associated with molecules like proteins which have assumed a tertiary structure. In other words, the molecule has some flexibility but will not significantly change shape or significantly lose configuration or conformation at neutral pH or at physiological temperature, especially when combined with the strap molecules. The molecule generally retains its three dimensional structure. The brick molecules preferably have a non polar core, but are charged on at least part of their surface, preferably over the majority of their surface. The brick molecules are rigid structures, preferably proteins or peptides. The charge on the surface may be provided by amino acids within those proteins or peptides or may be provided by modifications made to the surface of the brick molecules for example by linking amino acids or glycosaminoglycans, polyethylinimines or other charged molecules to the brick molecule.

The brick molecules may have a variety of shapes, for example may be generally globular, cylindrical, rod shaped or barrel shaped.

In one embodiment, the brick molecule is a peptide oligomer, hi that case the peptide preferably forms an ampipathic alpha helix, that is to say an alpha helix in which the charged amino acid residues are arranged so that the charge is found on one side of the alpha helix. Such peptides are well known in the art. Examples of such peptides include peptides having the following amino acid sequences: GLFGAIAGFIENGWEGMIDG; and

WEAALAEALAEALAEHLAEALAEALEALAA

The brick molecule preferably comprises an oligomer comprising peptides having charged amino acids, especially negatively charged amino acids such as Glutamic Acid or Aspartic Acid, every 3rd to 4th residue, hi particular the peptide preferably comprised the following sequence: GLFEALLELLESLWELLLEA.

When the brick is made up of a peptide oligomer, the brick is preferably barrel shaped or cylindrical. The oligomer is preferably a nonamer or decamer.

Particularly useful as peptides forming brick molecules are fusogenic peptides, as these enable the nanoparticles to escape from endocytic vesicles.

In another embodiment, the brick is a protein. In particular, the brick may be a histone or histone-like protein or other strongly cationic protein. Histones are well known in the art.

Alternatively, the protein brick may be a negatively charged protein such as fetuin.

hi a further embodiment, the brick is a naturally occurring protein with little or no surface charge that has been modified by the addition of charged amino acids or other charged molecules to its surface, the charged residues being linked to the protein by, for example, disulphide bonds.

The surface charge on the brick components may be evenly distributed or may be arranged in certain areas. It is preferably reasonably evenly distributed such that there are no large uncharged areas.

The strap components are flexible components which connect the bricks. The straps are preferably elongate in at least one direction and may be elongate in two directions. For example, the strap molecules may be linear, that is elongate in only one direction.

Alternatively, the molecules may be sheet like, elongate in two dimensions, but not in the third.

When the strap component is a peptide it preferably remains unfolded, rather than forming a secondary structure such as an alpha helix.

The strap may comprise repeating units. When the strap is a peptide such units may contain polar amino acids residues. For example, the chain may solely comprise charged amino acids residues, such as lysine, arginine, aspartate and glutamate. Alternatively charged amino acids may be interspersed with less polar amino acids or non polar amino acids. Examples of peptide chains useful as strap components include polylysine, polyarginine, polyaspartate, polyglutamate, a peptide chain comprising alternating lysine or arginine and histidine residues. Particular repeating units of interest include repeating blocks or units of lysine, arginine or histidine residues, especially lysine residues, especially K₁₆, linked by disulphide bonds in cystine residues. So the strap may contain repeating units of [C K₁₆C]. The strap may, for example contain between 4 and 10 of such units. The strap preferably includes at least 5 charged areas, when it is a peptide, it preferably includes at least 5 more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 12, more preferably at least 16 charged amino acid residues.

Specifically those charged residues are oppositely charged to the brick components. Preferred strap molecules include: K₁₆

KHKHKΉKΉKHKΉKΉKHKHKHKHKHKHKHKHKH GLFEALLELLESLWELLLEADP-K_I6

Commercially available polylysine containing, for example, 100 to 200 lysine residues per chain

K₁₆- ICRRARGDNPDDRCT K₁₆-IGRRARGDNPDDRCT

K₁₆-ICRRARGENPDDRCT K₁₆-CPPASYRGDSCQE K₁₆- H]₆

K₁₆-CSIPPEVKFNKPFVFLI and K_(6-16) FNKPFVFLI

K₁₆PVKRRLFG

The charge on the strap is preferably distributed along its length. This helps to prevent the strap folding, especially when it is a peptide.

Other strap molecules include anionic peptides such as (GIu)₁₆ and polyaspartate, heparins, hydrocarbons, poly(ethyleneimine), cationic lipids, and polyamidoamine dendrimers

As stated, the brick and strap components are oppositely charged. The brick may be a cation and the strap an anion, or the brick may be anionic and the strap cationic.

When the brick is anionic, especially an anionic peptide oligomer, the strap is preferably a peptide comprising at least five lysine or arginine residues. In particularly the strap preferably comprises a series of at least five, more preferably at least eight, even more preferably at least twelve lysine or arginine residues. Alternatively the strap preferably comprises lysine or arginine residues interspersed, especially alternating, with histidine residues.

When the brick is cationic, especially a cationic protein such as a histone, the strap is preferably (GIu) i₆ or polyaspartate.

The nanoparticles of the invention are useful for delivering compounds, especially for therapeutic purposes, either one of the brick and strap components or molecules attached to the brick or the strap. For example small molecules may be attached to the brick or the strap. Such molecules may be encapsulated by the nanoparticle and, hence, prevented from degradation or other chemical modification before arrival at the target site. Such small molecules may include therapeutic compounds. Alternatively one or both of the brick or strap or a metabolite of one of these may be a therapeutic agent.

The brick and strap components may also comprise additional residues, peptides or other molecules, especially those having a therapeutic or diagnostic use. In particular, the components may also comprise a biologically active peptide, especially a peptide encoding a binding region or a ligand for an enzyme or receptor.

Each nanoparticle comprises a plurality of each of the components. The components in each particle may be the same or may be different. In each nanoparticle the bricks may all be the same, or may be different. Equally the straps may all be the same, or may be different. Also, where the components comprise biologically active regions, or wherein biologically active or diagnostic molecules have been attached to the components, the regions or molecules may have different biological activities. More than one biologically or diagnostically active molecule may be included in each nanoparticle, or nanoparticles containing different molecules may be used together.

Each nanoparticle can contain any suitable ratio of straps to bricks as long as there are sufficient straps or bricks to form a nanoparticle. Preferably, the ratio of straps to bricks is between about 5:1 and about 1:10. More preferably, the ratio of straps to bricks is between about 1:1 and about 1:5, even more preferably, between about 1:2 and about 1:3 and, most preferably, about 1:2.5. The ratio of bricks to straps required may be determined by considering the natural stoichiochemistry of the brick molecules. The surface charge of the brick molecules may be considered and an appropriate ratio of strap molecules to brick molecules selected.

Additionally, targeting molecules may be attached to the brick or strap components, to target the nanoparticles to sites of interest.

In particular, an attached target may be a peptide motif. Many peptide motifs have been defined to target different cell types and motifs are well known in the art. Other cell targets may also be used. For example, the RGD motif is often used to target integrins, and mannose sugar residues used to target the asialoglycoprotein receptor on hepatocytes.

The nanoparticles could be used to deliver compounds to sites of interest by, for example, endocytosis, where the nanoparticles are taken up by cells and are able to escape the endosome by virtue of one of the components. Alternatively, the nanoparticles may access sites by transcytosis being taken up by cells and ejected on the other side. In this way, it may be possible for the nanoparticles to cross the blood brain barrier to treat Alzheimer's disease or to access the interior of, for example atherosclerotic plaques.

Accordingly, there is provided a nanoparticle according to the invention for use in therapy.

In particular, a nanoparticle according to the invention for the delivery of therapeutic or diagnostic agents.

