CAPSULES AND PARTICLES AND USES THEREOF

Abstract

Provided are antifouling particles and uses thereof in methods of anti-biofouling.

Claims

1.-61. (canceled)

62. A process for forming particles of an antifouling material, the process comprising contacting an antifouling material, in a non-particulate form, the material having at least one surface binding moiety, at least one antifouling moiety, and optionally at least one amino acid moiety with an aqueous medium under conditions permitting transformation of said material into particles having porosity dependent on the acidity of the aqueous medium.

63. The process according to claim 62, further comprising a step of isolating the particles from the aqueous medium.

64. The process according to claim 62, wherein the medium having a pH between 7 and 10, or between 7 and 9.

65. The process according to claim 62, wherein the medium having a pH between 2 and 5, or between 2 and 4, or between 2 and 3.

66. The process according to claim 62, wherein the particle porosity is characterized by a plurality of pores having pore densities of between about 10 pores/mm.sup.2 and about 10 pores/100 m.sup.2.

67. The process according to claim 66, wherein the pore density is between about 10 pores/mm.sup.2 and about 10 pores/90 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/80 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/70 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/60 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/50 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/40 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/30 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/20 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/10 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/5 m.sup.2, between about 10 pores/mm.sup.2 and about 10 pores/2 m.sup.2 or between about 10 pores/mm.sup.2 and about 10 pores/1 m.sup.2.

68. The process according to claim 66, wherein the pore density is between about 20 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 30 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 40 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 50 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 60 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 70 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 80 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 90 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 100 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 110 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 120 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 130 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 140 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 150 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 160 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 170 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 180 pores/mm.sup.2 and about 10 pores/100 m.sup.2, between about 190 pores/mm.sup.2 and about 10 pores/100 m.sup.2 or between about 200 pores/mm.sup.2 and about 10 pores/100 m.sup.2.

69. The process according to claim 62, wherein the average pore diameter is between 2 and 50, between 2 and 40, between 2 and 35, between 2 and 30, between 2 and 25, between 2 and 20, between 2 and 15, between 2 and 10, between 10 and 100, between 10 and 90, between 10 and 80, between 10 and 70, between 10 and 60, between 10 and 50, between 10 and 40, between 10 and 30, or between 10 and 20 micrometer.

70. Particles of a material having at least one surface binding moiety, at least one antifouling moiety, and optionally at least one amino acid moiety, the particles being porous.

71. The particles according to claim 70, entrapping, associating, encaging, containing or holding one or more active or non-active materials in cavities or pores present in the particles or on outer surfaces of the particles.

72. The particles according to claim 70, wherein the material is of the general Formula I:
J-L-X-B wherein J is a surface binding moiety, X is an antifouling moiety, L is a covalent bond or a linker moiety linking J and X, B may be absent or an amino acid moiety, and each of - represents a bond.

73. The particles according to claim 72, wherein, where L is present, it is bonded to each of J and X via covalent bonds or non-hydrolysable bonds.

74. The particles according to claim 72, wherein, where L is absent, J and X are bonded to each other via a covalent bond or non-hydrolysable bond.

75. The particles according to claim 72, wherein the surface binding moiety is selected from the group consisting of one or more 3,4-dihydroxy-L-phenylalanin (DOPA), DOPA containing moiety, dopamine and trihydroxyphenylalanine.

76. The particles according to claim 72, wherein the antifouling moiety is a fluorine (F) atom or a group comprising at least one fluorine atom.

77. The particles according to claim 76, wherein the antifouling moiety is a fluorinated carbon group.

78. The particles according to claim 72, wherein the material is selected from the group consisting of: J-X-B, J-B-X, X-B-J, X-J-B, B-X-J and B-J-X; wherein each of B, J, X and - are selected as defined in claim 72.

79. The particles according to claim 78, wherein B is at least one amino acid sequence promoting adherence of cells, optionally selected from the group consisting of RGD (Arg-Gly-Asp); KQAGDV; YIGSR; REDV; IKVAV; RNIAEIIKDI; KHIFSDDSSE; VPGIG; FHRRIKA; KRSR; NSPVNSKIPKACCVPTELSAI; APGL; VRN; and AAAAAAAAA.

