Peptide capsules for cytosolic delivery of cargo molecules

Abstract

The present invention provides a peptide capsule for delivery of a cargo molecule into a cell, the peptide capsule comprising a peptide shell and a cargo molecule, wherein the peptide shell is formed from a plurality of self-assembling peptides, the self-assembling peptides derived from Ostrinia furnacalis cuticles and each comprising an amino acid sequence given in SEQ ID NO: 1, 2 or 3. Also provided therein is an isolated self-assembling peptide consisting of SEQ ID NOs: 4-25, 43 and 44, a pharmaceutical composition comprising a peptide capsule, and uses thereof.

Description

[0001] PEPTIDE CAPSULES FOR CYTOSOLIC DELIVERY OF CARGO MOLECULES

[0002] FIELD OF INVENTION

[0003] The present invention generally relates to peptide capsules for the delivery of a cargo molecule into a cell. The present invention also provides compositions comprising the peptide capsules of the invention. The peptide capsules and compositions disclosed herein find utility as medicaments.

[0004] BACKGROUND

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] In the natural process of evolution, organisms have developed sophisticated strategies to adapt to their harsh environments by utilizing simple raw materials and optimizing their functional properties. One such strategy is the incorporation of functional gradients in the assembly of biomaterials, which involve changes in local chemical composition, arrangement of building units, and distribution within materials. These gradients can be observed at various length scales in biological materials. However, achieving precise control over gradients remains a formidable task in artificial self-assembly systems.

[0007] Artificial creation of such biomaterials often necessitates the engineering of a gradient interface, which can be a challenging endeavour. Particularly, solvent plays critical role in creating such gradients in numerous self-assembly systems. By regulating the interactions between the solvent and the components of the assembly, a range of nano-sized structures can be formed, such as particles, fibers, films, or vesicles. These assembly processes often exhibit dynamic and non-equilibrium characteristics. Despite their importance, the mechanisms underlying these assembly processes are often not well-understood. Nanoprecipitation, a common solvent mediated assembly technique, leverages mixed solvents to steer precipitation-based self-assembly, resulting in the production of polymer nanoparticles. The technique involves adding an excess of a poor solvent to a polymer solution, which triggers the formation of solid polymer nanoparticles through precipitation. This process has been successfully scaled up for industrial applications in fields as diverse as pharmaceuticals and agriculture.

[0008] Biomacromolecules are promising for the treatment of various diseases. However, although biomacromolecules often have good water solubility, they often have low cell membrane permeability. In addition, traditional drug loading methods are often not suitable for loading hydrophilic molecules. At present, the means of delivering biological macromolecules are often limited to inorganic nanoparticles, synthetic polymers, etc. Although these methods have started clinical trials, they have also exposed certain defects. The manufacturing method may be complex, and often inorganic nanoparticles have poor biocompatibility. Moreover, due to the wide molecular weight distribution of biomolecules, some carriers have high specificity, and are only suitable for specific types of biomacromolecules.

[0009] Therefore, there exists a need for improved or alternative systems for cytosolic delivery of cargo molecules.

[0010] SUMMARY OF INVENTION

[0011] The present invention provides a peptide capsule for delivery of a cargo molecule into a cell, the peptide capsule comprising a peptide shell and a cargo molecule, wherein the peptide shell is formed from a plurality of self-assembling peptides, the self-assembling peptides each comprising an amino acid sequence given in SEQ ID NO: 1 , 2 or 3, wherein:

[0012] SEQ ID NO: 1 is:

[0013] NX2X3X4VPPS [NX1°X11VPPS]nwherein, n is an integer from 2 to 6;

[0014] X2is N, K, R, H, D or E;

[0015] X3is Y, F or absent;

[0016] X4is Y or F; each X10is independently N, K, R, H, D or E; and each X11is independently Y or F;

[0017] SEQ ID NO: 2 is:

[0018] [X1X2AAPAX7'8]nX9X1OAAPAX15'1Swherein, n is an integer from 2 to 6; each X1is independently W or F; each X2is independently N, K, R, H, D or E; each X7'8is independently NQ, PS, or NH;

[0019] X9is W or F;

[0020] X10is N, K, R, H, D or E; and X15'16is absent, NQ, PS, or NH;

[0021] SEQ ID NO: 3 is:

[0022] [VX2X3APV]nwherein, n is an integer from 3 to 8; each X2is independently H or Y; and each X3is independently S, K, R, H, D or E.

[0023] The present invention also provides an isolated self-assembling peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-25, 43 and 44.

[0024] The present invention also provides a pharmaceutical composition comprising a peptide capsule of the present invention and a pharmaceutically acceptable carrier.

[0025] The present invention also provides an in vitro method of delivering a cargo molecule into a cell, said method comprising contacting the target cell with the peptide capsule or the pharmaceutical composition of the present invention. The peptide capsule and pharmaceutical composition of the present invention also find utility in treating a disease or disorder, find utility in the manufacture of a medicament for the treatment or prophylaxis of a disease or disorder, and find utility in a method of treating or preventing a disease or disorder in a patient.

[0026] Preferred but optional features are set out in the dependent claims. Additional aspects and embodiments of the peptide capsules and methods of the present invention will be apparent from the following description, drawings and claims. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment.

[0027] BRIEF DESCRIPTION OF DRAWINGS

[0028] Fig. 1 depicts the fourth molting and capsule hardening process of the Ostrinia furnacalis (Asian corn borer).

[0029] Fig. 2 depicts de novo sequencing and screening of capsule forming peptides from 0. furnacalis. (a) Images of an O. furnacalis larva (I) and its head capsule 0 min (ii) and 75 mins (iii) after the fourth molting. The head capsule inside the dashed box was used for proteomic analysis, (b) Heat map showing the abundance of identified proteins in the O. furnacalis head capsule transcriptome, created by the software Heml. (c) Screening of insect cuticle peptide (ICP) sequences from cuticular proteins. The shortest repeating unit required a minimum of five residues, and the units were repeated at least three times, allowing a spacer consisting of at most two residues between each repeating units, (d) Sequences of the selected ICPs. The repetitive and modular domains present in each peptide are indicated as follows: WNAAPA (SEQ ID NO: 32), NNY(Y)VPPS (SEQ ID NO: 33), VHSAPV (SEQ ID NO: 34), SSGGGGGF (SEQ ID NO: 35), AVSYSAP (SEQ ID NO: 36), GGGHSSGGLSL (SEQ ID NO: 37), GHEGHDH (SEQ ID NO: 38), GPEFGDH (SEQ ID NO: 39), YSEPAK (SEQ ID NO: 40), QSHGH (SEQ ID NO: 41). According to the final assembly structure, the nine peptides are divided into four categories; WA30, NS36 and W30 can form hollow capsules; QH33 can form solid particles; GH42 and YK34 have good solubility in both water and acetone; while SF32, AP36 and GL33 cannot dissolve in water, (e) Schematic illustration of the peptide capsule preparation process. The assembly structure was fixed by the cross-linking agent (IPDI) dissolved in acetone, and the organic solvent can be completely removed through the subsequent dialysis process, (f) Transmission Electron Microscopy (TEM) images of peptide nanostructures: WA30 (left) and NS36 (middle) capsules, and QH33 (right) nanoparticles. The insets provide enlarged views. Only WA30 and NS36 capsules shown in the insets were briefly stained for 15 seconds with a 0.3% uranium acetate solution prior to high-res TEM imaging to enhance contrast.

[0030] Fig. 3 depicts amino acid content of the Ostrinia furnacalis (Asian corn borer) head shells.

[0031] Fig. 4 depicts a grand average of hydropathicity (GRAVY) plot of insect cuticle peptides through ProtScale by using Kyte & Doolittle scale.

[0032] Fig. 5 depicts representative AFM and 3D reconstruction images of WA30 (a, e and h), NS36 (b, f and i) and VV30 (c, g and j) cross-linked peptide capsules and QH33 (d) cross-linked peptide particles, all scale bar are indicated in the images, respectively.

[0033] Fig. 6 depicts (a) Cryo-TEM images of WA30, NS36 and VV30 cross-linked peptide capsules and cross-linked QH33 particles, and (b) Cryo-TEM images of WA30, NS36 and VV30 crosslinked capsules wall.

[0034] Fig. 7 depicts circular dichroism (CD) spectra of ICPs in aqueous solutions under different concentration. Fig. 8 depicts the CD spectra of (a) WA30, (b) NS36, (c) VV30 and (d) QH33 with low concentration under different acetone to water ratios (from 5:1 to 9:1).

[0035] Fig. 9 depicts the assembly of ICPs in water-acetone mixed solvent, (a) Schematic illustration of the experiment setup and solvent gradient-mediated assembly of ICP capsules, (b) Ternary phase diagram showing the phase separation condition of solutions with acetone, water, and NS36 peptide. The shaded area indicates the phase separation region, (c) Optical (left) and fluorescence (right) images of peptide assemblies (2 mg / mL) formed after 10 minutes of microdroplet formation at a 9:1 volume ratio of acetone to water, (d) Fluorescence distribution micrograph of freshly prepared NS36 droplets infiltrated with fluorescein isothiocyanate (FITC) and Rhodamine-B (Rh-B), taken after the final mixed solution was prepared under a 9:1 volume ratio of acetone to water and a final peptide concentration of 4 mg / mL. (e-f) Representative images (e) and fluorescence recovery after photobleaching (FRAP) curve (f) of freshly prepared droplets of NS36 (2 mg / mL) under a 9:1 volume ratio of acetone to water, (g-h) Representative images (g) and FRAP curve (h) of fully matured NS36 capsules (2 mg / mL) taken 10 minutes after microdroplet formation. Data are presented as mean values ± SD from three repeated experiments, (i) Time-varying confocal micrograph showing the transformation of NS36 capsules from the microdroplet state. Scale bar is 5 pm.

[0036] Fig. 10 depicts liquid-liquid phase separation (LLPS) phase diagram of WA30 (a), VV30 (b) in Water / Acetone Systems.

[0037] Fig. 11 depicts size distributions of (a) NS36 and (b) WA30 LLPS droplets with generation time, and (c) the influence of adding crosslinking agents (IPDI) on the size of ICPs capsules.

[0038] Fig. 12 depicts size distribution analysis of (a) WA30, (b) NS36, (c) VV30 and (d) QH33 capsules or particles with different crosslinker to peptide molar ratio though Dynamic Light Scattering (DLS).

[0039] Fig. 13 depicts (a) fluorescence distribution micrograph of freshly prepared NS36 (top) and WA30 (bottom) coacervate droplets infiltrated with FITC (2 mg mL-1in 9 pL acetone) and Rhodamine-B (18 mg mL1in 1 pL water), final mixed solution under 9:1 vol ratio of acetone to water and final peptide concertation at 4 mg mL1. Three experiments were repeated independently with similar results. Scale bar is 10 pm. (b) Fluorescence microscope image of FITC distribution in the NS36 (top) and WA30 (bottom) capsules systems changing with time. Scale bar is 10 pm. Fig. 14 depicts optical (left) and fluorescence (right) micrograph of peptide LLPS droplets and capsules (2 mg mL1) at 9:1 vol ratio of acetone to water within and after LLPS formation (representative images of three independent experiments). Scale bar is 5 pm.

[0040] Fig. 15 depicts representative confocal images during FRAP of freshly prepared coacervate droplet and capsules of WA30 (a and b) and NS36 (c and d) under 9:1 vol ratio of acetone to water. Scale bar for a) and b) is 5 pm, for c) and d) is 1 pm.

[0041] Fig. 16 depicts the LLPS droplets formed by FITC and RhB labeled NS36 fuse with each other after forming phase separation, (a) Image under 60x objective lens, (b) Partial zoom in image.

[0042] Fig. 17 depicts transition of ICP secondary structure during the self-assembly process, (a) CD spectra of ICPs (0.5 mg / mL) in aqueous solutions, (b) 2D1H-15N HSQC spectrum of NS36 with individual amino acids labeled, (c) Single representative energy-minimized structure of NS36 from NMR measurement, (d) Simulation snapshots of single peptide WA30 (left), NS36 (middle), and QH33 (right) in water. The backbone atoms of each peptide were aligned to the corresponding NMR structures (transparent) using VMD RMSD calculator (WA30: 3.17 A; NS36: 6.96 A; QH33: 3.36 A), (e) Fluorescence spectra of ICP transformation structure information in gradient-composed mixed solvents monitored through thioflavin-T (ThT) staining in gradiometric composed mixed solvents, (f) Kinetic analysis of |3-sheet evolution during capsules formation based on ThT staining monitoring, (g) The amide I ATR-FTIR spectra of NS36 (2 mg / mL) under different water-to-acetone volume ratios. All data are presented as means ± SD binding assay from n = 3 independent assays.

[0043] Fig. 18 depicts FTIR deconvolution analysis of the amide I of WA30, NS36, VV30 and QH33 in aqueous solution under 10 mg mL'1, data are means ± SD from n = 3 independent assay.

[0044] Fig. 19 depicts (a) expression of isotopically substituted NS36 by the E. coli, (b) target proteins constructed by five NS36 tandem purified by His-tag, channel 1 and 2: total protein; and channel 3 and 4: target protein, (c) MALDI-TOF spectra of NS36 with supernumerary ring structure (i) and an additional alanine (ii), and (d) MALDI-TOF spectra of NS36 (i) and isotopically substituted NS36 (ii).

[0045] Fig. 20 depicts (a) target proteins constructed by five WA30 tandem purified by His-tag, channel 1 and 2: total protein; channel 3 and 4: target protein, (b) MALDI-TOF spectra of WA30 with supernumerary ring structure ([M+H]+: 3324.65) and an additional alanine ([M + H]+: 3209.20). (c) Target proteins constructed by five VV30 tandem purified by His-tag, channel 1 and 2: total protein; channel 3 and 4: target protein, (d) MALDI-TOF spectra of VV30 with supernumerary ring structure ([M+H]+: 3126.50) and an additional alanine ([M+H]+: 2998.81).

[0046] (e) MALDI-TOF spectra of VV30 ([M+H]+: 3275.7) and isotopically substituted VV30 ([M+H]+: 2925.9).

[0047] Fig. 21 depicts bar diagram representations of NOE connectivities of (a) WA30, (b) NS36 and

[0048] (c) QH33.

[0049] Fig. 22 depicts (a) chemical shift deviations (Ca) plot of NS36 and (b) chemical shift deviations (Ha) plot of NS36.

[0050] Fig. 23 depicts (a) 2D1H-1H NOESY spectrum of WA30 peptide displaying Ha region and noesy connectivities (only Ha spins of WA30 are marked for clarity), (b) Chemical shift deviations (Ha) plotof WA30. (c) Representative energy minimized structure of WA30 showing TT-TT interactions of W9-W17 while the other side chains of Trp (W1 and W25) remain exposed.

[0051] (d)1H-1H NOESY spectrum of QH33 peptide displaying Ha region and noesy connectivities (only Ha spins of QH33 are marked for clarity), (e) Chemical shift deviations (Ha) plot of QH33.

[0052] (f) Representative energy minimized structure of QH33 with sidechains of His labelled by residue number.