Also provided by the invention is a method for producing a nanoparticle comprising combining charged rigid peptide or protein brick components with oppositely charged flexible strap components. When the components are combined, the nanoparticles preferably self assemble.

The invention also provides a method of preparing substantially spherical nanoparticles comprising the step of: a) Mixing a cationic peptide having at least 5 lysine residues with an anionic fusogenic peptide having an amino acid sequence having substantial homology to the amino acid sequence:

GLFEALLELLESLWELLLEA

The peptides may be mixed in a salt solution (containing sodium chloride, magnesium chloride, magnesium sulphate, sodium sulphate or another salt). Preferably, the peptides are mixed in a salt solution. Preferably, when the salt is NaCI, the saline solution has a concentration of between 10 and about 30OmM, more preferably, between 10 and about 25OmM, more preferably still, between 10 and about 20OmM, even more preferably, between 10 and about 15OmM and, most preferably, between about 5OmM and about 15OmM. When the salt is magnesium chloride, magnesium sulphate or sodium sulphate, the solution preferably has a concentration of between 5mM and 25mM, more preferably between 5mM and 15mM.

The peptides may be mixed at any suitable temperature. Generally, the higher the temperature at which the peptides are mixed, the larger the size of the nanoparticles that are formed when mixed in a saline solution. Preferably, the peptides are mixed at a temperature of greater than 0°C and less than 8O⁰C.¹ To produce larger nanoparticles, the peptides are preferably mixed at a temperature of between about 4O⁰C and about 60°C.

To produce smaller nanoparticles, the peptides are preferably mixed at a temperature of between about 0⁰C and about 20⁰C.

The size of the nanoparticles may also be dependent on the initial concentration of the brick components. Generally, the higher the initial concentration of the brick component, the larger the size of the nanoparticles formed. Preferably, the initial concentration of the brick component is between about lμg/ml and about 5mg/ml and, more preferably, between about 10 μg/ml and about lmg/ml.

The method may also comprise the step of modifying the surface of the nanoparticles. The surface may be modified by, for example, cross-linking surface amino groups with HPMA. This may be used to stabilise small particle size (~150 run) of particles formed in the absence of salt. It can also be used to attach targeting moieties.

DEFINITIONS

Nanoparticle As used herein, the term nanoparticle means a particle that is between lOOnm and 1200nm, preferably between lOOnm and 700nm, more preferably between lOOnm and 500nm, even more preferably between lOOnm and 300nm, most preferably between lOOnm and 200nm in diameter. Diameter refers to the average diameter, though the nanoparticles produced in accordance with the invention are substantially spherical. Nanoparticles comprising the same components are similar in size, preferably having a polydiversity of less than 1.00, more preferably less than 0.5, more preferably less than 0.35. Rigid The term rigid is used herein to mean the rigidity usually displayed by a protein that has folded into its tertiary or quaternary structure.

Flexible The term flexible means the flexibility usually seen in a peptide chain having negative or positive charges distributed along its length, such as polylysine. The peptide can bend but cannot fold tightly due to repulsion between the charged areas.

Peptide The term peptide is used herein to mean a polymer of amino acids and include polypeptides. The term includes peptides comprising non-natural amino acids. The polymer preferably contains between 15 and 200 amino acids. The peptide may be naturally occurring or artificially created. Further the term includes naturally occurring peptides that have been modified. The term peptide does not exclude modifications such as glycosylation, acetylation or phosphorylation

Protein The term protein as used herein means a folded peptide or polypeptide chain having a stable configuration. The protein may be naturally occurring or artificially created. Further the term includes naturally occurring proteins that have been modified. Also the term may include modified proteins such as glycoproteins or lipoproteins; however, it is preferred that the term protein does not include glycoproteins or lipoproteins.

Amino acid The term amino acid is well known in the art. Herein, it means naturally occurring and non-naturally occurring, modified or artificial aminoacids.

The invention will now be described in detail by way of example only with reference to the drawings in which:

Figure 1 shows the results of particle formation by (Lys)j₆ peptides and the fusogenic peptide. a: Dynamic light scattering. A solution of the (Lys)₁₆ peptide at 15 μg/ml (3.7 μM) and the fusogenic peptide (Table 2) at 10 μg/ml (4.1 μM) (+/- charge ratio of 2.9:1) in PBS

¹ Is there an upper limit to the temperature range? Presumably, if the peptides become too hot they will denature and lose their secondary structure and the salt bridges will break down. Should there be a lower limit below was examined 30 minutes after mixing the peptides. A uniform population of particles with low polydispersity is produced. Solutions of the (Lys)i6 peptide alone and the fusogenic peptide alone do not have any particles (data not shown), b: Zeta potential of particles formed in 0.15 NaCl, 10 mM Tris, pH 7.4 with the fusogenic peptide at a constant 10 μg/ml (4.1 μM) and the (Lys)i₆ peptide (from left to right on the graph) at 7.5,

10, 15, 30, 60 and 120 μg/ml (1.9 to 30.0 μM) (+/- charge ratios of 1.5 to 23:4). c, d: Transmission electron microscopy. The (LyS)₁₆ .molossin peptide (Table 2) (carrying a biotin molecule on the amino terminal lysine) at 15 μg/ml (2.4 μM) and the fusogenic peptide at 10 μg/ml (4.1 μM) (+/- charge ratio of 1.9:1), were allowed to stand for 30 minutes before addition of streptavidin-gold. The gold-labelled particles were added to

T-24 cells, and the cells harvested 4 hours later. In c, a perfectly spherical gold-labelled particle can be seen in the extracellular fluid, close to the plasma membrane. The particle inside the cell has a less electron-dense halo, and the endocytic membrane has been completely disrupted. In d, a particle within an endocytic vesicle is shown. The endocytic membrane closest to the particle appears less dense than the rest of the endocytic membrane, and at one point (arrowed) the endocytic membrane is indented to the surface of the particle. The bars in c and d are 500 ran. e: Influence of salt. The (Lys)₁₆ peptide at 15 μg/ml and fusogenic peptide at 10 μg/ml were mixed in 1OmM Tris, pH 7.4, with various concentrations of salts as indicated. After 30 minutes, Zav was determined by dynamic light scattering. At 25mM MgSO₄ and 5OmM Na₂ SO₄ particles were smaller and fewer events were recorded (63 and 67 kilocounts per second, normally >100 kilocounts per second), f: Dynamic light scattering at the times indicated after mixing the (LyS)₁₆ peptide at 15 μg/ml and fusogenic peptide at 10 μg/ml in 10 mM Tris, pH 7.4, either in the absence of NaCl (group on left) or in 150 mM NaCl (group on right). In the middle group, the NaCl concentration was increased from zero to 150 mM at 60 minutes, as indicated, f: Influence of temperature on particle size. The (Lys)₁₆ peptide at 15 μg/ml and the fusogenic peptide at 10 μg/ml in either PBS or 10 mM Tris, were mixed and maintained at the temperatures indicated. After 30 minutes, Zav was determined by Dynamic light scattering.