80. The particles according to claim 72, wherein the material is selected from the group consisting of: ##STR00006## ##STR00007##

81. A medical device or implant having at least a surface region thereof coated with a plurality of particles according to claim 70.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0178] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0179] FIGS. 1A-C show Scanning Electron Microscopy (SEM) pictures of self-assembled structures of a peptide of the invention: FIG. 1Ain tris buffer, at pH 8.5;

[0180] FIG. 1Bin HCl 1M; and FIG. 1Cpolymerized particles.

[0181] FIGS. 2A-C provide optical microscope images of free fluorescent doxorubicin and encapsulated doxorubicin in particles of a peptide of formula I: FIG. 2A-no peptide;

[0182] FIG. 2Bin tris buffer, pH8.5; and FIG. 2C in HCl 1M.

[0183] FIGS. 3A-B provide Transmission Electron Microscope (TEM) of a peptide of Formula I, which is self-assembled in water in the presence of gold nanoparticles.

[0184] FIG. 4 demonstrates an antifouling assessment by protein adsorption.

[0185] FIGS. 5A-G provide the following: FIGS. 5A-B show Scanning Electron Micrographs of (FIG. 5A) symmetric spheres formed under acidic conditions, 1M HCl and (FIG. 5B) spikey spheres formed under basic conditions, 10 mM tris buffer, pH=8.5. FIGS. 5C-E show contact angles of (FIG. 5C) a bare glass slide, (FIG. 5D) a glass slide coated with the symmetric spheres, and (FIG. 5E) a glass slide coated with the spikey spheres. FIGS. 5F-G show FT-IR spectra of the peptide assemblies: (FIG. 5F) Symmetric spheres, and (FIG. 5G) spikey spheres.

[0186] FIGS. 6A-B provide assessment of the antifouling activity of the modified surfaces. (FIG. 6A) The graph plots the adsorbed amounts of BSA on the modified surfaces. (FIG. 6B) The plot shows the amount of bacteria on the modified surfaces. Error bars represent standard deviations (n=9).

[0187] FIGS. 7A-G provide the following: FIG. 7A-C show fluorescence microscopy micrographs of (FIG. 7A) doxorubicin on a bare substrate, (FIG. 7B) spheres self-assembled under an acidic condition in the presence of doxorubicin, and (FIG. 7C) spheres self-assembled under a basic condition in the presence of doxorubicin. FIGS. 7D-G show doxorubicin release from the peptide assemblies: fluorescence spectrum of doxorubicin released from (FIG. 7D) symmetric spheres and (FIG. 7E) spikey spheres. Fluorescence intensity at 590 nm as a function of time for doxorubicin released from (FIG. 7F) symmetric spheres and (FIG. 7G) spikey spheres.

[0188] FIG. 8 plots the number of CFUs resulting from surfaces modified with either the peptide assemblies, GOx, or a combination of the assemblies and the GOx. Error bars represent standard deviations (n=24).

[0189] FIGS. 9A-H show self-assembly of the peptide in different Tris concentrations. FIGS. 9A-D show the coverage of the surface by the peptide structures, and FIGS. 9E-H show the morphology of the structures. FIGS. 9A and 9B1 mM, FIGS. 9B and 9F5 mM, FIGS. 9C and 9D15 mM, and FIGS. 9D and 9H20 mM.

[0190] FIGS. 10A-F show self-assembly of the peptide at different pH values: FIG. 10A pH=1, FIG. 10B pH=2, FIG. 10C pH=3, FIG. 10D pH=4, FIG. 10E pH=5, and FIG. 10F pH=6. The average diameter of the spheres formed in acidic medium decreases with increasing pH until they merge to produce a film.

[0191] FIGS. 11A-H show self-assembly of the peptide at different pH values: FIG. 10A pH=6.5, FIG. 10B pH=7, FIG. 10C pH=7.5, FIG. 10D pH=8, FIG. 10E pH=9, FIG. 10F pH=9.5, FIG. 10G pH=10, and FIG. 10H pH=10.5. At a neutral pH the surface is mostly covered with undefined aggregates. When the pH is increased, spikey spheres are formed. The higher the pH, the denser each sphere becomes.