[0053] Fig. 24 depicts the kinetic analysis of ICPs WA30 (a), NS36 (b) and VV30 (c) transformation structure information monitored though ThT staining in gradiometric composed mixed solvents, (d) Fluorescence spectra of ICPs transformation structure information monitored though ThT staining after capsules formation within different time scale, (e) Structural changes of crosslinked capsules after re-dialysis into the aqueous environment, (f) Fluorescence optical micrograph of peptide capsules staining with 0.5 mg mL'1ThT. Scale bar is 5 pm.

[0054] Fig. 25 depicts (a) the amide I ATR-FTIR spectra of WA30 (2 mg / ml) under different water to acetone ratios, data are representative from n = 3 independent assays, (b) The proportion of WA30 secondary structure based on FTIR deconvolution fitting; data are means ± SD from n = 3 independent fitting.

[0055] Fig. 26 depicts (a) the amide I ATR-FTIR spectra of NS36 (2 mg / ml) under different water to acetone ratios, data are representative from n =3 independent assays, (b) The proportion of NS36 secondary structure based on FTIR deconvolution fitting; data are means ± SD from n = 3 independent fitting. Fig. 27 depicts (a) the amide I ATR-FTIR spectra of V30 (2 mg / ml) under different water to acetone ratios, data are representative from n = 3 independent assays, (b) The proportion of VV30 secondary structure based on FTIR deconvolution fitting; data are means ± SD from n = 3 independent fitting.

[0056] Fig. 28 depicts (a) the amide I ATR-FTIR spectra of QH33 (2 mg / ml) under different water to acetone ratios, data are representative from n = 3 independent assays, (b) The proportion of QH33 secondary structure based on FTIR deconvolution fitting; data are means ± SD from n = 3 independent fitting.

[0057] Fig. 29 depicts the amide I ATR-FTI spectra of (a) WA30, (b) NS36, (c) VV30 and (d) QH33 under 1 / 9 (water to acetone ratio) with and without crosslinker.

[0058] Fig. 30 depicts (a) evolution of mean square displacement (MSD) for water, acetone, and the system, (b) Partial density plot showing the distribution of acetone along the reaction coordinate with 2 ns intervals, (c) Partial density plot showing the distribution of water along the reaction coordinate with 2 ns intervals.

[0059] Fig. 31 depicts exploring the driving forces of ICP self-assembly through Molecular Dynamics (MD) simulations, (a-c) Simulation snapshots of the self-assembly of QH33 (a), WA30 (b) and NS36 (c), in the mixed solution with acetone (63% (w / w) acetone). Four peptides were simulated for each case. Insets: zoom-in views of the detailed inter-peptide interactions, (d) The number of H-bonds between a peptide and the solvent molecules (water and / or acetone) plot against the concentration of acetone, (e) Free energy profile of transferring a peptide from pure water to the target chemical environment (cacetone) showing different trend for QH33 versus WA30 and NS36. The dashed lines mark the quadratic fitting of each corresponding AGiFE with intercepts setting to zero at caCetone = 0. Data are means ± SD calculated with the Multistate Bennett Acceptance Ratio (MBAR) method, (f) A graphical schematic showcasing dynamic processes and driving force for gradient-mediated self-assembly of capsule forming peptides.

[0060] Fig. 32 depicts (a) root-mean-square deviation (RMSD) of a peptide as a function of acetone concentration, (b) Radius of gyration (Rg) of a peptide as a function of acetone concentration.

[0061] (c) Solvent accessible surface area (SASA) of a peptide as a function of acetone concentration.

[0062] (d) Number of hydrogen bonds between a peptide and solvent molecules (water and / or acetone) as a function of acetone concentration. Data represent means ± standard deviation (SD) obtained from simulation trajectories. Fig. 33 depicts (a) RMSD of the peptide assembly as a function of acetone concentration, (b) Rg of the peptide assembly as a function of acetone concentration, (c) SASA of the peptide assembly as a function of acetone concentration, (d) Number of hydrogen bonds between the peptide assembly and solvent molecules (water and / or acetone) as a function of acetone concentration. Data represent means ± standard deviation (SD) obtained from simulation trajectories.

[0063] Fig. 34 depicts (a) gel electrophoresis of different percentage acetone treatment on Gox. (b) The effect of acetone on the fluorescence intensity of enhanced Green Fluorescent Protein (EGFP). (c) The effect of acetone treatment time on the activity of different protein and peptide cargoes.

[0064] Fig. 35 depicts encapsulating protein and drug by ICP capsules, (a) Schematic representation of encapsulating Doxorubicin (DoX) and EGFP simultaneously into the ICP capsules, (b-c) EGFP (b) and DoX (c) encapsulation efficiency of the ICP capsules. Data are displayed individually (hollow dots of the same color as the bar) and as the mean ± SD (column with error bar) from n = 5 independent measurements, (d) Representative fluorescence images demonstrating the successful delivery of FITC-labelled ICP capsules into Hela cells. All delivery results were confirmed by n = 3 independent assays.

[0065] Fig. 36 depicts (a) fluorescence microscope images of NS36 capsules dispersed in acetone and waterwith loading FITC. (b) Schematic diagram of FITC distribution inside NS36 capsules.

[0066] Fig. 37 depicts recruitment efficiency of biomacromolecules (a) and small molecules (b) by NS36 capsules and (c) WA30 and VV30 capsules; the mean ± SD (column with error bar) of n = 3 independent measurements.

[0067] Fig. 38 depicts intracellular drug, protein and mRNA delivery into cells using ICPs capsules, (a) Cytotoxicity assessment of unloaded ICPs nanocapsules. Data are displayed as mean ± SD of n = 5 independent measurements, (b) Flow cytometry analysis of EGFP delivery mediated by ICPs capsules into HeLa cells after a 2-hour (2 h) incubation period. Two groups were included as controls: untreated cells (negative control, NC) and cells treated solely with EGFP without any carriers (blank), (c) Concentration-dependent cytotoxicity of DoX and DoX- loaded ICPs capsules. Data are presented as mean ± SD of n = 3 independent experiments. Two-sided Student’s t-test results are shown: N.A. = 0.1960, *P = 0.0063, ***P = 0.0007 compared to DoX alone at 10 g / ml. (d) 3D confocal microscopy images of Hela cells treated with Doxorubicin Hydrochloride (DoXHCI)-loaded NS36 capsules for 12 h. This delivery result was confirmed in n = 3 independent assays, (e) Confocal microscopy images of Hela cells treated with FITC-labeled BSA and DoXHCI co-loaded NS36 capsules for 4 h. This delivery result was confirmed in n = 3 independent assays, (f-g) Confocal microscopy images (f) and flow cytometry analysis (g) of MCF7 cells transfected with EGFP mRNA loaded in CC-NK3S- CC-RGD capsules. Control 1 , untreated MCF7 cells; Control 2, MCF7 cells directly treated with EGFP mRNA.

[0068] Fig. 39 depicts (a) size distributions of ICPs capsules in phosphate buffered saline (PBS), and (b) zeta potentials of ICPs capsules in different pH buffer and the mean ± SD of n = 3 independent measurements.

[0069] Fig. 40 depicts (a) size distributions of NS36 capsules (with different peptide to crosslinker ratio) in PBS. (b) Zeta potentials of NS36 capsules in PBS and the mean ± SD of n = 3 independent measurements, (c) The fluorescence-activated cell sorting (FACS) results of Hela cells internalization of the different size of NS36 capsules.

[0070] Fig. 41 depicts confocal microscopy images of different cell line internalization of the NS36 capsules.

[0071] Fig. 42 depicts the FACS results of different cell line internalization of the NS36 capsules. The cells with the PE intensity greater than 104were considered dead, (a) FACS results for Hela cells; (b) FACS results for MCF7 cell; and (c) FACS results for U2OS cells

[0072] Fig. 43 depicts (a) fluorescence microscopy and TEM images of the release of RhB labeled BSA loaded on NS36 and WA30 capsules; (b) breaking the capsules structure by adding 500 ng mb1protease in vitro to release internal macromolecular cargoes and the mean ± SD (error bar) of n = 3 independent measurements; and (c) DoXHCI (10 pg mL1) releasing dynamics of NS36 (c), WA30 (d), W30 (e) and QH33 (f) capsules under different pH, the mean ± SD of n = 3 independent measurements.

[0073] Fig. 44 depicts confocal microscopy images of Hela cells treated with FITC-labeled BSA and DoXHCI co-loaded NS36 capsules for 24 h. The delivery result was confirmed by n = 3 independent assays. The DoXHCI was showed by green, and the lysosome was stained by lysoTracker. The DoXHCI were generally co-localized with lysosome and nucleus. Scale bar for original image is 20 pm, and for partial enlarged 3D reconstructed image is 10 pm. The partially enlarged 3D reconstructed image showed the release of DoX from the lysosome which was co-located with peptide capsules. Fig. 45 depicts the FACS analysis of peptide capsules co-delivery of DoX and EFGP to HeLa cell for 4 h. After 4 h of internalizing the NS36 and VV30 capsules, the two experimental groups showed similar DoX intensity, while EGFP intensity showed significant differences. This phenomenon may be due to the proton sponge effect by the VV30 peptide facilitating endosomal / lysosomal escape, whereas the NS36-capsules may remain in the lysosomes. The low pH environment of the endosomes / lysosomes will cause fluorescence quenching of EGFP and result in lower signal intensity, meanwhile the EGFP loaded in VV30 which escaped into the cytoplasm did not have this concern. In this figure, “NC” is the negative control.

[0074] Fig. 46 depicts cell internalization of different peptide capsules. The NS36 capsules entered lysosome after being internalized by HeLa cells, and confocal fluorescence images show high fluorescence co-localization of lysosome and NS36 capsules. However, the capsules of VV30 were predominantly not co-localized with lysosomes after internalization. This may be due to a proton sponge effect by the His residues present in the VV30 peptide, which may facilitate endosomal / lysosomal escape of the VV30 capsules.

[0075] Fig. 47 depicts breaking the capsules structure by adding glutathione (GSH) in vitro to release BSA-RhB; the mean ± SD (error bar) of n = 3 independent measurements.

[0076] Fig. 48 depicts (a) zeta-potential and (b) DLS size distribution of different capsules made by NS36 and NS36 mutations.

[0077] Fig. 49 depicts intracellular plasmid and mRNA transfection though NS36 mutations nanocapsules. (a) Size distributions of NS36 mutations capsules in PBS. (b) The internalization of NS36 mutations capsules by a HeLa cell mass, (c) The effect of different organic solvents on the stability of EGFP nucleic acid, (d) Confocal microscopy images and FACS analysis of HeLa cells treated with EGFP plasmid loaded CC-N3KS-CCRGD capsules, (e) Confocal microscopy images and FACS analysis of HeLa cells treated with EGFP mRNA loaded CC- N3KS-CCRGD capsules, (f) Confocal microscopy images and FACS analysis of U2OS cells treated with EGFP mRNA loaded CC-N3KS-CC-RGD capsules.

[0078] Fig. 50 depicts (a) confocal microscopy images and (b) FACS analysis of HeLa cells treated with FITC labeled CC-N3KS-CC-RGD capsules.

[0079] Fig. 51 depicts cytotoxicity test of only ICPs vesicles and peptide without any cargo, data are means D (column with error bar) of n=3 independent measurements. Fig. 52 depicts confocal microscope images showing expression of GFP protein in HeLa cells. Cells were incubated with GFP mRNA loaded N3KS vesicles for 4 h in optiMEM medium, then optiMEM was changed back to Dulbecco’s modified Eagle medium (DMEM) and cells were cultured for overnight. Cells were washed with 1X PBS and fixed with 4% paraformaldehyde (PFA) before imaging.

[0080] Fig. 53 depicts GFP expression in Hela cells analysed by FACS: the dot plot shows live / dead cells from PI (detected by PE-CF594 channel) and cells with or without GFP expression (by FITC channel). Left panel is HeLa cells alone and right panel is HeLa cells with addition of GFP-mRNA loaded capsules formed from the N3KS peptide.

[0081] Fig. 54 depicts confocal microscope images showing expression of MCF7 protein in HeLa cells. Cells were incubated with GFP mRNA loaded N3KS vesicles for 4 h in optiMEM medium, then optiMEM was changed back to DMEM and cells were cultured for overnight. Cells were washed with 1X PBS and fixed with 4% PFA before imaging.

[0082] Fig. 55 depicts GFP expression in MCF7 cells analysed by FACS: the dot plot shows live / dead cells from PI (detected by PE-CF594 channel) and cells with or without GFP expression (by FITC channel). Left panel is HeLa cells alone and right panel is HeLa cells with addition of GFP-mRNA loaded N3KSvesicles.

[0083] Fig. 56 depicts confocal microscope images showing GFP expression in U2OS cells. Cells were incubated with GFP mRNA loaded N3KS vesicles for 4 h in optiMEM medium, then optiMEM was changed back to DMEM and cells were cultured for overnight. Cells were washed with 1X PBS and fixed with 4% PFA before imaging.

[0084] Fig. 57 depicts GFP expression in U2OS cells analysed by FACS: the dot plot shows live / dead cells from PI (detected by PE-CF594 channel) and cells with or without GFP expression (by FITC channel). Left panel is Hela cells alone and right panel is Hela cells with addition of GFP- mRNA loaded capsules formed from the N3KS peptide.

[0085] FIG. 58 depicts TEM images of the following peptide capsules: (a) N5DS (SEQ ID NO: 10); (b) N6FS (SEQ ID NO: 11); (c) 4D-WA30 (SEQ ID NO: 14) (d) 5F-WA30 (SEQ ID NO: 16); (e) 5K-VV30 (SEQ ID NO: 18); and (f) Y-VV30 (SEQ ID NO: 44).

[0086] FIG. 59 depicts cell internalization of CC-5K-VV30-CCRGD (SEQ ID NO: 25) peptide capsules. The peptide capsules entered lysosome after being internalized by HeLa cells, and confocal fluorescence images show high fluorescence co-localization of lysosome and peptide capsules.

[0087] DESCRIPTION

[0088] As discussed in more detail below, the present inventors have identified repeat peptide sequences present in proteins found in the cuticle of Ostrinia furnacalis (O. fumacalis) that display self-assembling properties and can form capsules. The peptide capsules formed by the identified peptides are surprisingly effective at encapsulating cargo molecules and delivering the encapsulated cargo molecules into cells. The present inventors have also identified various amino acid substitutions to the repeat peptide sequences which can alter the properties of the peptide capsules formed, such as the capsule size and the charge of the capsule, which in turn allows for modification of the intracellular delivery properties of the capsules and enables encapsulation of a wide variety of cargo molecules.