Figure 2 shows the results of structural studies. a: Circular dichroism studies on the fusogenic peptide and the alanine homologue of the fusogenic peptide (Table 1, line 2) are shown. Based on model secondary structures °C? from Gratzer et al¹⁹, the fusogenic peptide shows an α-helix conformation, while the alanine homologue corresponds to a random coil, b: Distribution of negative charges (white area) on the surface of an α-helical model of the fusogenic peptide. The peptide backbone was folded into a canonical α-helix (dihedral angles φ,ψ,ω = -62,-41,180), and sidechain conformational energy was minimised using molecular dynamics in VMD²³ and the companion NAMD²⁴ software utilities while the backbone was held constant, c —f: Analytical ultracentrifugation of the fusogenic peptide, c: Molecular weight distributions fit individually to sedimentation velocity experiments for 50 (solid), 250 (dashed) and 700 (dotted) μg/ml fusogenic peptide concentrations. All distributions are maximum at a molecular mass of 22.485 kDa. d: Residuals between sedimentation velocity data and distribution fits shown in (a) for 50, 250 and 700 μg/ml fusogenic peptide in the bottom, middle and top panels, respectively. Root mean square deviations (RMSD) between the data and the fits are 0.004, 0.004, and 0.006, and the runs test Z (a measure of the randomness of the residuals, where smaller values indicate less systematic error)²⁵ are 8.9, 5.1 and 30.0 for 50, 250 and 700 μg/ml fusogenic peptide, respectively, e: Data

(symbols), fits (lines), and residuals (inset) of the global analysis incorporating all sedimentation velocity and equilibrium data. Every third scan of the 50 μg/ml is shown; fits to sedimentation velocity data for other time points as well as 250 and 700 μg/ml fusogenic peptide concentrations are similar, f: Data for 35k rpm (circles) and 50k rpm (squares) sedimentation equilibrium experiments with global analysis fits (lines) and residuals (inset).

Figure 3 shows properties of peptide particles. a - c : Second round particle formation. Particles were formed using the (Lys)₁₆ peptide at 120 μg/ml and the fusogenic peptide at 10 μg/ml in PBS. m (b) and (c), and additional

10 μg/ml of fusogenic peptide was added at 28 minutes. Dynamic Light Scattering was performed at 30 minutes (a and b) and 45 minbutes (c). d-f: Order of peptide addition. Peptide particles were formed in PBS by the standard method of adding 10 μl of fusogenic peptide at 1 mg/ml to 990 μl of (LyS)₁₆ peptide at 15 μg/ml (f), or by adding 15 μl of (Lys)i₆ peptide at 1 mg/ml to 990 μl of fusogenic peptide at 10 μg/ml (d), or by mixing equal volumes of (Lys)₁₆ at 30 μg/ml with fusogenic peptide at 20 μg/ml. g,h: Polymer coating of peptides, g: The (Lys)i₆ peptide at 15 μg/ml and the fusogenic peptide at 10 μg/ml were mixed in 10 mM NaCl, 20 mM Hepes, pH 7.8. HPMA was added after 30 minutes to 0.1 or 0.5 mg/ml. Dynamic light scattering was performed after 30 minutes and 20 hours, as indicated. At 20 hours, the NaCl concentration was increased to 150 mM, and dynamic light scattering was performed 30 minutes later, h: Peptide particles were formed and exposed to HPMA as in c, except that 20 mM Hepes, pH 7.8 was used without salt. Zeta potential was measured at 20 hours.

Figure 4 shows the particles formed at various peptide molar ratios; a - d : Dynamic light scattering 30 minutes after mixing peptides in PBS. The fusogenic peptide (Table 1) at a constant 10 μg/ml (4.1 μM) was mixed with the (LyS)₁₆ peptide at various concentrations. At a (Lys)₁₆ concentration of 3.75 μg/ml (0.9 μM) (+/- charge ratio of 0.7) there was no particle formation (data not shown), a : (LyS)₁₆ at 7.5 μg/ml (1.9 μM) (+/- charge ratio of 1.5); b : (Lys)₁₆ at 30 μg/ml (7.49 μM) (+/- charge ratio of 5.8); c : (Lys)₁₆ at 120 μg/ml (30 .0 μM) (+/- charge ratio of 23.1); and d : (Lys)₁₆ at 240 μg/ml.

Figure 5 shows the results of particle formation in conditions of low or absent salt ions

Dynamic light scattering 30 minutes after mixing the fusogenic peptide at 10 μg/ml and the (Lys)16 peptide at 15 μg/ml in pure water (zero point) or in dilutions in water of 10

Figure 6 shows the results of varying salt concentration on particle size. Dynamic light scattering at 30 minutes and 60 minutes after mixing of the (LyS)₁₆ peptide at 15 μg/ml (3.7 μM) and the fusogenic peptide at 10 μg/ml (4.1 μM) (+/- charge ratio of 2.9:1) in 10 mM Tris, pH 7.4, with NaCl at the concentrations indicated.

Figure 7 is a schematic drawing showing the interaction of the bricks (10) and straps (12).

Figure 8 shows particle formation at various anionic/cationic peptide ratios. Dynamic light scattering 30 minutes after mixing of the fusogenic peptide at 10 μg/ml and the (Lys)16 peptide at 7.5 (a), 30 (b), 120 (c) and 240 (d) μg/ml in PBS.

Figure 9 shows particle formation in conditions of low or absent salt ions. Dynamic light scattering 30 minutes after mixing the fusogenic peptide at 10 μg/ml and the (Lys)16 peptide at 15 μg/ml in pure water (zero point) or in dilutions of 10 mM Tris pH 7.4 in water. The 10 mM Tris was adjusted to pH 7.4 with HCl.The pH of the solution of peptides in pure water was 5.9, and was 7.3, 7.3, 7.1, 6.9 and 6.6 for the solution of peptides in 10 mM, 5 mM, 2.5 mM, 1.25 mM and 0.63 mM Tris.

Figure 10 shows the influence of time on particle size. Dynamic light scattering 30 minutes and 60 minutes after mixing the fusogenic peptide at 10 μg/ml and the (Lys)16 peptide at 15 μg/ml in 10 mM Tris, pH 7.4, with NaCl at the concentrations indicated.

Figure 11 shows Zeta potential after polymer coating. The (Lys)16 peptide at 15μg/ml and the fusogenic peptide at 10 μg/ml were mixed in 10 mM NaCl, 20 mM Hepes, pH 7.8. HPMA was added after 30 minutes to 0.1 or 0.5 mg/ml. Zeta potential was measured at 20 hours.

Figure 12 shows the results of forming particles using non-peptide polycations such as PEI with fusogenic peptide.

Figure 13 shows that flow cytometry can be used to count peptide particles

EXAMPLES EXAMPLE 1

SUMMARY

Mixing aqueous solutions of a (Lys)₁₆ peptide and a 20 amino acid peptide of net charge - 5 (GLFEALLELLESLWELLLEA) yields almost perfectly spherical particles of -120 nm to -800 nm diameter (normally -600 nm diameter). Particle formation is probably a consequence of the unusual properties of the anionic peptide: it forms an α-helix in aqueous solution, all five anionic glutamates are on one side of the helix, and the peptide exists entirely as a discrete oligomer of 9 - 10 peptides, probably a decamer. A rigid oligomer of the anionic peptide, with 45 - 50 negative charges, almost certainly represents the core component of these nanoparticles. Cells internalise the particles by an endocytic process, and free particles are frequently seen in the cytosol, presumably because of the acid-dependent fusogenic properties of the anionic peptide. These particles might represent a novel class of self-assembled nanoparticles, and have potential for the targeted delivery of single or multiple therapeutic moieties directly to the cytosol. Self-assembly of nanoparticles from monomelic components is being pursued for a variety of objectives in the physical and biological sciences¹'². The properties of monomelic components which can self-assemble vary widely e.g.^3"5. However, one would not expect the mixing of two small water-soluble peptides of opposite net charge to result in their self-assembly into regular nanoparticles. Nevertheless, we have found that a (Lys)i₆ peptide plus a 20 amino acid peptide of net charge -5 (GLFEALLELLESLWELLLEA) produce virtually perfect spheres of -600 run in physiological salt solutions.

The anionic peptide has acid-dependent fusogenic properties⁶'⁷, and is based on the amino acid sequence of the fusogenic region of influenza virus haemagglutinin ' . The biological function of influenza haemagglutinin is acid-dependent membrane fusion (influenza is an enveloped virus) resulting in the release of viral contents into the cytosol following endocytosis of the virus. The anionic peptide has previously been demon- strated to promote the endocytic escape of non- viral vector/DNA complexes⁶'⁷'¹⁰'¹ ' .