[0192] FIG. 12 shows the reduction in the fluorescence intensity of Doxorubicin upon the self-assembly of the peptide.

[0193] FIGS. 13A-C show Doxorubicin encapsulation/adsorption. FIG. 13A Doxorubicin in Tris buffer, FIG. 13B the solution becomes opaque with peptide addition, and FIG. 13C stained peptide assemblies precipitate during the night, leaving a clear solution above them.

[0194] FIGS. 14A-D show Gentamicin release from the peptide assemblies after overnight incubation. FIGS. 14A and 14B, agar plates on which buffer incubated with bare surfaces was taken. FIG. 14C, zones of inhibition formed after applying buffer that was incubated with surfaces modified with spikey spheres. FIG. 14D, zones of inhibition formed after applying buffer that was incubated with surfaces modified with symmetric spheres.

DETAILED DESCRIPTION OF EMBODIMENTS

[0195] Various peptides of the invention have been formed into particulate form and have been used in a plurality of applications, such as, inter alia, biofouling, encapsulation of medicinal materials, encapsulation of nanoparticles, etc. The results presented herein demonstrate such uses and the particles broader utility as carriers or materials from which the material may be released or prevented from being released.

Example 1: Preparation of Particles of the Invention

[0196] Preparation of porous particles: A fresh stock solution was prepared by dissolving the peptide in pure ethanol to a concentration of 100 mg/mL. The peptide stock solution was then diluted to a final concentration of 1 mg/mL in 10 mM tris buffer.

[0197] Preparation of symmetrical spheres: A fresh stock solution was prepared by dissolving the peptide in pure ethanol to a concentration of 100 mg/mL. The peptide stock solution was then diluted to a final concentration of 1 mg/mL in 1M hydrochloric acid (HCl).

[0198] As FIGS. 1A, 1B and 1C provide, the peptide of the invention can self-assemble in an aqueous medium into either spherical or porous particles depending on the pH of the solution with a diameter of tens of micrometers. The morphology of the structures formed in solution of basic pH (pH=8.5) was highly porous (FIG. 1A). Conversely, in acidic pH (1M HCl) the peptide self-assembled into symmetrical spheres (FIG. 1B).

[0199] In addition, spherical particles were formed also by polymerization (FIG. 1C).

Example 2: Encapsulation of Doxorubicin in the Self-Assembled Structures of Peptide

[0200] Encapsulation of an active material, e.g., doxorubicin, in a particle of the invention has also been achieved.

[0201] Doxorubicin loaded particles were prepared by dilution of a peptide stock solution (100 mg/mL) to a concentration of 1 mg/mL in aqueous medium (either tris buffer or HCl) containing doxorubicin at a concentration of 0.05 mg/mL.

[0202] FIG. 2A shows Optical Fluorescent Microscope image of free doxorubicin. Fluorescent microscope image of the peptide structures self-assembled in the presence of doxorubicin, at high pH, is shown in FIG. 2B. Fluorescent microscope image of the peptide structure self-assembled in the presence of doxorubicin, at low pH (1M HCl), is shown in FIG. 2C.

Example 3: Encapsulation of Gold Nanoparticles in the Self-Assembled Structures of Peptide

[0203] Further prepared were particles of a material of formula I in the present of gold particles in the aqueous medium.

[0204] Gold nanoparticles loaded particles were prepared by dilution of a peptide stock solution (100 mg/mL) to a concentration of 1 mg/mL in aqueous medium (either tris buffer or HCl) containing gold nanoparticles at a concentration of 100 ug/mL.

[0205] Pictures of Transmission Electron Microscope (TEM) show that the peptide having Formula I self-assembled in water in the presence of gold nanoparticles, at a concentration of 2 mg/mL (FIG. 3A) and at a concentration of 1 mg/mL (FIG. 3B).