[0089] Thus, in a first aspect, the present invention provides a peptide capsule for delivery of a cargo molecule into a cell, the peptide capsule comprising: a peptide shell and a cargo molecule, wherein the peptide shell is formed from a plurality of self-assembling peptides, the self-assembling peptides each comprising an amino acid sequence given in SEQ ID NO: 1 , 2 or 3, wherein:

[0090] SEQ ID NO: 1 is:

[0091] NX2X3X4VPPS [NX10X11VPPS]nwherein, n is an integer from 2 to 6;

[0092] X2is N, K, R, H, D or E;

[0093] X3is Y, F or absent;

[0094] X4is Y or F; each X10is independently N, K, R, H, D or E; and each X11is independently Y or F;

[0095] SEQ ID NO: 2 is:

[0096] [X1X2AAPA X7 8]n X9X10AAPAX16'16wherein, n is an integer from 2 to 6; each X1is independently W or F; each X2is independently N, K, R, H, D or E; each X7-8is independently NQ, PS, or NH;

[0097] X9is independently W or F;

[0098] X10is independently N, K, R, H, D or E; and

[0099] X15 15is absent, NQ, PS, or NH;

[0100] SEQ ID NO: 3 is:

[0101] [VX2X3APV]nwherein, n is an integer from 3 to 8; each X2is independently H or Y; and each X3is independently S, K, R, H, D or E.

[0102] The amino acid sequences disclosed herein are shown with the N-terminus to the left, and where sequences are set out across multiple lines, the N-terminus is to the top left. Unless indicated otherwise, the amino acid residues in the sequences are L-amino acids.

[0103] In the context of the present disclosure, the term “amino acid” encompasses any naturally occurring amino acid or unnatural amino acid. It includes an amino acid in any chiral configuration. The amino acid may, especially, be a naturally occurring alpha-amino acid. The amino acid may, especially, be a naturally occurring L amino acid. The amino acid may, especially, be a naturally occurring alpha L-amino acid.

[0104] Within a polypeptide chain, the amino acids are linked by peptide bonds between the carboxyl group of one amino acid and the amine group of the next amino acid in the chain. An individual amino acid is called a “residue” or “amino acid residue” once it is linked in a polypeptide chain.

[0105] The amino acid sequences listed in the application are shown using standard letter abbreviations for amino acids.

[0106] For the avoidance of doubt, the peptide capsules of the present invention may also be referred to herein as “I CP capsules” or “Insect Cuticle Peptide capsules”.

[0107] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0108] In SEQ ID NO: 1 , n is an integer from 2 to 6, for example n may be 2 to 5, or 3 to 5. In certain embodiments, the self-assembling peptides may each comprise an amino acid sequence given in SEQ ID NO: 1 , wherein n is 4.

[0109] In SEQ ID NO: 2, n is an integer from 2 to 6, for example n may be 2 to 5, or 2 to 4. In certain embodiments, the self-assembling peptides may each comprise an amino acid sequence given in SEQ ID NO: 2, wherein n is 3.

[0110] In SEQ ID NO: 3, n is an integer from 2 to 8, for example n may be 3 to 8, or 4 to 6. In certain embodiments, the self-assembling peptides may each comprise an amino acid sequence given in SEQ ID NO: 3, wherein n is 5.

[0111] In certain embodiments, the self-assembling peptides that form the peptide shell each comprise an amino acid sequence given in SEQ ID NO: 4, 12, or 17. The amino acid sequences given in SEQ ID NO: 4, 12, and 17 correspond to the amino acid sequences referred to in the Examples section herein as NS36, WA30 and VV30, respectively. These peptide sequences may also be referred to herein as wtNS36, wtWA30 and wtVV30, wherein “wt” is an abbreviation for the term “wild-type”. The peptide sequences NS36, WA30 and VV30 have been identified by the present inventors as being present in cuticle proteins of O. furnacali, and found to be surprisingly effective at forming peptide capsules that can encapsulate a cargo and deliver the encapsulated cargo into a cell.

[0112] The term “wt” is an abbreviation for the term “wild-type”. As used herein, the term "wild-type" refers to a sequence such as a polynucleotide sequence or amino acid sequence that is found in nature. For example, in the context of a peptide, a wild-type peptide is one which has an amino acid sequence that corresponds to an amino acid sequence of a peptide or protein found in nature.

[0113] The present inventors have also found that by including one or more basic amino acids (e.g. K, R, or H) at selected positions within the self-assembling peptides, it is possible to form peptide capsules that are smaller than those formed from corresponding peptides without the one or more basic amino acid substitutions, for example the peptide capsules including one or more basic amino acids may have an average diameter of less than about 1 pm (e.g. less than 200 nm). Peptide capsules with an average diameter of less than about 1 pm may be particularly effective at delivering a cargo molecule into a cell. Without wishing to be bound by theory, the presence of one or more basic amino acids is also believed to have a proton sponge effect which facilitates release of the peptide capsule from endosomes and / or lysosomes into the cytoplasm. The positive charge may also improve encapsulation of negatively charged and / or hydrophilic cargo molecules, such as cargo molecules comprising or consisting of nucleic acids.

[0114] Thus, in certain embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 1 , wherein in SEQ ID NO: 1:

[0115] X2is N, K, R, or H, for example N or K;

[0116] X3is Y, F or absent, for example Y;

[0117] X4is Y or F, for example Y; each X10is independently N, K, R, or H, for example independently N or K; and each X11is independently Y or F, for example Y.

[0118] For example, the self-assembling peptides may each comprise an amino acid sequence given in SEQ ID NO: 1, wherein in SEQ ID NO: 1 , at least one (e.g. 1 , 2, 3, 4, or 5) of each X10and X2is K, R or H (e.g. K). Thus, in certain embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8 and 9. For example, the self-assembling peptides may each comprise the amino acid sequence given in SEQ ID NO: 7.

[0119] In certain other embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the plurality of self-assembling peptides comprises i) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 1 , wherein in SEQ ID NO: 1 , X2is N and each X10is N; and ii) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 1, wherein in SEQ ID NO: 1 , X2is N, K, R, or H (e.g. K), and each X10is independently N, K, R, or H (e.g. K), wherein at least one (e.g. 1, 2, 3, 4, or 5) of each X10and X2is K, R or H (e.g. K). Thus, for example, the plurality of self-assembling peptides may comprise self-assembling peptides with the amino acid sequence given in SEQ ID NO: 4, and one or more self-assembling peptides with an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8 and 9.

[0120] In certain other embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 2, wherein in SEQ ID NO: 2: each X1is independently W or F, for example W; each X2is independently N, K, R, or H, for example independently N or K; each X7’8is independently NQ, PS, or NH; X9is W or F, for example W;

[0121] X10is N, K, R, or H, for example N or K; and

[0122] X15 18is absent, NQ, PS, or NH, for example absent.

[0123] For example, the self-assembling peptides may each comprise an amino acid sequence given in SEQ ID NO: 2, wherein in SEQ ID NO: 2, at least one (e.g. 1 , 2, 3, 4, or 5) of each X2and X10is K, R or H (e g. K). Thus, in certain embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 13.

[0124] In certain other embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the plurality of self-assembling peptides comprises i) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 2, wherein in SEQ ID NO: 2, each X2is N and X10is N; and ii) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 2, wherein in SEQ ID NO: 2, each X2is independently N, K, R, or H, and X10is N, K, R, or H (e.g. K), wherein at least one (e.g. 1, 2, 3, 4, or 5) of each X2and X10is K, R or H (e.g. K). Thus, for example, the plurality of selfassembling peptides may comprise self-assembling peptides with the amino acid sequence given in SEQ ID NO: 12, and self-assembling peptides with an amino acid sequence given in SEQ ID NO: 13.

[0125] In other certain embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 3, wherein in SEQ ID NO: 3: each X2is independently H or Y, for example H; and each X3is independently S, K, R or H, for example independently K.

[0126] For example, the self-assembling peptides may each comprise an amino acid sequence given in SEQ ID NO: 3, wherein in SEQ ID NO: 3, each X2is H, and at least one (e.g. 1 , 2, 3, 4, or 5) of each X3is K, R or H (e.g. K). Thus, in certain embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the selfassembling peptides each comprise an amino acid sequence given in SEQ ID NO: 18.

[0127] In certain other embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the plurality of self-assembling peptides comprises i) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 3, wherein in SEQ ID NO: 3, each X3is S; and ii) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 3, wherein in SEQ ID NO: 3, each X2is H and at least one (e.g. 1 , 2, 3, 4, or 5) of each X3is independently K, R, or H (e.g. K). Thus, for example, the plurality of self-assembling peptides may comprise self-assembling peptides with the amino acid sequence given in SEQ ID NO: 17, and self-assembling peptides with the amino acid sequence given in SEQ ID NO: 18.

[0128] The present inventors have also found that by including one or more acidic amino acids (e.g. D and E) at selected positions within the self-assembling peptides, it is possible to form peptide capsules that are smaller than those formed from corresponding peptides without the one or more acidic amino acids substitutions, for example the peptide capsules including one or more acidic amino acids may have an average diameter of less than about 1 pm (e.g. less than 200 nm). This can provide peptide capsules that are particularly effective at delivering a cargo molecule into a cell. The negative charge may also improve encapsulation of positively charged and / or hydrophilic cargo molecules.

[0129] Thus, in certain embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 1 , wherein in SEQ ID NO: 1:

[0130] X2is N, D or E, for example N or D;

[0131] X3is Y, F or absent, for example Y;

[0132] X4is Y or F, for example Y; each X10is independently N, D or E, for example independently N or D; and each X11is independently Y or F, for example Y.

[0133] For example, the self-assembling peptides may each comprise an amino acid sequence given in SEQ ID NO: 1, wherein in SEQ ID NO: 1, at least one (e.g. 1 , 2, 3, 4, or 5) of each X10and X2is D or E (e.g. D). Thus, in certain embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NOs: 10.

[0134] In certain other embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the plurality of self-assembling peptides comprises i) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 1, wherein in SEQ ID NO: 1 , X2is N and each X10is N; and ii) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 1, wherein in SEQ ID NO: 1 , X2is N, D or E (e.g. D), and each X10 is independently N, D or E (e.g. D), wherein at least one (e.g. 1, 2, 3, 4, or 5) of each X10and X2is D or E (e.g. D). Thus, for example, the plurality of selfassembling peptides may comprise self-assembling peptides with the amino acid sequence given in SEQ ID NO: 4, and self-assembling peptides with the amino acid sequence given in SEQ ID NO: 10. In certain other embodiments, the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 2, wherein in SEQ ID NO: 2: each X1is independently W or F, for example W; each X2is independently N, D or E, for example independently N or D; each X7'8is independently NQ, PS, or NH;

[0135] X9is W or F, for example W;

[0136] X10is N, D or E, for example N or D; and

[0137] X15’16is absent, NQ, PS, or NH, for example absent.

[0138] For example, the self-assembling peptides may each comprise an amino acid sequence given in SEQ ID NO: 2, wherein in SEQ ID NO: 2, at least one (e g. 1 , 2, 3, 4, or 5) of each X2and X10is D or E (e.g. D). Thus, in certain embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 15.

[0139] In certain other embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the plurality of self-assembling peptides comprises i) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 2, wherein in SEQ ID NO: 2, X2is N and each X10is N; and ii) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 2, wherein in SEQ ID NO: 2, each X2is independently N, D or E, and X10is N, D or E (e.g. D), wherein at least one (e.g. 1 , 2, 3, 4, or 5) of each X2and X10is D or E (e.g. D). Thus, for example, the plurality of self-assembling peptides may comprise self-assembling peptides with the amino acid sequence given in SEQ ID NO: 12, and self-assembling peptides with an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 15.

[0140] In certain other embodiments, the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 3, wherein in SEQ ID NO: 3: each X2is independently H or Y, for example H; each X3is independently S, D or E, for example independently S or D.

[0141] For example, the self-assembling peptides may each comprise an amino acid sequence given in SEQ ID NO: 3, wherein in SEQ ID NO: 3, each X2is H, and at least one (e.g. 1 , 2, 3, 4, or 5) of each X3is D or E (e.g. D). In certain other embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the plurality of selfassembling peptides comprises i) self-assembling peptides comprising the amino acid sequence given in SEQ ID NO: 3, wherein in SEQ ID NO: 3, each X3is N; and ii) self- assembling peptides comprising the amino acid sequence given in SEQ ID NO: 3, wherein in SEQ ID NO: 3, at least one (e.g. 1 , 2, 3, 4, or 5) of each X3is independently D or E (e.g. D).

[0142] In certain embodiments, the self-assembling peptides that form the peptide shell of the peptide capsule may be cross-linked. It has been demonstrated that by cross-linking the selfassembling peptides, it is possible to form peptide capsules that have a smaller diameter than when the self-assembling peptides are not cross-linked. For example, to form peptide capsules with an average diameter of less than about 1 pm (e.g. less than 500 nm, less than 200 nm), the self-assembling peptides may be cross-linked using a cross-linking agent.

[0143] The term “cross-linking agent” as used herein refers to a molecule in which at least two chemically reactive groups are connected to one another via a linker. Examples reactive groups include sulfhydryl-reactive groups (e.g. maleimides, pydridyl disulfides, a-haloacetyls, vinyl sulfones, sulfato alkyl sulfones, amine-reactive groups (e.g. succinimidyl esters, carbodiimides, hydroxymethyl, phosphine, imidoesters, PFP esters, and aldehydes, isothiocyanates), carboxy-reactive groups (e.g. amines), hydroxyl-reactive groups (e.g. isocyanates), unselective groups (e.g. aryl azides), and photo-activatable groups (e.g. perfluorophenyl azide). These reactive groups may form covalent linkages with amine, thiol, carboxyl or hydroxyl groups present in the self-assembling peptides. Particularly suitable cross-linking agents for forming cross-links between the self-assembling peptides disclosed herein include, but not limited to, diisocyanates and disuccinimidyl esters, for example isophorone diisocyanate or disuccinimidyl suberate.

[0144] In certain embodiments, the self-assembling peptides each further comprise 1 or 2 cysteine residues or 1 or 2 selenocysteine residues at the N-terminus or the C-terminus, or each further comprise 1 or 2 cysteine residues or 1 or 2 selenocysteine residues at each of the N-terminus and C-terminus. For example, the self-assembling peptides may each comprise 1 or 2 cysteine residues at the N-terminus and 1 or 2 cysteine residues at the C-terminus. By incorporating cysteine residues or selenocysteine residues into the self-assembling peptides, it is possible to form cross-links between the self-assembling peptides that form the peptide shell of the peptide capsule, thereby reducing the size of the peptide capsules as compared to when the self-assembling peptides are not cross-linked. If cysteine residues are incorporated into the self-assembling peptides, the self-assembling peptides are cross-linked by one or more disulfide bonds. If selenocysteine residues are incorporated into the self-assembling peptides, the self-assembling peptides are cross-linked by one or more Se-Se bonds.

[0145] In certain embodiments, the self-assembling peptides may comprise an integrin-binding motif. The term “integrin-binding motif’ as used herein is a peptide sequence that can facilitate cell attachment of the peptide capsule through binding to integrins present in the membrane of a cell. An example of a suitable integrin-binding motif for use herein is the amino acid sequence RGD. The present inventors have found that by including the amino acid sequence RGD into the amino acid sequence of the self-assembling peptides it is possible to facilitate internalisation of the peptides capsules into a cell.