The delivery to the cytosol of membrane-impermeable substances (such as biologically active peptides and proteins) could have wide-ranging experimental and clinical applications, and represents an entirely novel approach to therapeutics. However, traversing the plasma membrane represents a considerable problem. "Cell-penetrating" peptides were originally considered to translocate directly across the plasma membrane and have been extensively investigated^12"14. These are small, usually arginine-rich peptides, which occur either as natural sequences (e.g. in the TAT protein of human immunodeficiency virus type-1)¹⁵ or as synthetic polyarginines¹⁶. Such peptides, and the cargo molecules to which they are attached, are now believed to be internalised by endocytosis¹⁷'¹⁸. This raises potential problems for optimisation of efficiency, in addition to problems of in vivo stability (especially susceptibility to proteases) and targeting.

The peptide nanoparticles described in this paper represent an alternative approach, specifically targeted for delivery to the cytosol via the endocytic pathway. From a structural point of view, they are of interest as their formation is almost certainly a consequence of the unusual physical properties of the fusogenic peptide, and they probably represent a novel class of nanoparticle based on a rigid polyanionic core component. From a therapeutic point of view they offer the possibility of carrying multiple peptide components in predetermined proportions to the cytosol, involving both cellular targeting and the exposure to multiple active compounds. The possibility of protecting chemically labile groups within the particle might represent an additional advantage.

RESULTS

General properties of the peptide particles

A typical Dynamic Light Scattering (DLS) profile 30 minutes after mixing the (Lys)₁₆ peptide and the fusogenic peptide under the standard conditions is given in Fig. Ia. A uniform population of particles with a Zav of 731 nm and a positive surface charge (zeta potential of +3.0 mV) can be seen. It is important to note that both peptides are readily soluble in pH neutral aqueous solutions, and that neither peptide alone gives any particles in solution.

hi order to visualise the particles in a natural state, while at the same time evaluating their interaction with cells in culture, we exposed the T-24 cell line to peptide particles, and then fixed the cells in situ for electron microscopy. Cells sometimes contain electron- dense inclusions, and so the peptide particles were positively identified by using biotinylated (LyS)₁₆-molossin peptide for particle formation, and labelling the particles with avidin-gold. Fig. Ic shows a perfectly spherical nanoparticle of -1,200 nm diameter in the extracellular fluid close to the plasma membrane of the cell. The halo of 10 nm gold particles positively identifies the structure as a peptide nanoparticle, and the relatively large size is probably a consequence of the length of time from particle formation to analysis (see later). Such a perfect sphere suggests an ordered assembly of components. Moreover, the restriction of the 10 nm gold particles to the surface of the sphere suggests a structure impermeable to the diffusion of avidin-gold (molecular weight of avidin is 68 kDa and diameter is ~5-6 nm assuming spherical geometry).

The cell protrusions on both sides of the extracellular particle in Fig. Ic suggest that it is in the process of cellular intemalisation by macropinocytosis. Of particular interest, in view of the presence of the acid-dependent fusogenic peptide in the particle, is that the peptide particle inside the cell (still surrounded by some gold particles) is not enclosed by a membrane (Fig. Ic). Many intracellular particles were in fact surrounded by a membrane, as shown in Figure Id, indicative of cellular intemalisation by an endocytic process, and suggesting dissolution of the endocytic membrane for the internalised particle in Figure Ic. It is interesting that the endocytic membrane closest to the intracellular particle in Figure Id appears less dense than the rest of the endocytic membrane, and that at one point (arrowed) the endocytic membrane is indented to the surface of the particle. The internalised particle in Figures Ic and Id have a less dense halo, suggesting loss of material from the particle surface.

The mean diameter of 19 particles in electron microscopy sections was 854 ran, (range 545 to 1273 nm, standard deviation 231 nm). The stoichiometry of (Lys)i₆ to fusogenic peptide within the particles was -1:2.5, irrespective of buffer and the initial ratio of peptides (Table 1). This represents a small excess of positive charge (+/- charge ratio 1.3:1).

The influence of salt ions

No particles were formed with peptides in pure water. Peptides in 1OmM Tris, pH 7.4 formed particles which were much smaller (Zav -100 - 150 nm) than in PBS, and the particles became progressively smaller in dilutions of 1OmM Tris (Fig. 10). Addition of salt promoted particle formation (Fig. Ie). At 15mM NaCI the Zav was 234 nm, while 15mM MgCI₂, Na₂SO₄ gave Zavs of 466, 541 and 499 nm respectively. Increasing NaCI concentration resulted in increasing particle size up to a plateau of Zav of -600 nm at 50 to 15OmM NaCI. A small increase of 20OmM NaCI completely abolished particle formation. Particle formation was inhibited or abolished at somewhat lower ionic strengths of the divalent cations: 5OmM Na₂SO₄, 5OmM MgCI₂ and 25mM MgSO₄.

Temperature markedly increased the kinetics of particle formation in the presence of salt (Fig. If, left), but not in its absence. (Fig. If, right).

Particle formation and charge at various peptide molar ratios For these studies we used the fusogenic peptide at a constant 10 μg/ml (4.1 μM) in PBS and the (LyS)₁₆ peptide at concentrations from 3.75 μg/ml to 240 μg/ml in PBS. The use of (Lys)₁₆ at 3.75 μg/ml (0.9 μM) corresponded to a net excess of negative charges (+/- charge ratio of 0.7:1). Interestingly, no particles were formed (data not shown). (Lys)₁₆ at 7.5 μg/ml (1.9 μM) (+/- charge ratio of 1.5:1) gave particles of Zav 427 nm (Fig. 4a). Particles were formed at all other (Lys))₆ concentrations evaluated, i.e. up to 240 μg/ml. Particle formation with (Lys)i₆ at 30 μg/ml (7.49 μM) (+/- charge ratio of 5.7:1) and 120 μg/ml (30 μM) (+/- charge ratio of 23.1:1) is shown in Figures 4b and 4c. Particles tended to get larger (~ 800 run) as the (Lys)i₆ concentration increased. (Zav range -400 to -900 nm) (Fig. 4). The surface charge of the particles increased with increase in the

(Lys)i₆ concentration up to 60 μg/ml (Fig. Ib).

The zeta potential of the particles is shown in Figure Ib. The particles formed with (Lys)i₆ at 7.5 μg/ml had a net negative charge, although the +/- charge ratio of the peptides at this concentration was 1.5:1. One possibility is that the (Lys)₁₆ peptide was less efficiently incorporated into the particles than the fusogenic peptides at this molar ratio. The positive charge of the particles increased with each increase in the concentration of (LyS)₁₆₎ up to 60 μg/ml.

Particle formation with peptide homologues

In order to obtain some insight into the requirements for particle formation, we evaluated various homologues of the cationic and anionic peptides (Table 1).

Firstly, we evaluated a homologue of the fusogenic peptide, where all amino acids except glutamates were replaced by alanines (combination 2). This alanine homologue has the same net charge (-5) as the fusogenic peptide, but no particles were formed. Small peptides rarely adopt secondary structure in solution, but we have previously noted that the fusogenic peptide forms an α-helix in aqueous solution⁷. CD studies (Fig. 2a) confirm that the fusogenic peptide forms an α-helix, and demonstrate that the alanine homologue has no secondary structure. Because the five glutamates of the fusogenic peptide are spaced at intervals of 3 - 4 amino acids, all the negative charges are on one side of the fusogenic peptide α-helix (Fig. 2b). This is likely to be critically important for the structural studies reported in a later section.

Secondly, a cationic peptide with alternating lysine residues, the (Lys - His)i₆ peptide

(combination 3), formed excellent particles with low polydispersity. However, the (Lys - PrO)₁₆ peptide (combination 4) did not form particles. The α-nitrogen of proline (uniquely among amino acids) is covalently linked to its side chain, forming a five- membered ring. This limits the rotational freedom of the Lys-Pro bond and could constrain the ability of the (Lys-Pro)₁₆ peptide to adopt an optimal configuration for interaction with the negatively charged glutamates on the fusogenic peptide.