Example 5: The Particles Resists Fouling

[0206] FIG. 4 demonstrates normalized adsorption of Bovine Serum Albumin (BSA) onto titanium substrate coated with the peptide particle, compared to a bare titanium substrate. A reduction in protein adsorption onto titanium surface due to the presence of the spheres particle can be clearly seen.

Example 6: Self-Assembly of the Tripeptide into Spherical Particles in Either Acidic or Basic Aqueous Solutions

[0207] To initiate the self-assembly of the tripeptide, the peptide was dissolved in either an acidic (1M HCl) or basic (Tris buffer, 10 mM, pH=8.5) solution. To characterize the structures formed by the tripeptide under different conditions, Scanning Electron Microscopy (SEM) analysis was obtained. The results revealed the formation of different structures in acidic or basic solutions. In acidic medium, the peptide self-assembles into symmetric spheres with an average diameter of 2.00.4 m. In contrast, in basic medium, the peptide self-assembles into porous spheres with an average diameter of 263 m. These assemblies are termed herein spikey spheres (FIGS. 5A-B).

[0208] By applying these structures onto a bare substrate the chemical and physical characteristics of the substrate's surface can be altered. The chemical characteristics of the surface will change due to the chemical nature of the peptide (hydrophobic and fluorinated), and the physical properties will change due to the new microtopography gained by these structures. Both features play a major role in the design of efficient antifouling materials.

[0209] To coat a surface with these peptide assemblies, the peptides were self-assembled and drop-casted the peptide solution on clean glass slides. After the samples dried in air, the samples were dipped in Triple Distilled Water (TDW) to remove excess and non-adhered peptide from the surface. Finally, they were dried under nitrogen. To determine whether the peptide structures indeed alter the features of the surface, the contact angle of the coated surfaces were measures. Whereas substrates covered with the symmetric spheres exhibited an increase in the contact angle, from 36 for bare glass, to 75 for coated glass, the substrates coated with the spikey spheres exhibited a decrease in the contact angle to 16 (FIGS. 5C-E). The increase in the contact angle in the case of the symmetric spheres indicates an increase in hydrophobicity. This could be attributed to the topography and to the hydrophobic features of the peptide.

[0210] In the case of the spikey spheres, the surface became more hydrophilic. This can be explained by the porosity of the structures. In fact, while measuring the contact angle, the inventors could detect that the water droplet was almost completely soaked by the surface.

[0211] To obtain information on the secondary structures of the peptide assemblies, Fourier Transform Infrared Spectroscopy analysis (FT-IR) have been performed. The symmetric spheres formed in HCl had a single peak at 1631 cm.sup.1, which corresponds to a -sheet structure. The spikey spheres had a different spectrum, consisting of three distinctive peaks at 1627 cm.sup.1, 1660 cm.sup.1, and 1677 cm.sup.1. These peaks may suggest a more complex structure that may combine both -helical and -sheet secondary structures. The different spectra are correlated with the different microstructures formed by the peptide.

[0212] To better understand how the ionic strength of the solution affects the self-assembly process, we self-assembled the peptide in different Tris concentrations, ranging from 1 mM to 20 mM and analyzed the morphology of the structures using SEM (FIG. 9) The results indicated that at a concentration of 1 mM, only a few spherical structures formed. However, their appearance was different from the structures obtained in 10 mM Tris. In addition, the majority of the surface was covered with amorphous aggregates. Upon increasing the concentration to 5 mM, spikey spheres were formed, but their dispersion on the surface was different. Whereas the spikey spheres formed in 10 mM Tris tend to appear in large clusters, the spheres formed in 5 mM Tris appeared in small clusters of only a few assemblies. Moreover, amorphous aggregates could be detected between the ordered structures, whereas the in-between spacing in the case of 10 mM Tris was free from aggregates. In higher concentrations, such as 15 mM and 20 mM, the amorphous aggregates were dominant. In addition, very few spheres formed, but they were denser and larger than with 10 mM Tris. Accordingly, we decided that the optimal buffer concentration was 10 mM Tris.