[0146] In certain exemplary embodiments, the self-assembling peptides may comprise 2 cysteine residues at the N-terminus, 2 cysteine residues at the C-terminus, and also an integrin-binding motif. In such embodiments, the self-assembling peptides may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 19-25.

[0147] The self-assembling peptides disclosed herein may be synthesised using methods known in the art. For example, they can be prepared by chemical synthesis methods (e g. FMOC solid phase peptide synthesis), or by recombinant protein production techniques, for example, by recombinant expression of the self-assembling peptides in bacterial, yeast, insect, fungal, plant or mammalian cells, or by cell-free in vitro protein expression methods using cell lysates and / or purified cell extracts. Thus, also provided herein is a polynucleotide encoding a selfassembling peptide, as defined herein. Also provided is an expression vector comprising a polynucleotide encoding a self-assembling peptide as defined herein, and a host cell (e.g. an E. coliceW) comprising a polynucleotide encoding a self-assembling peptide, as defined herein, or comprising an expression vector comprising a polynucleotide encoding a self-assembling peptide as defined herein.

[0148] As disclosed herein the peptide capsule of the present invention comprises a peptide shell cargo molecule. The peptide shell is formed by self-assembling peptides as disclosed herein above. The cargo molecule is typically encapsulated by the peptide shell. The cargo molecule may be a therapeutic agent, a diagnostic agent, or a therapeutic and diagnostic agent. The cargo molecule may be a biomacromolecule, a hydrophobic drug or a hydrophilic drug. For example, the cargo molecule may be a small molecule compound (e.g. a compound with a molecular weight of less than 900 Da), a peptide, a protein, or a nucleic acid.

[0149] More specific examples of cargo molecule that may be encapsulated by the peptide capsules of the present invention include, but not limited to, a cytotoxic drug, a cytostatic agent, a toxin, a radioisotope or radionuclide, a nucleotide, an RNA, a DNA, an antibiotic, an immunosuppressive agent, a fluorophore, a dye, and / or a protein. For example, in certain embodiments, the cargo molecule may be selected from the group consisting of rhodamine (e.g. rhodamine B), doxorubicin, paclitaxel, green fluorescent protein, an mRNA, an enzyme (e.g. p-galactosidase), and a proapoptotic protein (e.g. SMAC). Typically, the peptide capsule of the present invention has a diameter of less than about 10 pm, for example less than about 1 pm (e.g. less than about 500 nm, or less than about 200 nm). In embodiments wherein the self-assembling peptides include one or more basic amino acid (e.g. K, R, or H) or acidic amino acid (e.g. D or E) substitutions (with respect to the wildtype peptide sequence), the peptide capsule may have an average diameter of less of less than about 1 pm (e.g. less than about 500 nm, or less than about 200 nm). Peptide capsules with an average diameter of less than about 1 pm may be particularly effective at delivering a cargo molecule into a cell. The diameter of the peptide capsules may be reduced further by cross-linking the self-assembling peptides using a cross-linking agent or by including one or more cysteine or selenocysteine residues into the self-assembling peptide sequence, as disclosed herein above. In certain examples described herein, the peptide capsules may have an average diameter of less of less than about 500 nm, for example less than about 200 nm.

[0150] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, within 1%, within 0.5%, within 0.1 %, within 0.05%, within 0.01%, within 0.005%, or within 0.001% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0151] In exemplary embodiments disclosed herein, the peptide capsule of the present invention comprises: a peptide shell and a cargo molecule, wherein the peptide shell is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-25.

[0152] For example, the peptide capsule of the present invention comprises: a peptide shell and a cargo molecule, wherein the peptide shell is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-11 and 19-24.

[0153] Or, for example, the peptide capsule of the present invention comprises: a peptide shell and a cargo molecule, wherein the peptide shell is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-16.

[0154] Or, for example, the peptide capsule of the present invention comprises: a peptide shell and a cargo molecule, wherein the peptide shell is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18 and 25.

[0155] Or, for example, the peptide capsule of the present invention comprises: a peptide shell and a cargo molecule, wherein the peptide shell is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 , 16, 43, and 44.

[0156] For the avoidance of doubt, the self-assembling peptides disclosed herein which form the peptide shell of the peptide capsule, may each consist of an amino acid sequence given in SEQ ID NO: 1 , 2 or 3; and which optionally includes one or more of: i) 1 or 2 cysteine residues or 1 or 2 selenocysteine residues at the N-terminus or the C-terminus; ii) 1 or 2 cysteine residues or 1 or 2 selenocysteine residues at the N-terminus and the C-terminus; and ill) an integrin-binding motif.

[0157] In certain exemplary embodiments, the peptide shell of the peptide capsule is formed from a plurality of self-assembling peptides, wherein the self-assembling peptides consist of an amino acid sequence selected from the group consisting of 4-25, 43, and 44.

[0158] The present invention also provides an isolated self-assembling peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-25, 43, and 44. In certain embodiments, the isolated self-assembling peptide consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-25, 43, and 44.

[0159] The term "isolated" as used herein refers to any material that is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally- occurring peptide or protein in a living animal is not isolated, but the same peptide or protein, when separated from all or part of its original environment, is isolated.

[0160] The peptide capsules may be prepared from the self-assembling peptides described herein by precipitation of the self-assembling peptides by first dissolving them in water and adding a solvent such as acetone to precipitate the peptides, thereby forming capsules. For example, the self-assembling peptides described herein may be first dissolved in water and precipitated using acetone to form the peptide capsules of the present invention. Hydrophilic cargo molecules are loaded into the capsules when the self-assembling peptides form condensed droplets (also referred to herein as liquid-liquid phase separation (LLPS) droplets or capsules) in the water / solvent mixture, while hydrophobic cargo molecules are enriched in capsules through volatilization of the organic solvent after capsule formation. Typically, the solvent is acetone, and the ratio of acetone-to-water (v / v) required for the self-assembling peptides to form the peptide capsules is from about 2 to about 4 (e.g. from about 2.2 to about 3.8). Specific methods for the formation of the peptide capsules of the present invention are described in the Examples section herein. The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0161] The present invention provides a composition comprising the peptide capsule of the present invention and at least one binder, carrier or excipient. A composition of the invention comprising a peptide capsule of the present invention may comprise one or more the peptide capsules of the present invention. Preferably, it comprises more than one peptide capsule. In such embodiments, the peptide capsules may be the same, or they may be different. The peptide capsules may be different due to the amino acid sequence of the self-assembling peptides, the cargo molecule, or both the amino acid sequence of the self-assembling peptides and the cargo molecule.

[0162] Preferably, the composition of the invention is a pharmaceutical composition. For example, the composition may be a pharmaceutical composition comprising a peptide capsule according to the present invention and at least one pharmaceutically acceptable binder, carrier or excipient.

[0163] The pharmaceutical composition of the invention may be suitable for subcutaneous, intradermal, intramuscular, intravenous (bolus or infusion), intratumorally, intraarticular, intra lymphatic administration or in the form of an aerosol suitable for intranasal administration, although the most suitable route may depend upon, for example, the type of disease or disorder that is being treated. Preferably, the pharmaceutical composition of the invention is suitable for intradermal, intra-muscular, or subcutaneous injection; or the pharmaceutical composition is in the form of an aerosol suitable for intranasal administration.

[0164] Suitable pharmaceutically acceptable binders, carriers or excipients for including in the pharmaceutical formulation of the present invention may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable binders, carriers or excipients may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice. As disclosed herein, the present inventors have found that peptide capsules of the present invention are surprisingly effective for the delivery of cargo molecules, such as drug molecules, into a cell. Typically, the cell is a eukaryotic cell, for example a mammalian cell, such as a human cell. The mammalian cell (e.g. human cell) may be involved in a disease or disorder, for example the cell may be a cancer cell.

[0165] Thus, as will be appreciated the peptide capsule and pharmaceutical composition of the present invention may find utility as a medicament. Thus, the present invention further provides a peptide capsule and pharmaceutical composition as disclosed herein for use as a medicament, wherein the peptide capsule comprises a cargo molecule that is useful in the treatment or prevention of a disease or disorder, or encodes a bioactive substance useful in the treatment or prevention of a disease or disorder. The peptide capsule and pharmaceutical composition of the present invention also find utility in a method of treating or preventing a disease or disorder in a patient, said method comprising administering a pharmaceutically effective amount of the peptide capsule or pharmaceutical composition to the patient. The peptide capsule and pharmaceutical composition of the present invention may also find utility in the manufacture of a medicament for the treatment or prophylaxis of a disease or disorder

[0166] As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

[0167] The term “pharmaceutically effective amount” refers to an amount of the peptide capsule or pharmaceutical composition of the present invention which confers a therapeutic effect on the treated patient (e g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect). The amount of the peptide capsule or pharmaceutical composition that may administered to a subject will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, the function, potency or immunogenicity of cargo molecule, or the bioactive substance encoded by the cargo molecule (e g. a bioactive substance encoded by a nucleotide cargo molecule, such as an mRNA). In any event, a medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. However, typically, the dosage administered is one that may be considered to be a “therapeutic amount” or “efficacious amount”, wherein the terms “therapeutic amount” or “efficacious amount” as used herein may refer to the administration of an amount of the peptide capsule or pharmaceutical composition of the present invention that is capable of preventing a disease or improving the condition of a subject suffering from a disease.

[0168] The pharmaceutical formulation as disclosed above may be administered by any suitable route, but may particularly be provided in the form of a solution suitable for intradermal, intramuscular, or subcutaneous injection or in the form of an aerosol suitable for intranasal administration.

[0169] Further aspects of the present invention are defined in the following numbered clauses:

[0170] §1 . A peptide comprising one of the following amino acid sequences:

[0171] (i) WNAAPANQWNAAPAPSWNAAPANHWNAAPA (SEQ ID NO: 12);

[0172] (ii) NNYYVPPSNNYVPPSNNYVPPSNNYVPPSNNYVPPS (SEQ ID NO: 4);

[0173] (iii) VHSAPVVHSAPVVHSAPVVHSAPVVHSAPV (SEQ ID NO: 17);

[0174] (iv) NKYVPPS NNYVPPS NNYVPPS NNYVPPS NNYVPPS (SEQ ID NO: 5); or

[0175] (v) CCNKYVPPSNNYVPPSNNYVPPSNNYVPPSNNYVPPSCCRGD (SEQ ID NO: 20).

[0176] §2. A nanovesicle or nanocapsule comprising: peptides comprising the following amino acid sequences:

[0177] (i) WNAAPANQWNAAPAPSWNAAPANHWNAAPA (SEQ ID NO: 12);

[0178] (ii) NNYYVPPSNNYVPPSNNYVPPSNNYVPPSNNYVPPS (SEQ ID NO: 4);

[0179] (iii) VHSAPVVHSAPVVHSAPVVHSAPVVHSAPV (SEQ ID NO: 17);

[0180] (iv) NKYVPPS NNYVPPS NNYVPPS NNYVPPS NNYVPPS (SEQ ID NO: 5); or

[0181] (v) CCNKYVPPSNNYVPPSNNYVPPSNNYVPPSNNYVPPSCCRGD (SEQ ID NO: 20).

[0182] §3. The nanovesicle or nanocapsule according to clause 2, wherein the nanovesicle or nanocapsule is crosslinked by a crosslinking agent.

[0183] §4. The nanovesicle or nanocapsule according to clause 3, wherein the crosslinking agent is isophorone diisocyanate and disuccinimidyl suberate.

[0184] §5. The nanovesicle or nanocapsule according to clause 2, wherein the peptide comprising amino acid sequence (v) is crosslinked by disulfide bonds.

[0185] §6. The nanovesicle or nanocapsule according to any of the preceding clauses, wherein the nanovesicle or nanocapsule further comprises one or more of biomacromolecules, hydrophobic drugs and hydrophilic drugs. The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The applicant reserves the right physically to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0186] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0187] EXAMPLES

[0188] Materials and methods:

[0189] Peptides, resins and Fmoc-protected amino acids used in solid-phase peptide synthesis were purchased from GL Biochem. / V,M'-diisopropylcarbodiimide was purchased from Tokyo Chemical Industry (TCI). Acetic acid, A / , / V-diisopropylethylamine, 1- [bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, piperidine, tetrahydrofuran, / V, / V-dimethylformamide, dichloromethane, trifluoroacetic acid, bovine serum albumin, lysozyme, and rhodamine-B (Rh-B) were obtained from Sigma-Aldrich. LysoTracker Red DND-99, Opti-MEM, and Hoechst 34580 were purchased from Thermo Fisher Scientific. Other organic solvents including ethyl acetate, hexane and diethyl ether were purchased from Aik Moh Paints & Chemicals Pte Ltd. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline buffer (PBS 10X), were purchased from Gibco. Penicillin-Streptomycin solution was purchased from Cytiva. Live cell imaging solution was purchased from Invitrogen. Trans-Blot Turbo 0.2-pm nitrocellulose transfer packs, 4-20% Criterion TGX stain-free protein gel and Clarity and Clarity Max Western ECL substrate were purchased from Bio-Rad. Enhanced Green Fluorescent Protein (EGFP), and designed proteins were expressed in Escherichia coli BL21 strain and purified with Ni-NTA His Bind resin. Hela cell lines were obtained from ATCC. Doxorubicin (DoX), doxorubicin hydrochloride (DoXHCI) and paclitaxel (PTX) were purchased from Sigma-Aldrich.

[0190] Peptide synthesis and purification

[0191] The wild-type peptides were customized from GL Biochem. All those peptides were purified using preparative high-performance liquid chromatography (HPLC, Column: Gemini-NX 5 pm C18 110 A, 4.6*250 mm) in a gradient of acetonitrile and water with 0.1% trifluoroacetic acid (TFA). The purified peptides were tested by positive mode electrospray ionization mass spectrometry (ESI-MS) and semi analytical and preparative HPLC. For the other mutations, the classical Merrifield solid-phase peptide synthesis (SPPS) technique was used. Wang resin was swollen in N,N-dimethylformamide (DMF) first before use. All N-terminal protected amino acid (Fmoc-AA-OH) was dissolved in DMF before coupling, then 1- [bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b] pyridinium 3-oxide- hexafluorophosphate (HATU, 2 equiv., 1.12 mmol) and N,N-Diisopropylethylamine (DIPEA, 5 equiv., 2.80 mmol) were added into the solution. After 5 min of reaction, the mixture was added to the Wang resin in DMF at room temperature for more than 1 h coupling reaction with bubbling nitrogen flow. The resin was washed by DMF three times before use. The amino acids were coupled by Liberty Blue Automated Microwave Peptide Synthesizer from the C- terminal to N-terminal. For deprotection of the N-terminal and the side chain, 20% of piperidine and DMF mixture was added to the filtered resin for 1 h at room temperature under nitrogen flow, then the resin was washed by DMF three times to remove the piperidine. After all amino acids coupling was finished, the peptide needed to be cleaved from the resin by using a cocktail containing 95% of TFA, 2.5% of H2O and 2.5% of triisopropylsilane (TIPS). After cleavage for 2 h, the resin was removed by filtration, then the supernatant was evaporated to half the original volume by bubbling nitrogen flow. Crude peptides were precipitated by adding ten times the volume of cold diethyl ether to the supernatant. The precipitate was collected by centrifugation and was fully dissolved with 10% acetic acid and 90% deionised (DI) water. The HPLC (1260 prime II Infinity, Agilent Technologies) equipped with a C8 column (Zorbax 300SB-C8, Agilent Technologies) was used to purify those mutation peptides by using a gradient mixture of acetonitrile and water. The purified peptides were isolated by lyophilization (Labconco) from HPLC elutes.