Thirdly, we evaluated a single 38 amino acid-peptide consisting of the fusogenic peptide at the amino terminus and (Lys)j₆ at the carboxy terminus (combinations 5 and 6). It was in principle possible that the composite (LyS)₁₆-fusogenic peptide would by itself form nanoparticles in solution, but no particles were seen (data not shown). When the fusogenic peptide was added to the (LyS)₁₆-fusogenic peptide, good particles with low polydispersity were formed (combination 5). This suggests that the fusogenic moiety of the (LyS)₁₆-fusogenic peptide was not available in a configuration that permitted particle formation. This is supported by the fact that adding (LyS)₁₆ to the (Lys)i ₆-fusogenic peptide did not form particles (combination 6).

Fourthly, commercially available polylysine chains (>100 lysines) form excellent particles (combination 7).

Fifthly, we wished to see if adding amino acids to (LyS)_1O would affect particle formation. This is important as additional amino acids could serve valuable functions, in particular targeting of the particles or the delivery of therapeutic moieties. Cyclised and linear peptides of 30 or 31 amino acids, comprising (LyS)₁₆ at the amino terminus and various integrin-binding moieties at the carboxy terminus, all formed excellent particles (combinations 8 - 11). The (Lys)]₆ - (His)₁₆ peptide (combination 12) formed particles which were larger and more diverse in size. The cationic peptide : anionic peptide molar ratio was 0.6:1, and the +/- charge ratio (assuming that none of the histidines are protonated) was 1.9:1, which is similar to the preceding peptide combinations. It is possible that the (His)₁₆ chain represents a relatively hydrophobic sequence, especially if none or few of the histidine residues are protonated. This would depend on the pKa of the imidazole groups of the histidines in this peptide. In free histidine, the pKa of the imidazole group is 6.0 (so that <5% are protonated at pH 7.4), but in proteins the pKa can be as high as 7. Combinations 13 and 14 involve cationic peptides with (Lys)₁₆ at the amino terminus and sequences from human alpha 1 -antitrypsin which bind to the serpin- enzyme complex receptor²³'²⁴. These particles also tended to be larger and more diverse in size, possibly a consequence of the hydrophobicity of the alphal -antitrypsin peptides. Finally, combinations 15 to 19 involve the shorter alphal -antitrypsin peptide with (Lys)₁₂₎ (Lys)g, (Lys)₆, (Lys)₄, and (Lys)₂. Particle formation is seen down to (Lys)₆ (combination 17), although polydispersity with (Lys)₆ is high. No particles were seen with (Lys)₄ and (Lys)₂. The cationic peptide : anionic peptide molar ratio increased progressively from 0.6:1 with (Lys)₁₆ (combination 13) to 2.0:1 (combination 19), while the +/- charge ratio fell progressively from 2.0:1 with (Lys)₁₆ to 1.2:1 with (Lys)₂. As the +/- charge ratio in combinations 18 and 19 was relatively low, we evaluated particle formation with the (Lys)₄ and the (Lys)₂ peptides at 30 μg/ml, with the fusogenic peptide at the standard 10 μg/ml. This gave +/- charge ratios of 3.2:1 for the (Lys)₄ peptide and 2.4 for the (Lys)₂ peptide, but particles nevertheless were not formed. Thus the critical variable was almost certainly the length of the lysine chain, with particle formation requiring a minimum of six lysines.

Oligomeric structure of the pre-assembly fusogenic peptide Electrospray time-of- flight mass spectrometry confirmed a peptide of molecular mass of

2300.6 Daltons (theoretical 2300.5 Daltons). There were no apparent impurities, but interestingly there was a species of 4601.7 molecular mass consistent with a fusogenic peptide dimer (data not shown).

Sedimentation velocity data for 50, 250, and 700 μg/mL fusogenic peptide were individually fit to a continuous distribution of molecular masses. Each data set indicated a single, monodisperse molecular mass of 22.485 kDa irrespective of concentration (Fig. 2c and Fig. 2d). This persistence over a 20-fold concentration range suggests a single, non- dissociating oligomeric species down to the minimum detection limits of the analytical ultracentrifuge UV optics.

Subsequent global analysis of sedimentation equilibrium data of 1 mg/mL fusogenic peptide at 2 speeds, together with sedimentation velocity data at the three concentrations described above, was performed. The fit to a single, non-dissociating species model (Fig. 2e and Fig. 2f) indicated a molecular mass of 21.849 ± 0.043 kDa with a global reduced χ² of 0.224, providing further evidence of a single oligomeric species consisting of 9 or 10 monomers (calculated molecular masses of 20.715 and 23.017 kDa respectively). The persistence of a dimer through mass spectrometry manipulations suggests that the fusogenic peptide oligomer may be constructed from dimers, in which case a decamer is the likely structure. Furthermore, solution NMR spectra (data not shown) indicate two equal populations of α-helical peptides, which would also support an even-order decameric species.

Additional hydrodynamic parameters based on sedimentation equilibrium and velocity data were: sedimentation coefficient, S = 1.814 ± 0.001; factional ratio, f/f_o = 1.14 (assuming decamer); and cylinder model dimensions (length/diameter) = 1.22 (assuming decamer). Frictional ratios were computed for a calculated molecular mass of 23.017 kDa (decamer), where the frictional coefficient of the sedimenting species is normalized by the frictional coefficient of a sphere with the same molecular mass. For the cylinder model, the length and diameter dimensions were determined for a model cylinder with the same frictional ratio as the decamer.

The influence of salt on the size of peptide particles No particles were formed with peptides in pure water (pH 5.9) (Fig. 5). In 10 mM Tris, pH 7.4 without added NaCl, particles were much smaller than in PBS, remaining stable over time at -125 nm diameter (Fig. Ie, left). When dilutions of 10 mM Tris pH7.4 were used particles of progressively smaller size were formed (Fig. 5).

The (Lys)₁₆ peptide at 15 μg/ml (3.7 μM) and the fusogenic peptide at 10 μg/ml (4.1 μM) were mixed in 10 mM Tris pH 7.4 at NaCl concentrations from zero to 0.5 M (Fig. 6) Particles formed in the absence of salt were relatively small, at -125 nm in diameter, and particle size was stable with time. Particle size increased with increasing salt concentration, reaching a plateau of -700 nm at 50 mM to 150 mM NaCl. At high salt (0.5 M NaCl) no particles were formed, demonstrating the importance of internal electrostatic interactions in particle formation. In the presence of salt, particle size increased with time. For example, at 150 mM NaCl, particle size increased from -731 nm to -904 nm over 30 to 60 minutes.

If particles are formed in the absence of salt, and NaCl is added to 150 mM after 60 minutes, particle size increased to that normally seen at 150 mM, NaCl (Fig. Ie).

In the presence of salt, temperature had a marked effect on the kinetics of particle formation (Fig. If, left). In the absence of salt temperature had no effect (Fig. If, right). Nucleation and particle growth

The fusogenic peptide is consumed in particle formation within 30 minutes, leaving free

(Lys)i₆ (Table 2). Particles were formed with (Lys)₁₆ at 15 or 120 μg/ml, and additional fusogenic peptide added at 28 minutes. In the absence of added fusogenic peptide, there was (as expected) a single population of particles at 30 minutes (Fig. 3a). Fusogenic peptide at 28 minutes resulted in two populations of particles at 30 minutes (two minutes after addition of fresh fusogenic peptide), one presumptively of newly formed small particles (peak 307 nm) and the other presumptively the pre-existing particles, but larger (peak 875 nm) than in the absence of fresh fusogenic peptide (peak574 nm) (Fig. 3b). By

45 minutes, the two populations had merged (Fig. 3c).