[0213] To better understand how the pH value of the solution affects the self-assembly process, self-assembled peptides were achieved in solutions of different pH values, ranging from 1 to 10.5. Since the acidic solutions differ from the basic solution in their chemical composition, they cannot be compared. Interesting trends, however, could be observed in the two different pH series. SEM analysis revealed that lowering the acidity of the solution results in smaller spheres. FIG. 10 presents the alteration of spheres while the pH is increased from 1 to 6. The spheres became increasingly smaller, until no spheres could be detected, and a uniform coating was formed. With the basic medium, upon an increase in the pH value, the spikey spheres became denser and less spikey (FIG. 11). According to these results, we can conclude that by fine-tuning of the pH values, we can control the physical properties of the spheres and their appearance.

[0214] To determine the antifouling activity of surfaces decorated with these assemblies, we first investigated their resistance to protein adsorption. Bare glass slides and peptide-coated glass slides were incubated in a solution of Bovine Serum Albumin (BSA) at a concentration of 150 M, for two hours at 37 C. To determine the adsorbed amounts of BSA on the substrate, the Non-interfering protein Assay kit was used. The plot in FIG. 6A summarizes the results. The amount of protein adsorbed on the bare glass substrates was substantially higher than the amount of protein adsorbed on the peptid-coated glass substrates. The symmetric spheres, formed in acidic medium, reduced the amount of protein from 1.9 nmol/cm.sup.2 to 0.5 nmol/cm.sup.2. The spikey spheres formed in basic medium were found to be even more efficient, and reduced the amount of adsorbed protein, from 1.9 nmol/cm.sup.2 to 0.28 nmol/cm.sup.2. Previous reports showing the higher tendency of BSA to adsorb onto hydrophobic surfaces compared with hydrophilic surfaces support these results. It was also found that BSA undergoes denaturation and spatial rearrangements while adsorbing to surfaces. Its spatial orientation depends on the surface vacancy, which allows it to spread. Since the spikey spheres form non-smooth surfaces with complex topography, they may be more effective against the protein, compared with the spherical spheres.

[0215] To assess the extent of the bacterial attachment to the surface, bare and peptide-modified surfaces were incubated with Escherichia coli overnight to allow the adsorption of bacteria and the formation of biofilms on the substrates. After incubation, the bacteria were removed from the substrates by sonication, diluted 10-fold, plated, and the colonies forming units (CFUs) were counted. The surfaces coated with the different peptide assemblies exhibited antifouling activity, and the number of colonies counted from these samples was lower than the number of colonies from the bare substrates. The spikey spheres reduced the bacterial growth by 68%, but importantly, the spherical spheres reduced it by 85% (FIG. 6B). Similar results were also obtained for longer incubation times.

[0216] To improve the antifouling activity of the peptide assemblies, we decided to exploit their ability to adsorb or encapsulate and then release active compounds. To determine this capability, we self-assembled the peptide in the presence of 50 g/mL of the fluorophore molecule, doxorubicin. After a few minutes of incubating them together, we drop-casted the solution onto a glass cover slip and dried it under ambient conditions. The glass was then rinsed with water to remove the excess dye and non-adsorbed peptide. Fluorescence microscopy analysis revealed that glass slides casted with only doxorubicin exhibited a fully stained surface, whereas the slides casted with the peptide and dye exhibited clearly distinct fluorescent spheres. The drug specifically adsorbed onto the peptide assemblies, without affecting their structure (FIG. 7).

[0217] To further investigate the association of the drug with the peptide, we measured the fluorescence spectroscopy. Under the same conditions, the peptide was added to either acidic or basic medium containing the doxorubicin, and the fluorescence intensity at 590 nm before and after the addition was measured. Upon the addition of the peptide, the intensity of the dye markedly decreased (FIG. 12). This can be attributed to the lower amount of free dye in the solution, owing to its encapsulation/adsorption to the peptide assemblies.