[0192] The sequences of the peptides synthesised and tested herein are provided in Table 1.

[0193] Table 1. Peptide sequences

[0194]

[0195] Preparation and characterization of peptide capsules:

[0196] Peptides were dissolved in water (5 to 30 mg mL1concentration with 2.5 mg mb1as interval) at ambient temperature, whereas slightly excessive amount of isophorone diisocyanate (IPDI) were dissolved in acetone. The organic phase was poured directly into the peptide solutions with ratios from 9:1 to 1 :1 with vigorous stirring to generate capsules. After 6 h, equal volume of water was added to the reaction mixture to quench the reaction and the acetone in the reaction mixture was removed by gentle nitrogen flow. Then, the samples were dialyzed in water for 2 days and freeze-dried into powder.

[0197] The average hydrodynamic diameters (Dh) and Zeta potentials of peptide capsules were measured by Dynamic Light Scattering (DLS) on a Nano Particle Analyzer SZ-100 (HORIBA, Japan) at ambient temperature. The freshly prepared capsules were dispersed to a concentration of 2 mg mL1in water which had already adjusted pH which covered the range from 3.0 to 7.4 with 1 .0 interval by 0.01 M HCI or 0.01 M NaOH. Cursory morphological evaluation of the capsules was performed by AFM. All the AFM images were acquired by contact (tapping) mode on an Asylum Research Cypher S with silicon nitride hard tips. All capsules solution samples at 2 mg mL'1were dropped on freshly cleaved mica surface and dried under low vacuum drying oven at ambient temperature overnight. All images were fixed zero, flattened to remove background curvature, changed the stretch colour range, and cut into same size by Gwyddion 2.52; no further modification was made. Detailed morphological evaluation of the capsules was performed by TEM, those images were obtained on a Carl Zeiss Libra 120 Plus 120kV. Capsules samples were prepared by dropping 4 pL of dialysed sample solution with 2 mg mL1of the ICP on a 300-mesh carbon-coated copper grid. The loaded copper mesh was placed in a drying oven with ambient temperature for 24 h to let DI water evaporate. All images were taken by OLYMPUS CCD camera system under bright field with objective aperture inserted. The Cryo-TEM images were taken by the FEI Titan Krios 300kV TEM, all ICP capsules were prepared on the same condition as normal TEM.

[0198] Example 1. Screening of the Insect Cuticle Inspired Peptides

[0199] Tandem repeated peptides searching and sequence analysis

[0200] A mathematic program was constructed for repeated sequences that meet a specific criterion (the repeat unit contains 5 residues or more and the unit repeats at least 3 times).

[0201] Transcriptomic analysis of larvae cuticle and annotation of cuticle peptides

[0202] Laboratory colonies of 0. furnacalis were originally obtained from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. O. furnacalis were reared on an artificial diet at 26 to 28 °C and a relative humidity of 70% with a photoperiod of 16 h light and 8 h darkness. Total RNAs were extracted from the head capsule and integument of the fifth instar day 1 larvae, respectively, when the head capsules were not heavily tanned. Then, the total RNAs were used for mRNA preparation and cDNA library construction. This was followed by sequencing on an Illumina HiSeq 2500 platform using paired-end 150 bp reads. The raw reads were processed using Cutadapt (Martin, M., EMBnet.journal 2011 , 17, 10-12) and de novo assembled using Trinity (Haas, B. J. et al., Nat. Protoc. 2013, 8, 1494-1512) to obtain the unigenes. The unigenes were searched against the non-redundant protein database using BLASTx with a cutoff e-value of 1 x 10-5. The putative CP genes were identified on the basis of the NCBI reference sequence database (https: / / www.ncbi.nlm.nih.gov / qenbank / ).

[0203] Proteomic analysis of O. furnacalis head capsule

[0204] The 4thand 5thinstars O. furnacalis heads were grounded into powder with liquid nitrogen and treated with lysis solution (4% SDS, 100 mM Tris / HCI, pH 7.6, 0.1 M DTT) to extract protein. Protein digestion by trypsin was performed according to the filter-aided sample preparation (FASP) procedure described by Matthias Mann. The digest peptides of each sample were desalted on Empore™ SPE C18 Cartridges (Thermo Fisher Scientific, Waltham, USA), concentrated by vacuum centrifugation, and reconstituted in 40 L of 0.1 % (v / v) formic acid. The peptide content was estimated by UV light spectral density at 280 nm. Liquid chromatography with tandem mass spectrometry (LC-MS / MS) analysis was performed on a Q Exactive mass spectrometer that was coupled to Easy nLC (Thermo Fisher Scientific). The peptides were loaded onto a reverse phase trap column Acclaim PepMaplOO (100 m * 2 cm, nanoViper C18, Thermo Fisher Scientific) connected to the C18-reversed phase analytical column (10 cm long, 75 pm inner diameter, 3 pm resin, Thermo Fisher Scientific) in buffer A (0.1 % Formic acid in water) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1 % Formic acid) at a flow rate of 300 nL / min. The mass spectrometer was operated in positive ion mode. The MS raw data were combined and searched using the MaxQuant (version 1.6.14) software for identification and quantitation analysis.

[0205] Results and discussion

[0206] Insect cuticles are a rich source of self-assembling peptides. Insect cuticle is a composite material mainly composed of chitin and cuticle proteins, with complex and hierarchical structures. Cuticle proteins play critical roles in the assembly of the cuticle, while chitin serves as the reinforcement phase. The variations between 4th and 5th instar heads (Fig. 1) suggest rapid and intensive expression changes of cuticle proteins. We conducted a label-free, quantitative proteomic analysis on the head capsule of O. furnacalis, (Fig. 2a). This analysis yielded a total of 951 proteins, identified through a search in the SwissProt database. The normalized spectral abundance factor (NSAF), calculated based on spectral counting, was used to quantify each protein from the proteomic data sets. A subsequent transcriptomic analysis of O. furnacalis larvae cuticle revealed 233 cuticle proteins derived from unigenes (Fig. 2b and Table 2). These cuticle proteins possess molecular weights ranging from approximately 5 to 60 kDa. A comparative analysis with the amino acid compositions of eukaryote proteins revealed that cuticle proteins are heavily skewed towards Gly, Ala, and Vai, with a moderate preference for Leu, Pro, and Ser (Bogatyreva, N. S. et al., J. Bioinform. Comput. Biol. 2006, 4, 597-608) (Fig. 3). Given that functionally significant cuticle proteins often contain recurring functional domains, such as the chitin-binding Rebers and Riddiford (RR) domains (Willis, J. H., Insect Biochem. Mol. Biol. 2010, 40, 189-204), we conducted a further examination of the polypeptide sequences derived from the cuticle proteins. The screening criteria used herein for these sequences necessitated the presence of three or more repeating units, each comprising a minimum of five amino acids. By adhering to these screening criteria, nine repeated peptides were isolated from insect cuticle protein sequences (Figs. 2d and 4, and Table 3). Protein tandem repeats are frequently not perfect, containing mutations defined as spacers. The spacers found in our selected peptides are a result of their natural occurrence, not active selection. Tandem repeats in proteins may contain mutations in spacers accumulated during evolution, including substitutions, insertions, and deletions (Kajava, A. V., J. Struct. Biol. 2012, 179, 279-288). The screening algorithm used permits up to two amino acid variations between repeated sequences to account for this natural variability.

[0207] Table 2. Amino acid content of the Ostrinia furnacalis (Asian corn borer) head shells.

[0208] *O4L_3_Head = 4thinstars 0. furnacalis head; 05L_1_Head = 5thinstars 0. furnacalis head; and Eukaryotic = whole O. furnacalis body.

[0209] Table 3. Sequence information of insect cuticle peptides obtained through specific screening threshold.

[0210] Example 4. Mixed solvent induced self-assembly of insect cuticle inspired peptide

[0211] We investigated the assembly of the insect cuticle peptides (ICPs) in mixed solvents using various ratios of water and acetone, employing the nanoprecipitation method (Lebouille, J., et al., Colloids Surf. A: Physicochem. Eng. Asp. 2014, 460, 225-235) (Fig. 2e and see Example 2). In this method, acetone serves as a poor solvent for the peptides, enabling the induction of peptide assembly upon the addition of multiple acetone volumes into a concentrated peptide aqueous solution. The interaction between the solvent and peptides plays a vital role in controlling peptide assembly. Non-polar solvents, such as diethyl ether, result in extensive peptide precipitation, while solvents with high polarity, like dimethyl sulfoxide, lead to no observable assembly (Table 4). Five peptides, namely GL33, SF32, AP36, GH42, and YK34, were unable to form assembly structures due to either low solubility in water (GL33, SF32, and AP36) or high solubility in both acetone and water (GH42 and YK34, Table 5). In contrast, four peptides, namely WA30, NS36, VV30, and QH33, successfully formed nanostructures. We introduced isophorone diisocyanate (IPDI) as a crosslinking agent during the assembly process, serving to crosslink the N- and C-termini of the assembled peptides. It is noteworthy that the presence of IPDI ensures the structural integrity of the peptide assemblies in aqueous environment but does not affect the assembly structure of the peptides in the mixed acetone / water solution (Figs. 2f, 5 and 6a).

[0212] Table 4. The assembly phenomena of different solvents on insect cuticle peptide phase separation.

[0213] Table 5. Solubility of ICPs in water and acetone.

[0214] Peptides NS36, WA30, and VV30 demonstrated solvent-tunable secondary structures and suitable solubility in the mixed solvent (Figs. 7 and 8). We hypothesized that this characteristic would enable the modulation of these peptides assemble by adjusting the peptide-solvent interactions. I ntriguingly, we discovered that by altering the volume ratio of water and acetone during the assembly process, these three peptides could be coaxed to form hollow capsules. To our best knowledge, the generation of peptide capsules without templates has not been reported in the nanoprecipitation literatures. In contrast, peptide QH33 formed solid nanoparticles, which aligns with the expected outcome from a nanoprecipitation process. Example 5. Assembly of ICP capsules is mediated by microdroplet formation

[0215] The self-assembling process of the ICPs was investigated using fluorescence recovery after the photobleaching (FRAP), optical and fluorescence microscopy, and turbidity measurements. The following methods were used:

[0216] Fluorescence recovery after the photobleaching (FRAP)

[0217] For FRAP experiments, a laser scanning confocal microscope (AX / AX R with NSPARC) with a 60x objective was used for the FRAP studies. The selected peptide capsules were bleached with a 20% laser power at 450 nm with 50 ms. After that the collect peptide capsules was monitored for 80s to understand the fluorescence recovery. To prevent the impact of the photography process on capsule fluorescence, every 10 seconds take a photo.

[0218] Optical and fluorescence microscopy

[0219] Both optical and fluorescence microscopy observations were conducted using OLYMPUS IX73 inverted fluorescence microscopy. Freshly prepared peptide capsules solution was quickly dropped onto the surface of a grooved glass slide and sealed with a cover glass to prevent the evaporation of organic solvents. Figures then visualized at a magnification of *40. Software Lumenera corporation, Release 6.5, was utilized to analyze images.

[0220] Turbidity measurement

[0221] The turbidity measurement was conducted by the Agilent Cary 5000 UV-Vis spectrometers. The turbidity was calculate by testing the absorption at 650nm.

[0222] Results and discussion

[0223] Our experimental observations reveal the presence of two distinct timescales in the selfassembly process of ICPs without the presence of crosslinker. The assembly process is initiated by forming peptide-rich microdroplets (Fig. 9a). Immediately upon the addition of acetone, we witnessed the formation of liquid droplets for NS36, WA30, and VV30. The required minimum acetone-to-water volume ratios for phase separation, using peptide saturated aqueous solutions (Table 5), were 2.41, 3.65, and 3.76, respectively. By integrating microscopic observations and solution turbidity measurements, we identified the peptide concentrations and acetone-to-water volume ratios that resulted in the microdroplet formation for these three ICPs (Figs. 9b and 10).

[0224] The rapid formation of peptide-rich microdroplets upon the introduction of acetone into the peptide-water solution implies the operation of a fast timescale in the early stages of peptide assembly. Subsequently, a lengthier timescale, extending over several hundred seconds, is needed to transition the peptide droplets to more solid-like structures (Figs. 9e-h). Through optical and fluorescence microscopy imaging of FITC labelled ICPs, we observed that the droplet size continuously increased over several hundred seconds due to droplets coalescence (Fig. 9c). DLS measurements further confirmed this trend. The final microdroplet sizes were recorded as 0.9 ± 0.14, 2.3 ± 0.28, and 4.2 ± 0.87 pm for VV30, NS36, and WA 30, respectively. The size of the droplets and capsules can be tuned by adjusting the IPDI to peptide molar ratio. Higher crosslinker concentration leads to smaller capsules size. (Figs. 11 and 12). The wall thickness of representative WA30, NS36, and VV30 capsules, quantified through high-resolution Cryo-TEM, was found to be approximately 137.6 ± 12.1 nm, 62.1 ± 5.6 nm, and 7.8 ± 1.3 nm, respectively (Fig. 6b). The wall thickness is subject to variation depending on experimental parameters, including the ICP concentration, crosslinker quantity, and acetone-to- water ratio.

[0225] The swift formation of microdroplets is facilitated by the ICPs, given that water and acetone can be combined in any ratio. Acetone acted as a poor solvent for the peptides, leading us to speculate that the peptide-rich droplets are primarily water-based. This hypothesis was validated using fluorescent dyes with varying degrees of hydrophobicity. The water-soluble dye, Rh-B, was found to accumulate in the microdroplets, whereas the more hydrophobic FITC remained in the surrounding solution (Figs. 9d and 13). In essence, such phase separation results in a gradual transition zone rather than a sharply defined interface between the two phases. This zone is characterized by a steady evolution of acetone concentration, maintaining constant water-acetone diffusion as long as a concentration gradient exists (Reuvers, A. & Smolders, C., J. Membr. Sci. 1987, 34, 67-86) (Fig. 9a).