The standard procedure involves addition of fusogenic peptide at 1 mg/ml to (Lys)i₆ at 15 μg/ml. Adding (Lys)i₆ at 1 mg/ml to the fusogenic peptide at 10 μg/ml (Zav 336 nm, peak 297 nm) (Fig. 3d), or mixing equal volumes of (Lys)₁₆ at 30 μg/ml and fusogenic peptide at 20 μg/ml (Zav 364 nm, peak 361 nm) (Fig. 3e) gave smaller particles than the standard procedure (Zav 623 nm, peak 608 nm) (Fig. 3f)-

In the presence of salt, particle size increased slowly with time, e.g. from Zav of 731 nm to 904 nm over 30 to 60 minutes in 15OmM NaCI (Fig.10). If particles were formed in the absence of salt, and NaCI was added at 60 minutes, particle size increased dramatically to that normally seen with NaCI (Fig. 3g).

Polymer coating of peptide particles We attempted to stabilise the size of small particles formed in low salt by cross-linking surface amino groups with a multivalent crosslinker. Thirty minutes after formation of peptide particles (in 20 mM Hepes, 10 mM NaCl, pH 7.8), HPMA was added to 0.1, 0.5, 1 and 2 mg/ml. The nitrophenol group of HPMA targets primary amines (hence the use of Hepes buffer rather than Tris) and reacts with exposed NH₃ ⁺ groups on the surface of the peptide particles. Moreover, there are ~14 nitrophenol groups per HPMA molecule, resulting in cross-linking at multiple sites on the particle surface. Figure 3g demonstrates that peptide particles formed in 10 mM NaCl and treated with 0.1 mg/ml of HPMA are hardly affected at all when the NaCl concentration is increased to 150 mM. The use of 0.5 mg/ml of HPMA completely protects the particles from salt-induced size increase. Consistent with the interaction of HPMA with NH₃ ⁺ groups on the particles, HPMA- treated particles showed a progressive decrease of surface positive charge with increasing concentrations of HPMA. The peptide particles are only slightly positively charged with 0.1 mg/ml of HPMA, and have a net negative charge with 0.5 mg/ml of HPMA (Fig. 3h). Note that the zeta potential in the absence of salt (Fig. 3h, -20-25 mV) is much higher than in 150 mM NaCl (Fig. Ib, ~ 4 mV), as previously noted^11' HPMA particles showed a progressive decrease of surface positive charge with increasing HPMA concentration.

DISCUSSION The unexpected formation of spherical nanoparticles on mixing two small, water-soluble peptides is almost certainly a consequence of three unusual structural characteristics of the anionic fusogenic peptide. Firstly, although small peptides rarely adopt secondary structure in aqueous solution, the fusogenic peptide forms an cc-helix⁷. This provides a rigid, rod-like molecule which would facilitate the formation of a higher order structure. Secondly, the arrangement of the five glutamates at every third or fourth position of the amino acid sequence means that the negatively charged side chains are present on only one side of the helix. This might aid oligomerisation by providing hydrophobic or polar surfaces for peptide/peptide interactions, while allowing the negative charges to face the solution. Finally, the unexpected self-assembly of the fusogenic peptide into a discrete oligomer of 9-10 peptides provides a relatively large, rigid structure with 45-50 negative charges on the surface. These fusogenic peptide oligomers might form the core unit of the nanoparticle, the cationic peptides serving to bind the oligomers together in an overlapping fashion, bringing large numbers together for particle formation.

To our knowledge, nanoparticles of this nature have not previously been described. They might, therefore, form the prototype for a class of self-assembled particles where rigid molecules of high net charge are the core building block, with largely unstructured polymers of opposite charge to tether them together.

Our experimental observations suggest a structural model where the fusogenic peptide self-assembles via hydrophobic packing into a relatively rigid oligomer with 45-50 negative surface charges. These anionic oligomers then condense with unstructured polycationic peptide at a relatively constant stoichiometry to form the core of the supramolecular assemblies. The stoichiometry demonstrates an excess of positive charge by a factor of -1.3 within the particle, indicative that not all lysines within the condensed core are involved in inter-peptide salt bridges. A net absorption of negative ions during the first phase of particle growth would therefore be expected, and is consistent with the dependence of particle formation on the presence of salt counterions.

If the ratio of cationic to anionic peptide in the initial solution is less than the natural stoichiometry, particles do not form. If peptides are mixed at approximately the same ratio as the particle core, the surface charge of the particles is slightly negative, suggesting exposed anionic peptide oligomers. If cationic peptide is supplied in increasing excess, the surface charge of the particles becomes increasingly positive to a plateau, suggesting that the exposed anionic oligomers at the surface of the particles absorb more cationic peptide until they saturate.

The stoichiometry of peptide monomers within the particles corresponds to a 1.1 to 1.5- fold excess of positive charge, suggesting that not all primary amines form a salt bridge with carboxyl groups. A fraction of carboxy groups might similarly be left without a salt bridge to primary amines, even with a net excess of positive charge. In this circumstance, the absolute dependence of particle formation on salt counterions is consistent with the expectation that the particle core is electrically neutral.

Particle assembly appears to occur in two phases. Particles initially grow quickly while assembly is supplied by free peptide in solution, this phase being complete before 30 minutes. Thereafter, growth can occur for some particles only at the expense of others, either by simple disassembly of some particles to generate free peptide, or by collision- based exchange of material or particle merger.

A negative surface charge is observed for particles formed with the lowest concentration of (Lys)₁₆ (7.5 μg/ml) that sustains particles with 10 μg/ml fusogenic peptide. This mix ratio is roughly stoichiometric with the ratio of peptides within the particles. In this case (LyS)₁₆ is not in excess, and the negatively charged oligomer may be more exposed on the surface. Surface charge becomes increasingly positive as (Lys)₁₆ increases in the peptide mix, perhaps because the particle surface becomes increasingly saturated with (Lys)₁₆ after the depletion of fusogenic peptide oligomers. Irrespective of structural considerations, the fact that the particles incorporate an acid- dependent fusogenic peptide raises the interesting possibility of delivering membrane- impermeable therapeutic substances to the cytosol following endocytosis. We in fact show that the peptide particles do enter the cytosol, the sequence of events probably being electrostatic interaction of the cationic particles with the plasma membrane, macropinocytosis, acidification of the endocytic vesicle, activation of the fusogenic peptide, and dissolution of the endocytic membrane with release of the particle.

These particles might provide a versatile system for the targeted delivery to the cytosol of biologically active compounds which currently cannot be considered for therapeutic use because of their impermeability to the plasma membrane. It should be possible to incorporate several (LyS)₁₆-containing peptides at predetermined ratios for multiple functions, e.g. one peptide for targeting and one or more peptides for single or multiple (possibly coordinated) functional effects. It remains to be seen if moieties sensitive to enzymatic or chemical modification (e.g. proteolysis, oxidation) are protected from such reactions when present inside the particle. This would greatly increase the potential value of these particles for clinical or experimental applications.

METHODS Peptides

The sixteen cationic and two anionic peptides (listed in Table 1) were synthesised, cyclised via cysteines (where indicated) and purified by Cambridge Research Biochemical (Cleveland, UK). They were supplied as trifluoroacetate (TFA) salts in the form of a dry powder, and stored dessicated at -35°C. There is one TFA counterion for each positive charge in the peptide. Quantities of 1 - 2 mg were dissolved at 1 mg/ml in phosphate buffered saline (PBS) (137 mM NaCl, 1.5 mM KH₂ PO₄, 8.1 mM Na₂HPO₄, 2.7 mM KCl, 0.9 mM CaCl₂ 2H₂O, 0.5 mM MgCl₂ 6H₂O, pH 7.4) (Sigma-Aldrich, Dorset, UK); or 150 mM NaCl, 10 mM Tris, pH 7.4; or 10 mM Tris, pH 7.4 without salt; or 10 mM NaCl, 20 mM Hepes, pH 7.8 (which does not have primary amines) and stored in aliquots at -35°C. The (Lys)₁₆-molossin peptide (combination 8, Table 1) was additionally synthesised with a biotinylated lysine at the amino terminus. The high molecular weight polylysine (combination 7, Table 1) was from Sigma-Aldrich (Dorset, UK). _ _

25 Formation of peptide particles

The standard conditions involved diluting the LyS₁₆ peptide to 15 μg/ml (3.7 μM) in PBS and then adding the required volume of fusogenic peptide (Table 1) at 1 mg/ml in PBS to give a concentration of 10 μg/ml (4.1 μM), while constantly stirring. This represents a +/- charge ratio of 2.9:1. After 30 minutes at room temperature, the solution was analysed.