[0218] To determine whether the peptide assemblies can release the drug, we self-assembled the peptide in the presence of the drug and incubated it overnight. During the incubation, the peptide assemblies precipitated (FIG. 13), and the excess drug and medium could be easily removed. Then, we washed the precipitate and re-dispersed it in PBS (10 mM, pH=7.4). The re-dispersed peptide solutions were transferred to a dialysis device (MWCO 3 kDa), and the fluorescence intensity of the buffer outside the membrane was sampled for 9 days, to detect any traces of the drug. The release results are presented in FIG. 3. In both samples, the one containing the symmetric spheres and the one containing the spikey sphere drug traces could be detected and they increased with time. However, the release from the spheres formed in acidic medium was faster, and reached equilibrium after 6 days.

[0219] In the case of spheres formed in basic medium, a more controlled release pattern was observed with a linear release. After 9 days, not all of the dye was released. This means that the peptide assemblies can act as an active leaching surface by releasing antibacterial compounds. Based on these results, we attempted to combine the antifouling activity of the peptide with other active compounds against bacteria such as antibiotics and hydrogen peroxide.

[0220] To determine the release of the antibiotic gentamicin from the peptide assemblies, substrates coated with the peptide assemblies entrapping gentamicin were incubated in PBS overnight. Then, a 10 L drop of the buffer was taken and streaked on agar inoculated with E. coli. After allowing the bacteria to grow, we examined the plates for zones of inhibition. Indeed, clear zones were observed where the buffer drops were applied. However, these zones could not be detected in plates streaked with buffer drops taken at t=0 or drops taken from bare glass slides. The average radius of inhibition from the antibiotic released from the spheres was 0.840.07 cm, and from the spikey spheres it was 0.940.06 cm (FIG. 14). The difference in the size of the zone of inhibition probably resulted from the difference in size: the spikey spheres are larger in size than the symmetrical spheres and might entrap a larger amount of antibiotic.

[0221] The antifouling and antibacterial activity of peptide assemblies encapsulating Glucose Oxidase (GOx) were also tested. GOx is an enzyme that oxidizes glucose in the presence of oxygen into gluconolactone and hydrogen peroxide. Hydrogen peroxide interrupts with the bacterial cell wall, leading to bacterial death. Thus, it was assumed that incorporating the enzyme would improve the antibacterial activity of the surface. Since the enzyme activity is sensitive to pH, the peptide was assembled only under conditions that resulted in the spikey spheres, since they are formed under milder pH conditions. Bare glass slides and modified glass slides were incubated in the inoculums of E. coli, at an initial concentration of 10.sup.5 CFUs/mL, for one hour.

[0222] To prove that the peptide assemblies promote the anchoring of GOx to the substrates, we prepared slides on which we drop-casted only GOx. After incubation, the bacteria were removed from the surface by sonication, diluted 10-fold, plated, and counted for CFUs. FIG. 8 summarizes the results.

[0223] As observed before, substrates coated with the spikey spheres reduced the amount of bacteria by 70%. The surfaces that combined the peptide and the GOx exhibited the best activity. The number of bacteria dropped by an order of magnitude, from 2.310.sup.6 CFUs/mL to less than 510.sup.5 CFUs/mL. This proves that combining the peptide activity with the GOx activity improves the overall ability of the surface to resist fouling. It is important to note that although the enzyme was not anchored to the surface, it was found that glass slides coated with GOx alone slightly reduced the amount of bacteria. It is assumed that this is due to some residual enzyme molecules that could form some non-specific interactions with the surface.

Experimental

Materials

[0224] All chemicals, proteins, and bacteria were purchased from commercially available companies and used as supplied unless otherwise stated. The reported peptide was synthesized by a conventional solution-phase method as described before. Doxorubicin, gentamicin, BSA, and GOx were obtained from Sigma-Aldrich (Jerusalem, Israel). Eschrichia coli (ATCC 25922) was purchased from ATCC (Virginia, USA). Luria broth and tryptic soy broth were obtained from BD difco (New Jersey, USA). Nutrient agar was obtained from Merck (Darmstadt, Germany).

Stock Solution

[0225] To avoid any pre-aggregation, a new fresh stock solution was prepared for each experiment. The fresh stock solution was prepared by dissolving the peptide in pure ethanol (Gadot, Israel) to a concentration of 100 mg/mL.