[0226] Fluorescence microscopy observations indicated that the ICP microdroplets transitioned from homogeneous liquid droplets into capsules with hollow centers approximately 6 minutes after the initial mixing (Figs. 9e and 14). To gain further insight into this microdroplet mediated peptide self-assembly process, we carried out fluorescence recovery after photobleaching (FRAP) experiments using Alexa 488 labeled NS36 and WA30 (Figs. 9e-g and 15). However, the VV30 droplets were not suitable for FRAP measurement due to their exceedingly small diameter. Following photobleaching, both NS36 and WA30 droplets exhibited nearly 80% fluorescence recovery in 80 seconds, indicating the fluidic state of the microdroplets (Figs. 9f and 16). In contrast, after the formation of capsules, the fluorescence intensity of the capsule walls in NS36 and WA30 only recovered by 27.6% and 37.8%, respectively (Fig. 9h). This suggests that during the peptide capsule assembly process, the peptides in the droplets undergo a transition from a liquid state to a gel state (Taylor, N. O. et al., Biophys. J. 2019, 117, 1285-1300; and Ray, S. et a!., Nat. Chem. 2023, 15, 1306-1316). Example 6. p-sheet formation promotes the assembly of ICPs

[0227] To probe the phase separation mediated ICP assembly mechanism, we examined the ICP secondary structures during assembly using circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The secondary structure of peptides at different concertation was characterized using CD and Nuclear Magnetic Resonance (NMR) spectroscopy. Thioflavin-T (ThT) labeling and ATR-FTIR were used to monitor protein secondary structure changes during structural changes during capsule formation.

[0228] Secondary structure characterization during capsules formation

[0229] The secondary structure of peptides at different concentration before assembly was characterised using CD. All peptides were dissolved at 0.5 to 10 mg mb1concentration in DI water and loaded in a quartz cuvette with 0.2 mm path length. The data was acquired by an AVIV 420 Circular Dichroism spectrometer equipped with a temperature controller. Data were collected at 25 °C over a wavelength range of 185 to 260 nm with a 1 nm wavelength steps size. Each data was averaged over three scans and smoothed via OriginLab Pro 2021.

[0230] ATR-FTIR spectra of the peptides in the self-assembling microenvironment (water was replaced by deuterium oxide) were obtained using a Bruker Vertex 70 spectrometer equipped with a PIKE Technologies MIRacle Attenuated Total Reflectance CaF2crystal reflection accessory (Bruker). The data was collected at ambient temperature over the range of 400 to 4000 cm1with a resolution of 4 cm1. Each 10 pL sample solution was injected to the CaF2crystal surface and averaged over 64 scans. All data were further processed by OPUS 6.5.

[0231] ThT labelling strategies were used to demonstrate structural changes during capsule formation. ThT solution (1 mg ml / in stock, further diluted by a factor of 50 to 2 pg mL'1as working solution) was used to dissolve peptide, then incubated by dry bath at 10 °C. The fluorescence intensity was then tested immediately, after 6 h of crosslink reaction and lyophilization then re-dissolution by DI water, then recorded the fluorescence intensity. The ThT fluorescence data was collected by a Cary Eclipse Spectrophotometer (Agilent) at excitation 450 nm and emission 485 nm. The optical microscope and fluorescence labelled capsule images were captured by Olympus IX73 inverted microscope with a CoolLED-pE- 300-Ultra-LED-Fluorescence-Light-Source accessory.

[0232] NMR spectroscopy

[0233] The NMR experiments were carried out in 600 MHz spectrometer equipped with cryoprobe. Two dimensional1H-1H TOCSY (Total Correlation spectroscopy) and1H-1H NOESY (Nuclear Overhauser Spectroscopy) measurements were made for 0.5 mM of peptides (QH33, WA30 and NS36) in water, pH 4.5 with 10% D2O for deuterium lock and DSS for signal reference with 80 ms and 200 ms mixing times, respectively. Uniform13C and15N labelled NS36 peptide was prepared as discussed above and the backbone NMR spectra viz HNCA, HNCACB and CACBCONH along with1H-1H-15N NOESY-HSQC spectra were also acquired for further analysis.

[0234] The molecular level conformational landscape information was obtained by analyzing 2D1H-1H TOCSY and1H-1H NOESY experiments of QH33, WA30 and NS36. Of the three peptides, the number of NOE connectivities were observed in the order of WA30 > NS36 > QH33, proposing structural propensities in WA30 and NS36.

[0235] For WA30 peptide, all of the residues can be unambiguously assigned.1Hachemical shift deviations of non-proline residues exhibited negative trend suggesting alpha helical conformation. A closer observation of1H-1H NOESY spectrum revealed a number of i,i+3 and i,i+4 NOEs spanning over residues S16-A28, diagnostic of helical conformation. The three- dimensional structure of WA30 was then calculated using all of these medium range NOEs that culminated into partially folded a-helix in the C-terminus. The helical fold in the N-terminus was found to be interrupted by the presence of P13 and P15 residues. This flexibility in turn brought the side chain of the residues W9 and W16 closer to be involved in TT-TT interaction and the side chains of W25 were found to be exposed. Thus, it can be speculated that the TT- TT interaction of W9-W16 stabilizes monomeric conformation whereas the exposed W25 can interact with other monomers bringing about the formation of nanoparticles.

[0236] For NS36 peptide, the presence of two Pro residues sequentially affected the sequential walk of TOCSY and NOESY spectra. But since we were successful in expressing and purifying uniform isotope (13C,1SN) labelled peptide, 3D backbone experiments (HNCA, HNCACB and CACBCONH) assisted the complete assignment of the peptide. The 2D1H-15N HSQC spectrum was well dispersed over a range of 7.5-9.0 ppm. The positive and negative chemical shift deviations of13Ca and1Ha indicated helical propensity for NS36 for all non-proline residues. The diagnostic i,i+3 and i,i+4 peaks were also found to be present but sparsely located when compared to WA30. The three-dimensional structure calculated with all of the assignments resulted in flower like loop structure. A closer observation revealed the presence of TT-TT side chain interaction between Y11 and Y18 residues with flexibility support from P13 and P14 residues as highlighted with different shades of green. On the other hand, the side chains of rest of the Tyr residues were exposed which might interact with other Tyr residues when they form nanoparticles. For QH33 peptide, all of the residues can be assigned unambiguously, and the positive chemical shift deviations indicated an extended conformation. The 2D1H-1H NOESY spectrum of QH33 also displayed very limited NOE connectivities mainly between backbone. Hence the three-dimensional structure of QH33 displayed an extended conformation with all of the sidechains of His residues exposed.

[0237] Overall, the structural studies of monomeric conformations of WA30, NS36 and QH33 peptide explained the molecular mechanism behind sequence specific nanoparticle formation. In the case of WA30 and NS36 peptides, the helical landscape was mostly interrupted by the presence of consecutive or closely available Pro residues that in turn provided the flexibility for the aromatic residues (Trp in WA30 and Tyr in NS36) to engage in TT-TT side chain interactions. On the other hand, because of the absence of intervening Pro residues in QH33, there is no structural stability of monomers that can bring about the formation of higher order structures and thus nanoparticle formation.

[0238] All-atom Molecular dynamics (MD}

[0239] The GROMACS package (version 2018.2) was utilized for all-atom MD simulations to explore the secondary structure, association, and self-assembly of ICPs in solution. Two peptides capable of forming nano-capsules (NS36 and WA30) and a precipitant-forming peptide (QH33) were investigated computationally. The initial peptide configuration were obtained through liquid-phase NMR experiments. Following the protocol from our previous study (Guo, Q. et al., Nat. Commun. 2022, 13, 5771), the amber99ff99SB force field was employed for the MD simulations, including the assignment of partial charges and force field parameters (Hornak, V. etal., Proteins: Struct., Fund., Bioinf. 2006, 65, 712-725). The TIP3P water model was employed (Jorgensen, W. L. et al., J. Chem. Phys. 1983, 79, 926-935), and the general Amber force field (GAFF) parameters for acetone were adopted (Wang, J. et al., J. Comput. Chem. 2004, 25, 1157-1174). The same acetone force field was reported in literature for the study of biphasic mixing (Caleman, C. et al., J. Chem. Theory Comput. 2012, 8, 61-74). In the QH33 simulation, counterions (Na+) were introduced to neutralize the system. The LINCS algorithm was used to constrain hydrogen atoms (Hess, B. etal., J. Comput. Chem. 1997, 18, 1463-1472). To simulate a water-acetone mixed solution, the simulation box, with dimensions of 10 x 10 x 20 nm, was initially filled with water molecules in half of the simulation cell and pre-relaxed acetone molecules in the other half. The solvent system was relaxed through a short steepest-descent energy minimization, followed by a 300 ps NVT equilibrium simulation with a force constant of 1000 kJ / mol / nm applied to the heavy (non-hydrogen) atoms (oxygen for water and carbon / oxygen for acetone). Subsequently, a 300 ps NVT equilibrium simulation was conducted without restraints. The equilibrated structures were then subjected to a 50 ns molecular dynamics simulation to mix water and acetone, while monitoring the water / acetone concentration along the reaction coordinate. An in-house Tel script was employed to extract cubic boxes of water-acetone mixtures at the desired target concentrations, along with a pre-relaxed pure water box of the same size. The resulting six sub-systems (yAJvW = 0, 1 , 3, 5, 7, 9) were duplicated in the x, y, and z directions to expand the simulation boxes to a size of approximately 10 nm. These systems were then subjected to a 100 ns relaxation in the NPT ensemble to reach equilibrium. The relaxed systems were subsequently used to solvate the peptides using the GROMACS solvate command. The resulting simulation systems were sufficiently large to prevent peptide molecules from interacting with their periodic images. A time step of 2 fs was employed, and the systems were simulated in the NPT ensemble at 1 atm and 300 K. Periodic boundary conditions were imposed in all three directions. Temperature and pressure were maintained using the Nose- Hoover and Parrinello-Rahman algorithms, respectively (Hoover, W. G., Phys. Rev. A 1985, 31, 1695; and Parrinello, M. & Rahman, A., J. Appl. Phys. 1981 , 52, 7182-7190).

[0240] To investigate the self-assembly of peptides in different chemical environments, the system was duplicated in the x-direction to create dual-peptide systems and duplicated in the xy- directions to generate quadra-peptide systems. These multi-peptide simulations underwent the same equilibrium procedure as single peptides, with the only difference being longer relaxation times (ranging from 100 ns to 250 ns) to ensure sufficient interactions and preliminary peptide self-assembly. Temporal evolution of peptide-system interactions was analysed using various built-in GROMACS tools, including distance, rms, rmsf, gyrate, sasa, and dssp. Specifically, the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), surfactant accessible surface area (SASA), and secondary structures were examined. Hydrogen bonds were evaluated between peptides and between peptides and the solvent using a cutoff scheme of 0.35 nm and 30 degrees.

[0241] Results and discussion

[0242] Capsule-forming peptides WA 30, VV30, and NS36, exhibited distinct solution structures in water: WA 30 displayed a-helical characteristics, while W30 and NS36 revealed a mix of extended coil and p-turn structures. The nanoparticle forming QH33 showed a random coil structure (Greenfield, N. J., Nat. Protoc. 2006, 1, 2891-2899) (Figs. 17a and 18). Further 2D1H-1H TOCSY and1H-1H NOESY measurements confirmed these observations.1H-1H NOESY and 2D1H-16N HSQC spectra revealed that WA30 mainly contained helical conformation, evidenced by the i,i+3 and i,i+4 NOEs spanning over residues S16-A28, while QH33 mainly exhibited an extended conformation (Vogeli, B. etal., Nat. Struct. Mol. Biol. 2012, 19, 1053-1057). 2D1H-15N HSQC spectra of NS36 suggested a flower like loop structure (Figs. 17b-c, 19 and 21-23).

[0243] We also corroborated the solution conformations of ICP through atomistic simulations, using structures obtained from liquid-phase NMR experiments as initial configurations for aqueous MD simulations. The simulated peptide configurations showed stable RMSD, SASA, and Rg evolution, suggesting peptide stability within the simulation timescale in water, akin to the NMR experiments. The obtained configurations from MD exhibited a high degree of resemblance to the NMR structures, thereby validating the efficacy of MD simulations as a valuable tool for investigating the behavior of ICP (Figs. 17d).

[0244] Upon forming peptide capsules, the ICPs transition into p-sheet structures (Ulijn, R. V. & Smith, A. M., Chem. Soc. Rev. 2008, 37, 664-675). This is supported by the strong fluorescence displayed by the ICP capsules upon staining with ThT, which indicates the presence of cross- p-sheet structures in the capsule walls (Figs. 24d-e). To monitor the assembly kinetics of the ICPs within the microdroplets, we conducted a ThT assay to track fluorescence emission based on water to acetone ratio and time. No capsule formation was observed for NS36 at water content (vW / vA) higher than 1 :7, and fluorescence emission remained stable over time (Fig. 17e). In conditions leading to droplet formation (1 / 9 < vW / vA < 1 / 8), weak fluorescence was detected in the initial 150 seconds, then rapidly escalated, plateauing within 100-200 seconds - coinciding with the formation of peptide capsules (Fig. 17f). Thereafter, the ThT fluorescence intensity was continuously monitored for 6 h and remained steady even after six months (Table 6). WA30 and VV30 showed similar behavior, with an increase in acetone volume fraction leading to faster kinetics of p-sheet formation and higher p-sheet content (Figs. 26a, c). The formation of p-sheets during the capsule forming process was also confirmed by ATR-FTIR results (Figs. 17g and 25-28) and the addition of crosslinker did not significantly change the p-sheet content in the capsules (Fig. 29).

[0245] Table 6. The secondary structure stability monitoring of ICP capsules by ThT staining (fluorescence intensity (au)). Example 7. Uncovering the driving forces of ICPs self-assembly

[0246] Transfer Free Energy Calculation

[0247] To assess the free energy changes associated with peptide nano-capsulation and precipitation, we employed the Free Energy Perturbation (FEP) method to calculate the free energy differences between the prescribed thermodynamics states in the process. Specifically, we evaluated the transfer free-energy difference (AAG) by comparing the binding free energies of the peptide in various acetone concentrations from c = 0 (water) to c = 1 (pure acetone). The transfer free-energy difference can be calculated as

[0248] AAG = AGW— AGaw

[0249] Where AGWand AGaware the binding free energies of peptide in water and water-acetone mixture, respectively.

[0250] To initiate the FEP calculations, we utilized starting configurations derived from single peptide Molecular Dynamics (MD) simulations, ensuring a reliable foundation for our FEP analysis. A time step of 1 fs and a total of 25 intermediate states were used, aligned with previous studies (Qian, X. et al., Forces Meeh. 2022, 8, 100098). Each intermediate state was sampled for a duration of 4 ns. The Multistate Bennett Acceptance Ratio (MBAR) method was employed to compute the change in free energy associated with the annihilation process, using the open- source Alchemical Analysis script (Klimovich, P. V. et al., J. Comput. Aided Mol. Des. 2015, 29, 397-411).

[0251] Results and discussion

[0252] During the introduction of acetone into the peptide-water solution, a rapid phase separation occurs, resulting in the formation of water-rich droplets containing peptide molecules, surrounded by an acetone-rich phase. This process does not create a sharp interface between the two phases, but rather a gradient interface characterized by a transition zone with smoothly changing acetone concentration (Fig. 9a). Notably, simulation studies revealed the swift diffusion of acetone into the water-rich phase, with a diffusion coefficient on the order of 1 x IO5cm2 / s (Fig. 30 and Table 7). This diffusion process is instrumental in defining the concentration profile and location-specific chemical environments at the gradient interface. Consequently, it prompts the vital question of how such a dynamic gradient interface could influence the self-assembly and encapsulation behavior of ICPs, thus driving our efforts to unearth the driving forces at play. Table 7. The diffusion constant of water, acetone, and the system, determined from MD simulations of water-acetone diffusion.