The standard conditions were varied, as indicated in the text, in order to evaluate homologous peptides, peptide concentration, salt concentration and the influence of time and surface modification.

Analysis of particle size by dynamic light scattering (DLS).

DLS measurements were performed on a Zetasizer 3000 HS (Malvern Instruments Ltd., Malvern, UK). Data analysis used the non-negatively constrained least squares (NNLS) method, via software provided by Malvern Instruments. The intensity-weighted mean diameter, designated Zaverage (Z_av), and the polydispersity index²⁵'²⁶ were measured at least three times, with replicate measurements always agreeing within 10%. The average of these measurements is reported. The polydispersity index is a measure of the broadness of the size distribution, and ranges from 0 to 1 ; 0.08 or less is effectively monodisperse, and 0.7 or greater is very polydisperse.

Analysis of surface charge of particles (zeta potential)

The zeta potential²⁷ was determined using the Zetasizer 3000 HS (Malvern Instruments Ltd, Malvern, UK). Five measurements were taken, which always agreed within 10% of each other, and the average of these five measurements is reported. The zeta potential is the electric potential at the shear plane boundary between ions associated closely enough with the particle to move with it, and bulk solution ions which do not. Values were derived from the electrophoretic mobility of the particles using the Smoluchnowski approximation²⁷, via software provided by Malvern Instruments.

Determination of secondary structure by circular dichroism (CD) A volume of 250 μl of peptide at 1 mg/ml in PBS was scanned through wavelengths 200-

250 nm using a Jobin Yvon CD6 instrument and a path length of 0.1 cm. The results were recorded as molar ellipticity (with PBS background subtracted) against wavelength. Secondary structure was inferred from comparisons with model structures . Analytical ultracentrifugation

Sedimentation velocity experiments were performed on the fusogenic peptide at 50, 250, and 700 μg/ml in modified PBS (139 mM NaCl, 3.6 mM KH₂PO₄, 23.6 mM Na₂HPO₄, 2.6 mM KCl, 10% D₂O, pH 7.3). Experiments were performed at 50k rpm and 20⁰C, using Beckman XLI centrifuges equipped with an AnTi60 rotor and 1.2 mm 2-channel epon-fϊlled centrepieces. Radial absorbance data were collected in continuous scanning mode with 0.003 cm increments at 230 nm (50 μg/ml) or 295 nm (250 and 700 μg/ml) at 5 min intervals for a total of 80 scans without averaging. Varying wavelengths were chosen for the different concentrations to optimise absorbance data collection with respect to the dynamic range of the instrument. A partial specific volume for the peptide

(V) of 0.7819 cm³/g, buffer density of 1.019 g/cm³ and buffer viscosity of 1.028 cP were calculated using SEDNTERP²⁹. Sedimentation boundaries and equilibrium curves were fit using SEDPHAT³⁰ and the embedded algorithm for translation of sedimentation coefficients to molecular mass. Distributions of molecular mass were calculated over 100 mass increments between 0.3 and 50 kDa; resulting distributions were smoothed by maximum entropy regularisation³¹ to provide a mass profile at 95% confidence interval.

Sedimentation equilibrium data were collected for the fusogenic peptide at 1 mg/ml at 35k and 50k rpm, and at a wavelength of 300 nm. Data was collected at four hour intervals using a radial step size of 0.001 cm and averaging over 10 scans until the sample reached equilibrium (i.e. successive data curves were indistinguishable) at ~ 24 hours. Sedimentation equilibrium data was combined with velocity data for all concentrations and fit globally to a single molecular mass using SEDPHAT. Molecular mass and sedimentation coefficient errors are quoted at 95% confidence interval and were estimated by globally refitting the data after each of 1000 Monte Carlo iterations in which

Gaussian-random noise is added to the data.

Cell line

The T-24 cell line (European Collection of Cell Cultures, Salisbury, UK) is an adherent line originally derived from a human bladder carcinoma. It was maintained under mycoplasma-free conditions in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated foetal calf serum, 2 mM glutamine and Ix nonessential amino acids (Invitrogen, Renfrewshire, UK), referred to as culture medium. Electron microscopy

Peptide particles were formed by incubating biotinylated (Lys)₁₆-molossin at 15 μg/ml with the fusogenic peptide at 10 μg/ml in DMEM (without supplements). After 30 minutes at room temperature, streptavidin-gold (Agar Scientific, Standsted, UK) (10 nm gold particles) was added to a final dilution of 1 in 300 of the stock solution supplied.

The gold-labelled particles were added to cells after a further 10 minutes' incubation at room temperature.

The T-24 cells were seeded into 12 well plates at 2 x 10⁵ cells per well, each well containing a sterile 16 mm diameter glass coverslip. The cells were incubated overnight at 37°C in 5% CO₂/95% air. The culture medium was then removed, the cells washed once with DMEM without supplements, and 0.75 ml of the gold-labelled peptide particles was added to each well. After four hours at 37°C in 5% CO₂/95% air, the solution was removed and 1 ml of ice-cold 2.5% glutaraldehyde in 0.13 M phosphate buffer (pH 7.3) was added to each well. After two hours at 4⁰C, the glutaraldehyde was removed and replaced with 1 ml of ice-cold 0.25 M sucrose in 0.07 M phosphate buffer pH 7.3, and the plate was stored at 4⁰C until processing.

The cells were post-fixed in 1% osmium tetroxide for 30 minutes followed by dehydration with 10% ethanol for 10 minutes, 70% ethanol for 15 minutes and 100% ethanol for 15 minutes (3 times). The coverslip was then fixed onto a glass slide, covered with embedding resin (medium hardness) (TAAB, Reading, UK) and left for two hours at room temperature. A TAAB embedding capsule was filled with resin, inverted over the coverslip and placed in an embedding oven at 70°C for 24 hours to polymerise. The embedding capsule was then snapped off, removing the layer of cells from the coverslip.

Ultra-thin sections (0.75 - 2 μm) were cut using a Leica Ultracut machine (Leica Microsystems, Milton Keynes, UK). They were placed on 200 mesh Guilder Grids (EM Technologies Ltd, Ashford, UK) with a support film of 0.5% Pioloform (Agar Scientific Ltd) in chloroform. The grids were stained with uranyl acetate and lead citrate, and viewed on a Hitachi H7600 transmission electron microscope (Hitachi, Wokingham, UK).

Surface modification of peptide particles Peptide particles were formed in 20 mM Hepes, pH 7.8 using the (Lys)₁₆ peptide at 15 μg/ml (3.7 μM) and the fusogenic peptide at 10 μg/ml (4.1 μM). After 30 minutes at room temperature, the appropriate volume of a copolymer of N-(2-hydroxypropyl) methacrylamide and N-methacrylated glycylglycine 4-nitrophenol ester (HPMA) (at 50 mg/ml in 20 mM Hepes, pH 7.8) (a kind gift from Dr Simon Briggs and Professor Len

Seymour, Department of Clinical Pharmacology, University of Oxford, England) was added, to achieve final concentrations of 0.1 - 2 mg/ml. The HPMA has a molecular weight of 21,500 Daltons, a composition of 10.3 parts of the glycylglycine nitrophenol ester moiety to 89.7 parts of the hydroxypropyl methacrylamide moiety, with an average of 13.8 reactive nitrophenol groups per molecule.