[0253] We hypothesized that peptide-peptide interactions vary with changes in the mixed solvent. To investigate these interactions and determine the timescale guiding the initial peptide assembly stages, we carried out MD simulations of numerous isolated peptides at a variety of preequilibrated acetone concentrations, reflecting the experimental conditions (i.e., vA / vW = 0,1 , 3, 5, 7, 9). Initial contact of peptides is driven by TT-TT interaction between tryptophan (W) residues for WA30, hydrogen bonding networks for NS36, and stable hydrogen bond pair between the side chains of Glu and His for QH33. (Figs. 31a-c). We observed intriguing results relating to the timescale required for the formation of initial self-assemblies (Table 8). This timescale ranged from a few tens of nanoseconds to hundreds of nanoseconds, depending on both acetone concentration and peptide sequence. Importantly, we found that higher acetone concentrations were correlated with longer times required for the initial contact of NS36, influencing the early stages of peptide self-assembly.

[0254] Table 8. Simulation-derived timescales for peptide assembly initiation based on initial contact events. indicates that simulated quadra-WA30 separated at 20 ns.

[0255] We assessed the number of hydrogen bonds (H-bonds) between peptides and solvent at each simulated concentration. The total H-bonds between peptides and solvent decreased mildly at high acetone concentrations cacetone= 0.69, or vAMN = 9) for NS36 and WA30 (by 4.35% and 1.83%, respectively), and significantly for QH33 (by 21.22%) compared to the reference concentration (cacetone= 0.42 , or vMvW = 3). In pure acetone, the number of H-bonds decreased drastically for all three peptides (Figs. 31 d and 32). Additional analysis of SASA, RMSD, and Rg suggested that peptide assembly adopted more extended conformations as acetone concentration increased in the solvent. The exception was QH33 at concentrations above 0.42 (or vA / vW = 3), which coincided with the turning point in H-bond numbers between peptides and solvent molecules (Fig. 33).

[0256] In view of the different timescales between experiments and MD simulations, we performed simulations on the peptide-peptide interactions in the pre-equilibrated mixed solvent, noting that the diffusion of peptides in solvents has been reported to be significantly slower, with diffusion coefficients typically one to two orders of magnitude smaller compared to wateracetone diffusion (Nauman, J. V. et a / ., Biophys. J. 2007, 92, 4444-4450). Furthermore, we conducted FEP simulations to calculate the transfer free energy (TFE) of peptides at different concentrations across the water-acetone interface (Fig. 31e).

[0257] Our calculated TFE landscape reveal a consistent and incremental increase for the nanoparticle-forming peptide QH33 as the acetone concentration rises. This trend suggests an energetic disadvantage for QH33 as acetone molecules infiltrate the peptide containing water-rich phase. The incursion of acetone alters the chemical environment surrounding the QH33 peptides, driving their migration towards a more aqueous environment with higher water concentration. This relocation ultimately culminates in the precipitation of QH33 peptides, and such process conforms to nanoprecipitation principles.

[0258] Our investigation revealed important insights into the behavior of capsule forming peptides, NS36 and WA30. These peptides exhibit single-well TFE landscapes with global minima observed between pure water and pure acetone. These energy minima mark the energetically optimal concentrations for peptide localization at the forefront of water-acetone mixing. The free energy differences provide the driving force for peptides to spontaneously migrate towards the optimal concentration at the gradient interface, triggering peptide localization and facilitating the peptide capsule formation (Fig. 31f). It is worth noting, however, that not all peptides manifested this behavior - only those displaying local minima in their TFE landscapes showed the propensity for peptide capsule formation.

[0259] We underscored that these observations from MD simulations and FEP calculations are rare but not unexpected. In fact, acetone does not act strictly as a "poor solvent" for capsule forming peptides. Rather, the concentration of acetone plays a crucial role in the formation of capsules. Insufficient amounts of acetone fail to create necessary conditions for capsule formation, while excessive concentrations still allow the formation process to occur (Fig. 17e). The localization of the ICPs at the water-acetone interface, coupled with a high acetone concentration locally, facilitates the formation of -sheet structures occurring over an extended timescale. Furthermore, we propose that peptide localization at the gradient interface may impede the diffusion of acetone into the water phase. This barrier to water-acetone diffusion might account for the longer timescale (>100 seconds) for microdroplet to capsule transition observed in the experiments.

[0260] Example 9. ICP capsule based cytosolic delivery of drugs

[0261] Loading and releasing the small molecule cargoes and protein cargo

[0262] The small molecule cargoes Rh-B, FITC, DoX, DoXHCI, PTX and the macromolecule EGFP, Rh-B labelling BSA, EGFP-plasmid / mRNA, -Gal and Rh-B-Smac were used to measure the ability of repetitive peptide capsules to transport different types of molecules. The cargo molecule and the peptide were dissolved together in DI water and a sufficient amount of acetone with crosslinker solution was added dropwise. After 6 h of crosslinking, the acetone solution was removed by bubbling nitrogen flow. The repeat peptide capsules were washed three times by redispersing in DI water and collecting by centrifugation to remove unreacted crosslinker and unencapsulated molecular cargo. Molecular cargo loading was determined by the UV-Vis-NIR Cary 5000 (Agilent). The encapsulation efficiency was calculated as

[0263] Where mt and murepresent the mass of the small molecule cargoes used and the mass of the unloading cargoes, respectively.

[0264] The Rh-B labelling BSA release from the peptide capsules was conducted by measuring the diffusion of Rh-B through a dialysis membrane (10 kDa molecular weight cut off). In brief, the freeze-dried capsules (1 mg) were first redispersed into PBS (10 mL, pH 7.4, ionic strength 0.15 M) and dialysed against 200 mL PBS (pH 7.4, ionic strength 0.15 M). The protease (2 mg, bovine pancreas) was added to the capsules suspension after dialysing for 3 h. Each 20 minutes, a 200 L sample was taken from the outer dialysis PBS to measure the release of Rh-B labelled BSA by using a microplate reader using 560 nm / 610 nm for the excitation / emission wavelengths.

[0265] The small molecule cargo release from the peptide capsules was also conducted by measuring the diffusion of the small molecule cargo through a dialysis membrane (10 kDa molecular weight cut off). In brief, freeze-dried capsules (1 mg) were first redispersed into PBS (10 mL, pH 7.4, ionic strength 0.15 M) and dialysed against 200 mL PBS (pH 7.4, ionic strength 0.15 M). Each 1 h, a 200 pL sample was taken from the outer dialysis PBS to measure the release of small molecules by using a microplate reader using 560 nm / 610 nm for the excitation / emission wavelengths and Agilent Cary 5000 UV-Vis spectrophotometer using a spectra absorbance at 488 nm.

[0266] Cytotoxicity study

[0267] The methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of the DoX-loaded peptide-based capsules. 1 x 104HeLa cells in 100 pL of cell culture medium (High Glucose DMEM, 5% FBS, 1 % Penicillin-Streptomycin) were transferred into a 96-well plates and incubated for 24 h. The pH 7.20 1X PBS was used to wash each well twice and then replaced with 100 pL of Opti-MEM containing various concentrations of peptide-based capsules and the DoX-loaded capsules (various concentration of DoX, 0.5 mg mL-1peptide-based capsules). After 4 h of uptake, the Opti-MEM was fully removed and the cells were washed by pH 7.20 PBS twice before changing back to 100 pL of cell culture medium (High Glucose DMEM, 5% FBS, 1% Penicillin-Streptomycin). The 96-well plates containing HeLa cells was incubated for another 24 h before 10 pL of 5 mg mL"1MTT dissolved in pH 7.20 PBS was added to each well. After another 4 h of incubation, all medium was removed, and the cells were washed by pH 7.20 PBS twice. Then, 100 pL of DMSO was added to each well and mixed evenly through the pipette. The absorbance at 570 nm was measured by a microplate reader (Infinite M200 Pro, Tecan). The relative cell viability was calculated as: where As, Ab and Ancrepresent the absorbance of the sample cells, blank control, and negative control cells, respectively.

[0268] Delivery of the small molecule cargoes and protein cargoes

[0269] For delivery of protein and small molecule drugs like DoXHCI into HeLa cells, a total amount of 1 x 106HeLa cells was uniformly dispersed by Trypsin in 1 mL cell culture medium (High Glucose DMEM, 5% FBS, 1% Penicillin-Streptomycin), and then subcultured into a 350 mm2glass bottom petri dish. After incubation for 24 h, the medium was changed into 950 pL Opti- MEM with 1 % Penicillin-Streptomycin, then 50 pL of loaded protein capsules at 1 mg mb1was added. After 4 h of uptake, the Opti-MEM with capsules were fully removed and the cells were washed by pH 7.20 PBS twice before adding the fresh culture medium (High Glucose DMEM, 5% FBS, 1 % Penicillin-Streptomycin). The cells were incubated for another 12 h and washed again by pH 7.20 PBS. The medium was then replaced by 1 mL Live Cell Imaging Solution (invitrogen). The capsules uptake was evaluated by FACS (LSR Fortessa X20, BD Biosciences) and observed by a confocal microscope (Stellaris 5, Leica).

[0270] The internalization mechanism was studied by using LysoTracker staining. After cells uptake of FITC-labeled BSA carrying capsules, the cells were washed by pH 7.20 PBS twice before adding 1 mL culture medium (High Glucose DMEM, 5% FBS, 1% Penicillin-Streptomycin). After 2 h culture, the medium was replaced by 1 mL Opti-MEM containing 50 nM LysoTracker Red DND-99. The cells were further stained in the medium for 1 h. The staining-treated HeLa cells were washed by pH 7.20 PBS twice and and fixed with 5% formaldehyde solution. The medium then was replaced by 1 mL Live Cell Imaging Solution (invitrogen) and observed by a confocal microscope (Stellaris 5, Leica) though super resolution model.

[0271] Delivery of plasmid and mRNA

[0272] For delivery of plasmid and mRNA into different cells, a total amount of 1x 105HeLa, MCF7 and U2OS cells was uniformly dispersed by T rypsin in 1 mL cell culture medium (High Glucose DMEM, 5% FBS, 1% Penicillin-Streptomycin), and then subcultured into a 350 mm2glass bottom petri dish, respectively. After incubation for 24 h, the medium was changed into 950 pL Opti-MEM with 1% Penicillin-Streptomycin, then 50 pL of loaded plasmid or mRNA capsules at 1 mg mL1was added. After 4 h of uptake, the Opti-MEM with capsules were fully removed and the cells were washed by pH 7.20 PBS twice before adding culture medium (High Glucose DMEM, 5% FBS, 1 % Penicillin-Streptomycin). The cells were incubated for another 12 h and washed again by pH 7.20 PBS. Then, the medium was replaced by 1 mL Live Cell Imaging Solution (Invitrogen). The capsules uptake was evaluated by FACS (LSR Fortessa X20, BD Biosciences) and observed by a confocal microscope (Stellaris 5, Leica).

[0273] Results and discussion

[0274] The solvent gradient-mediated assembly process and hollow structure of the ICP capsules make them effective vehicles for co-delivering large hydrophilic biomolecules and small molecule drugs. Early in the phase separation process, hydrophilic biomacromolecules, such as nucleotides and proteins, prefer to reside in the water-rich droplet phase alongside the ICPs. As a relatively mild solvent, acetone did not significantly change the activity of biomacromolecule cargoes (Fig. 34). During the solvent exchange process, small hydrophobic molecules dissolved in acetone can be concentrated and trapped in the peptide capsules, finally accumulated on the capsule wall during dialysis in water (Figs. 35a and 36). We utilized EGFP and DoX as model compounds for large hydrophilic and small hydrophobic drugs to demonstrate this capability. All ICP capsules exhibited exceptionally high EGFP loading efficiency, ranging from 87.5% to 92.1% (Fig. 35b). Similar loading efficiencies were observed for other biomacromolecules, including DNA, RNA, |3-Galactosidase, and Smac (Tian, Y. et al., Adv. Healthc. Mater. 2022, 11, 2270066), using NS36 capsules (Figs. 37a-b). In contrast, the loading efficiency for small molecules was highly sequence-dependent; NS36 capsules displayed the highest loading efficiency (~ 54.8%) for DoX, whereas VV30 demonstrated the highest loading efficiency (-76.2%) for the more hydrophobic drug, paclitaxel (PTX) (Figs. 35c and 37c).

[0275] The cellular uptake of FITC-labeled peptide capsules in HeLa cells was confirmed through confocal microscopy (Fig. 35d). The peptide capsules demonstrated very low cytotoxicity, as indicated by the MTT assay, with over 90% cell survival for all three peptides even at a concentration of 400 mg / mL (Fig. 38a). We conducted a detailed test on the cellular activity of NS36 capsules and peptides taken up by Hela cells at different time scales. We found that there was no significant difference in the survival rate of Hela cells after treating by NS36 peptides or capsules for 4 or 12 hours (Fig. 51). Confocal microscopy images revealed that VV30 capsules exhibited the highest uptake efficiency, while WA30 capsules showed the lowest efficiency, likely due to its larger size (Tang, L. etal., Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 15344-15349) (Figs. 39-40). Furthermore, we conducted examinations on the uptake of NS36 capsules containing a compound in U2OS and MCF7 cells. Remarkably, our findings revealed insignificant cytotoxicity along with notable uptake efficiency for both cell lines.

[0276] To further investigate the versatility of the peptide capsules, the FITC-labeled NS36 capsules were tested on another two cancer cell lines, namely MCF7 and U2OS. Based on the fluorescence signals observed inside the cells, the low cytotoxicity and high uptake efficiency of NS36 capsules were confirmed in all three cell lines (Figs. 41-42). To evaluate the intracellular cargo delivery efficiency of the peptide capsules, EGFP protein was loaded as a model protein, and its internalization was quantitatively measured by FACS. VV30 and NS36 capsules also exhibited high internalization efficiency (Fig. 38b). Furthermore, the peptide capsules effectively delivered and released the hydrophilic drug DoXHCL Dose-dependent viability decrease confirmed the efficient uptake and action of DoXHCI in cells (Fig. 38c). In vitro experiments demonstrated that the peptide capsules could rapidly release drugs under lower pH conditions (Fig. 43). Confocal microscopy images illustrated the gradual release of DoXHCI from lysosomes to the nucleus after 12 h (Figs. 38d and 44). Additionally, the co- delivery of fluorescence-labeled BSA with DoXHCI was tested, revealing clear colocalization of DoXHCI and BSA in the lysosomes, along with released DoXHCI signal in the nucleus after 4 h of incubation (Figs. 38e and 45). These cell experiments demonstrate the high loading and cellular delivery efficiency of the peptide capsules.