EXAMPLE 2

Do particles form using non-peptide polycations such as PEI with fusogenic peptide

Methods PEI 2K (Sigma) was prepared at 10OmM in 5% dextrose. Fusogenic peptide was dissolved in 1OmM Tris/5% dextrose, pH 7.4 at lmg/ml.

Particles were formed by diluting PEI to the appropriate concentration in PBS. Then the required volume of fusogenic peptide at lmg/ml was added drop wise whilst constantly stirring. After 30 minutes at room temperature, the solution was analysed using Dynamic light scattering on the Zetasizer 3000 HS (Malvern Instruments Ltd, Malvern, UK). Data analysis used the non-negatively constrained least squares (NNLS) method, via software provided by Malvern Instruments. The intensity-weighted mean diameter, designated Zaverage (Zav), and the polydispersity index were measured at least three times. Results The results are shown in figure 12. The ratio of PEI 2K : fuso was (0.15mM : 4.1μM).

The particle ZAverage was 631 run, the polydispersity was 0.04 and the KCps was 193. Conclusion

Excellent particles are formed using a non-structured organic polycation with the fuosgenic peptide. These are essentially identical on DLS to those seen with (Lys)₁₆.

EXAMPLE 3

Do particles form with cationic proteins and polyanions. In this case, the cationic protein forms the rigid 'brick' and so it was mixed with an unstructured polyanion for particle formation.

Methods

Histone Ills (Sigma, molecular weight 21.5 kDa, lysine-rich fraction, approximately 25% lysines) and poly-L-aspartate (Sigma, molecular weight 5-15 kDa) were dissolved to lmg/ml in PBS.

Particles were formed by diluting the histone solution to the appropriate concentration in PBS. Then the required volume of poly-L-aspartate at lmg/ml was added dropwise whilst constantly stirring. After 30 minutes at room temperature, the solution was analysed using

Dynamic light scattering on the Zetasizer 3000 HS (Malvern Instruments Ltd, Malvern, UK). Data analysis used the non-negatively constrained least squares (NNLS) method, via software provided by Malvern Instruments. The intensity-weighted mean diameter, designated Zaverage (Zav), and the polydispersity index were measured at least three times.

Results

No particles were seen when histones were mixed with the fusogenic peptide. This presumably is because both components are rigid 'brick' structures.

Conclusion

Particle formation occurs with cationic proteins (cationic 'bricks') and polyaspartate (unstructured anionic 'straps').

EXAMPLE 4 Can flow cytometry be used to count peptide particles

Methods

Fusogenic peptide was labelled with Alexa 488 (Invitrogen). (Lys)₁₆ peptide was dissolved to lmg/ml in water. Particles were formed by diluting (Lys)i₆ to the appropriate concentration in PBS. Then the required volume of fusogenic peptide at 100 μg/ml was added dropwise whilst constantly stirring. After 30 minutes at room temperature, 100 μl Cytocount beads (Dako) was added per ImI peptide particle solution and the samples were analysed on the flow cytometer (FACScalibur, Becton Dickinson).

(Lys)i₆:fusogenic particles were formed at two different concentrations using Alexa 488 labelled fusogenic. 5000 events were counted on the flow cytometer. The samples contained a known number of beads and it was therefore possible to calculate the number of peptide particles/ μL, using the following formula;

Number of cells counted x Cytocount Concentration x Dilution Factor

Number of Cytocount Beads counted Results

The results are shown in figure 13.

Conclusion

The fluorescent peptide particles can be readily detected and counted using flow cytometry.

Table 1. Particle formation by (Lys)i6 and fusogenic peptide homologues

Cationic peptides were at 15μg/ml, and the anionic peptides at 10μg/ml, in phosphate buffered saline.

Detailed molar and charge ratios are given in the text, but in almost all cases the cationic peptide: anionic peptide molar ratio was 0.5 to 0.9: 1, and the +/- charge ratio was ~ 2: 1. Z average was measured 30 minutes after mixing the peptides.

a Fusogenic peptide, based on influenza virus haemagglutinin.⁶ Note that all amino acids are hydrophobic except for 5 glutamates. b Fusogenic peptide homologue. AU amino acids except glutamates have been replaced with alanines. c 38 amino acid peptide comprising of the fusogenic peptide with (Lys)i₆ at the carboxy terminus. d Commercial polylysine, 100-200 lysines per chain. (Sigma- Aldrich, Cat. No. P7890). e (LyS)₁₆ with cyclised peptide containing the 15 amino acid integrin-binding loop from the snake venom molossin.¹⁹"²⁰ f (Lys)i₆ with non-cyclised version of the molossin peptide (see combination 8), with the cysteine residues replaced by glycines. g (Lys)₁₆ with cyclised peptide homologous to the molossin peptide (see combination 8), with the integrin-binding RGD motif replaced by RGE.²¹ h (Lys)i₆ with cyclised peptide containing the 14 amino acid integrin-binding loop of human laminin.²² i (Lys)₁₆ with human alphal -antitrypsin sequence which binds to the serpin-enzyme complex receptor.²³'²⁴ j (Lys)₁₆ with a shortened version of the human alphal -antitrypsin sequence (see combination 13).

Claims

1. A nanoparticle comprising a plurality of polyvalent charged rigid brick molecules connected by a plurality of polyvalent oppositely charged flexible strap molecules, the brick molecules being proteins or peptides.

2. A nanoparticle according to claim 1, in which the brick and strap molecules are polyvalent.

3. A nanoparticle according to claim 1 or claim 2, in which the brick molecule is a peptide oligomer.

4. A nanoparticle according to claim 3, wherein the peptide has the following amino acid sequence: GLFE ALLELLES LWELLLEA.

5. A nanoparticle according to claim 1 or claim 2, in which the brick molecule is a protein, especially histone or a histone-like protein.

6. A nanoparticle according to any preceding claim, in which the strap molecules are linear or sheet like.

7. A nanoparticle according to any preceding claim, in which the strap molecules comprise repeating units.

8. A nanoparticle according to any preceding claim, in which the strap molecule is selected from:

KHKHKHKHKHKHKHKHKHKHKHKHKHKHKHKH GLFEALLELLESLWELLLEADP-K₁₆

Polylysine

K₁₆- ICRRARGDNPDDRCT K₁₆-IGRRARGDNPDDRCT K₁₆-ICRRARGENPDDRCT K₁₆-CPP AS YRGDSCQE Ki₆- H₁₆

K₁₆-CSIPPEVKFNKPFVFLI and K_(6-,₆₎ FNKPFVFLI K₁₆PVKRRLFG

(GIu)₁₆ polyaspartate heparin poly(ethyleneimine), a cationic lipid a polyamidoamine dendrimer

9. A nanoparticle according to any of claims 1 to 3, comprising an anionic peptide oligomer brick and a peptide strap comprising at least five lysine or arginine residues.

10. A nanoparticle according to any of claims 1 to 3, comprising a cationic peptide brick and a (GIu)₁₆ or polyaspartate strap.

11. A nanoparticle according to any preceding claim, further comprising targeting, therapeutic or diagnostic residues or other regions.

12. A nanoparticle according to any preceding claim, wherein the ratio of strap molecules to brick molecules is between about 1:1 and about 1:5.

13. A nanoparticle according to any preceding claim for use in therapy.

14. A nanoparticle according to any preceding claim for use in the delivery of therapeutic or diagnostic agents.

15. A method for producing a nanoparticle comprising combining charged rigid peptide or protein brick components with oppositely charged flexible strap components in solution.

16. The method of claim 15, further comprising modifying the surface of the nanoparticles.