[0277] Modification in the peptide sequences further enables the delivery of nucleic acids. In the case of EGFP delivery, the VV30 capsules escaped from the lysosome to the cytoplasm by triggering the proton sponge effect through the His residues on the peptide capsule surface (Midoux, P. etal., Br. J. Pharmacol. 2009, 157, 166-178) (see Fig. 46). This characteristic also allows these peptide capsules to deliver nucleic acids. We introduced two Cys residues at both the N- and C-termini of the NS36 sequence for cross-linking and stabilizing the capsule structure (Fig. 47). Additionally, three Asn residues were replaced with Lys to induce the proton sponge effect. The Lys residues reduced the size of the capsules by increasing the zeta-potential of the peptide-rich droplets prior to capsule formation (Fig. 48). To enhance targeted recognition, an RGD sequence was added at the C-terminal to enhance cell adsorption (Ruoslahti, E. & Pierschbacher, M. D., Science 1987, 238, 491-497). Based on these design principles, the CC-N3KS-CC-RGD capsules demonstrated improved delivery depth compared to NS36 capsules and successfully delivered EGFP plasmid and mRNA into MCF7 (Figs. 38f-g), HeLa, and U2OS cells (Figs. 38f-g and Figs. 52-57 and 49). The decrease of the fluorescence intensity of FITC labeled ICP capsules indicated that the peptide capsules can be degraded after cellular up-take (Fig. 50). For mRNA delivery, all three types of cells exhibited over 90% expression efficiency. The high cargo loading capacity and efficiency of the ICP capsules render them a potentially promising platform for immunotherapy and gene therapy.

[0278] Example 10. Mutation of the ICPs

[0279] Preparation process

[0280] 1. Fully dissolve 20 mg ICP in 1000 pl deionized water to reach a mass concentration of 20 mg / ml; this peptide solution is called solution A. The hydrophilic macromolecule to be loaded is dissolved by solution A, and the concentration was calculated by the required loading amount and loading rate (loading efficiency is around 90% by weight for WT peptide sequence).

[0281] 2. Fully dissolve 10 mg Isophorone diisocyanate (IPDI), or 17.5 mg disuccinimidyl suberate (DSS), or 18.2 mg Dithiobis(succinimidyl propionate) (DSP) into 1 ml acetone or tetra hydrofuran (THF); this crosslinker organic solution is called solution B. The small hydrophobic molecule drug to be loaded is dissolved by solution B and calculated according to the selected peptide type and loading rate. 3. Add 900 pl of solution B with hydrophobic cargoes into 100 pl of solution A with protein quickly. In the process of preparing vesicles, slow vortex or ultrasound is required to prevent droplet fusion. This process requires at least 2 h to ensure the completeness of crosslinking.

[0282] 4. All organic solvents were removed by volatilization or nitrogen blowing. In view of the high volatility of the selected organic solvent, the general process only takes 30 to 50 minutes.

[0283] 5. Add the same volume (900 pl) of deionized water into the final capsule suspension and dialyse in the 10 kDa dialysis bag for at least 48 h to completely remove the residual crosslinking agent and organic solvent. For the self-crosslinked NS mutant, the dialysis time can be reduced to 12 h.

[0284] Results and discussion

[0285] The cross-linking sites for different peptide sequences are different, and the common N- terminal C-terminal, as well as the exposed amino and carboxyl groups of the side chain, may be potential cross-linking sites. The existence of possible cross-linking sites in the side chain is entirely determined by the sequence itself. We added cysteine into the sequence, due to the ability of cysteine to form disulfide bonds through self-crosslinking. The crosslinking site is the double C sequence located at the N and C terminal.

[0286] By replacing hydrophilic but uncharged amino acid Asn with positively charged amino acid Lys in WT-NS36, the fusion of micro LLPS droplets can be easily controlled and thus can effectively reduce the size of peptide capsules to about 120 nm. It also provides the high reaction activity amino group, which provides the possibility for modification after capsule formation. The additional RGD sequence is a well-known cell adhesion motif - this adhesion sequence helps some special cancer cells, such as breast cancer cells to uptake the peptide capsules. The addition of Cys can provide self-crosslinking sites. Compared with traditional crosslinking agents, self-crosslinking has two significant advantages. First, the addition of cross-linking agents which are often toxic can be effectively reduced. Second, the disulfide bond formed by the self-crosslinking will be destroyed by glutathione (GSH) in the cancer cells, thus releasing the loaded cargoes.

[0287] Table 9 provides a summary of the effect of amino acid substitution on the ability of the NS36 peptide to form peptide capsules. Table 9. A table showing the effect of a selection of NS36 mutations on capsule formation.

[0288] A summary of results for the ICPs tested is included below (Table 10). Table 10. Summary of wild-type and mutant ICPs results.

[0289] Here, we unveil a new strategy for harnessing gradients in the design of biomimetic functional self-assembling peptides. Leveraging the proteome of insect cuticle rich in conservative domains as a source of self-assembling peptides, we have identified three biomimetic peptides with repetitive sequences that can form hollow peptide capsule structures without the need of any external templates. The peptide capsules formation is first initiated by the rapid formation of peptide-rich microdroplets, followed by a slower solvent gradient-mediated self-assembly process. The primary driving force for the initial phase separation is the affinity of the peptides for the water-rich environment. At a longer timescale, the diffusion between water and acetone creates a gradient interface, disrupting the metastable microdroplet state and prompting the localization and self-assembly of peptides in the vicinity of this gradient interface. These peptide capsules can be a versatile delivery platform with potential applications in various biomedical fields, such as drug delivery, immunotherapy, and gene therapy.

[0290] The present disclosure provides valuable insights into how solvent gradients influence the self-assembly and encapsulation behaviors of insect cuticle peptides (ICPs). The selfassembly process operates on distinct timescales, beginning with a rapid phase where fluidlike droplets followed by a slower p-sheet formation that results in solid-like structures. Using MD simulations and FEP calculations, we further delved into the interactions between peptides and the energetics at the gradient interface. The concentration gradient, created through solvent exchange, is the key driver for capsule formation. Our TFE landscape calculations uncover the fundamental mechanisms behind ICP localization and self-assembly. The capsule forming peptides show unique single-well TFE profiles with a global minimum in mixing concentration between water and acetone, guiding the peptides to localize at the gradient interface. High peptide concentration facilitates p-sheet formation in the localized zone, aiding the solidification of peptide capsules. Importantly, our experiments and simulations indicate that the initial secondary structure of peptides does not decisively determine capsule formation. Instead, the differing solvation free energy plays a predominant role as peptides dissolve in the constantly changing chemical environment created by the solvent mixture.

[0291] Forging a universal design principle for peptide sequences capable for gradient-mediated selfassembly is a critical goal for future study. This can be achieved by combining MD simulation with systematic point mutation of the ICPs. Secondly, while our simulation approach provides valuable insights into ICP initial contact and self-assembly, it does not capture the formation process of p-sheet structures. Simulating peptide diffusion, localization, and assembly in a co- diffusional system is beyond the current capabilities of all-atom molecular dynamics in terms of time- and length-scale. Alternative methods such as coarse-grained molecular dynamics, Monte Carlo simulations, and continuum-level modeling could be explored in future endeavors to study similar systems theoretically and computationally. The present disclosure demonstrates a straightforward solvent exchange procedure and an extensive peptide screening strategy, leading to encapsulation of diverse cargoes. The capsule formation mainly depends on the solvation free energy of peptides in the mixed solvent. By properly designing the energy landscape of the peptides through sequence programming, we have achieved the control of their phase and assembly behaviors. Similarly, the presence of concentration gradients in natural systems could play a pivotal role in directing the assembly and organization of proteins at biological interfaces. Moreover, we have demonstrated such peptide capsules can be customized as vehicles for targeted therapeutics and drug delivery. These revelations underscore the potential applicability of our study across a range of fields, such as nanomedicine, biomaterials, and sustainable manufacturing.

Claims

CLAIMS1. A peptide capsule for delivery of a cargo molecule into a cell, the peptide capsule comprising a peptide shell and a cargo molecule, wherein the peptide shell is formed from a plurality of self-assembling peptides, the self-assembling peptides each comprising an amino acid sequence given in SEQ ID NO: 1 , 2 or 3, wherein:SEQ ID NO: 1 is:NX2X3X4VPPS [NX1°X11VPPS]nwherein, n is an integer from 2 to 6;X2is N, K, R, H, D or E;X3is Y, F or absent;X4is Y or F; each X10is independently N, K, R, H, D or E; and each X11is independently Y or F;SEQ ID NO: 2 is:[X1X2AAPAX7'8]nX9X10AAPAX15'16wherein, n is an integer from 2 to 6; each X1is independently W or F; each X2is independently N, K, R, H, D or E; each X7'8is independently NQ, PS, or NH;X9is W or F;X10is N, K, R, H, D or E; andX15'18is absent, NQ, PS, or NH;SEQ ID NO: 3 is:[VX2X3APV]nwherein, n is an integer from 3 to 8; each X2is independently H or Y; and each X3is independently S, K, R, H, D or E.

2. The peptide capsule of claim 1 , wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 1, wherein n is 4.

3. The peptide capsule of claim 1 or 2, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 1 , and wherein in SEQ ID NO: 1 :X2is N, K, R, or H, for example N or K;X3is Y, F or absent, for example Y;X4is Y or F, for example Y; each X10is independently N, K, R, or H, for example independently N or K; and each X11is independently Y or F, for example Y.

4. The peptide capsule of claim 3, wherein in SEQ ID NO: 1 , at least one of each X10and X2is K, R or H.

5. The peptide capsule of claim 1 , wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, 6, 7, 8 and 9, for example, the self-assembling peptides each comprise the amino acid sequence given in SEQ ID NO: 7.

6. The peptide capsule of claim 1 or 2, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 1, and wherein in SEQ ID NO: 1 :X2is N, D or E, for example N or D;X3is Y, F or absent, for example Y;X4is Y or F, for example Y; each X10is independently N, D or E, for example independently N or D; and each X11is independently Y or F, for example Y.

7. The peptide capsule of claim 6, wherein in SEQ ID NO: 1 , at least one of each X10and X2is D or E.

8. The peptide capsule of claim 1, wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 10, for example, the self-assembling peptides each comprise the amino acid sequence given in SEQ ID NO: 10.

9. The peptide capsule of claim 1 , wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 2, wherein n is 3.

10. The peptide capsule of claim 1 or 9, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 2, and wherein in SEQ ID NO: 2: each X1is independently W or F, for example W; each X2is independently N, K, R, or H, for example independently N or K; each X7'8is independently NQ, PS, or NH;X9is W or F, for example W;X10is N, K, R, or H, for example N or K; andX15’16is absent, NQ, PS, or NH, for example absent.

11. The peptide capsule of claim 10, wherein in SEQ ID NO: 2, at least one of each X2and X10is K, R or H.

12. The peptide capsule of claim 1 , wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13, for example, the self-assembling peptides each comprise the amino acid sequence given in SEQ ID NO: 13.

13. The peptide capsule of claim 1 or 9, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 2, and wherein in SEQ ID NO: 2: each X1is independently W or F, for example W; each X2is independently N, D or E, for example independently N or D; each X7'8is independently NQ, PS, or NH;X9is W or F, for example W;X10is N, D or E, for example N or D; andX15 18is absent, NQ, PS, or NH, for example absent.

14. The peptide capsule of claim 13, wherein in SEQ ID NO: 2, at least one of each X2and X10is D or E.

15. The peptide capsule of claim 1 , wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14 and 15, for example, the self-assembling peptides each comprise the amino acid sequence given in SEQ ID NO: 14.

16. The peptide capsule of claim 1 , wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 3, wherein n is 5.

17. The peptide capsule of claim 1 or 16, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 3, and wherein in SEQ ID NO: 3: each X2is independently H or Y, for example H; and each X3is independently S, K, R or H, for example independently K.

18. The peptide capsule of claim 17, wherein in SEQ ID NO: 3, each X2is H, and at least one of each X3is K, R or H.

19. The peptide capsule of claim 1 , wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 18, for example, the self-assembling peptides each comprise the amino acid sequence given in SEQ ID NO: 18.

20. The peptide capsule of claim 1 or 16, wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NO: 3, and wherein in SEQ ID NO: 3: each X2is independently H or Y, for example H; and each X3is independently S, D or E, for example independently S or D.

21. The peptide capsule of claim 20, wherein in SEQ ID NO: 3, each X2is H, and at least one of each X3is D or E.

22. The peptide capsule of any one of the preceding claims, wherein the self-assembling peptides are cross-linked by a cross-linking agent.

23. The peptide capsule of claim 22, wherein the cross-linking agent is a diisocyanate or a disuccinimidyl ester, for example isophorone diisocyanate or disuccinimidyl suberate.

24. The peptide capsule of any one of the preceding claims, wherein the self-assembling peptides each further comprise 1 or 2 cysteine residues or selenocysteine residues at the N-terminus or the C-terminus, or each further comprise 1 or 2 cysteine residues or selenocysteine residues at each of the N-terminus and C-terminus.

25. The peptide capsule of claim 24, wherein the self-assembling peptides are cross-linked by one or more disulfide bonds or one or more Se-Se bonds.

26. The peptide capsule of any one of the preceding claims, wherein the self-assembling peptides comprise an integrin-binding motif.

27. The peptide capsule of claim 26, wherein the integrin-binding motif has the amino acid sequence RGD.

28. The peptide capsule of any one of claims 24 to 27, wherein the self-assembling peptides each comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-25.

29. The peptide capsule of any one of the preceding claims, wherein the peptide capsule has a diameter of less than about 1 pm.

30. The peptide capsule of any one of the preceding claims, wherein the cargo molecule is encapsulated by the peptide shell.

31. The peptide capsule of claim 1 , wherein the self-assembling peptides each comprise an amino acid sequence given in SEQ ID NOs: 4, 5, 12, 17 or 20.

32. The peptide capsule of any one of the preceding claims, wherein the cargo molecule is selected from the group consisting of a small molecule compound, a peptide, a protein, and a nucleic acid.

33. The peptide capsule of any one of the preceding claims, wherein the cargo molecule is a therapeutic agent or a diagnostic agent.

34. The peptide capsule of any one of the preceding claims, wherein the cargo molecule is a therapeutic agent.

35. An isolated self-assembling peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-25, 43 and 44.

36. The isolated self-assembling peptide of claim 35, wherein the isolated self-assembling peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 5, 12, 17 and 20.

37. A pharmaceutical composition comprising a peptide capsule of claim 34, and a pharmaceutically acceptable carrier.

38. An in vitro method of delivering a cargo molecule into a cell, said method comprising contacting the target cell with the peptide capsule of any one of claims 1 to 34, or the pharmaceutical composition of claim 37.

39. The peptide capsule of claim 34, for use in treating a disease or disorder.

40. Use of the peptide capsule of claim 34, in the manufacture of a medicament for the treatment or prophylaxis of a disease or disorder.

41. A method of treating or preventing a disease or disorder in a patient, said method comprising administering a pharmaceutically effective amount of the peptide capsule of claim 34 to the patient.

42. The peptide capsule of any one of claims 1 to 34, the pharmaceutical composition of claim 37, or the in vitro method of claim 38, wherein the cell is a eukaryotic cell.

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