Preparation of Porous Spheres

[0226] The peptide stock solution was diluted to a final concentration of 1 mg/mL in Tris buffer (10 mM, pH=8.5).

Preparation of Symmetric Spheres

[0227] The peptide stock solution was diluted to a final concentration of 1 mg/mL in 1M HCl solution.

High Resolution Electron Microscopy (HR-SEM)

[0228] A 30 L drop containing the peptide spheres was drop-casted on a glass cover slip and allowed to dry at room temperature (RT). The peptides on the glass were coated with gold using a Polaron SC7640 sputter coater. SEM images were taken using an extra high-resolution scanning electron microscope, Magellan TM400L, operating at 2 kV.

Fourier Transform Infrared Spectroscopy (FT-IR)

[0229] Peptide stock solution was diluted to a final concentration of 1 mg/mL in duterated media (Tris buffer in D20 or 1M DC1). Then, each peptide solution was deposited on a CaF2 plate and dried under vacuum. Infrared spectra were recorded using a Nicolet 6700 FT-IR spectrometer with a deuterated triglycine sulfate (DTGS) detector (Thermo Fisher Scientific, MA, USA). The measurements were taken using 4 cm.sup.1 resolution and an average of 2000 scans. The absorbance maximal values were determined by the OMNIC analysis program (Nicolet). Each spectrum was deconvoluted.

Contact Angle Measurements

[0230] Contact angle measurements were carried out using a Theta Lite optical tensiometer (Attension, Finland).

Preparation of Doxorubicin-Loaded Spheres

[0231] Doxorubicin-loaded spheres were prepared by diluting a peptide stock solution (100 mg/mL) to a concentration of 1 mg/mL in aqueous medium (either Tris buffer or HCl) containing doxorubicin at a concentration of 0.05 mg/mL.

Fluorescence Microscopy

[0232] A 30 L drop containing the doxorubicin-loaded sphere solution was drop-casted on a glass cover slip and allowed to dry at RT. A 30 L drop of doxorubicin in aqueous medium (HCl or tris buffer) was also drop-casted on a glass cover slip as a control. Images were taken using a fluorescence microscope (Carl Zeiss, Axio Vision). Samples were excited at 510 nm.

Drug Release Study

[0233] Doxorubicin-loaded spheres were prepared as described. After the self-assembly process, samples were left to precipitate overnight, the aqueous medium was decanted, and the peptide assemblies were re-dispersed in PBS (10 mM NaCl, pH=7.4, 150 mM). Then, 2 mL of the doxorubicin-loaded peptide were transferred into a dialysis bag (MWCO 3 kDa), and the bag was immersed in 45 mL of PBS, at RT. One mL of the buffer outside the dialysis bag was taken at different time intervals for 9 days, for fluorescence measurements. The volume of the solution was kept constant by adding 1 mL of the original PBS solution after each sampling. The fluorescence measurements were performed at RT using a fluorescence spectrometer (Edinburgh instruments FLS920). The emission spectra were collected from 500 nm to 750 nm, with an excitation wavelength of 480 nm.

Protein Adsorption

[0234] Fifty L of BSA solution (150 M in PBS) were applied onto the substrate in a Petri dish. The plate was placed in a humidified incubator at 37 C. for 2 hours. The substrates were then rinsed 3 times with PBS (pH=7.43, 10 mM NaCl, 150 mM), and transferred into test tubes with 1 mL of 2% (w/w) SDS. The samples were shaken for 60 minutes and sonicated for 20 minutes at room temperature to detach the adsorbed proteins. Protein concentrations in the SDS solution were determined using the Non-interfering protein assay (Calbiochem, USA) according to the manufacturer's instructions, using a microplate reader (Synergy 2, BioTek) at 480 nm. All measurements were performed in triplicate and averaged.

Preparation of Gentamicin-Loaded Spheres

[0235] Gentamicin-loaded spheres were prepared by diluting the peptide stock solution (100 mg/mL) to a concentration of 1 mg/mL in aqueous medium (either Tris buffer or HCl) containing gentamicin at a concentration of 0.5 mg/mL.

Preparation of Surfaces Coated with Gentamicin-Loaded Spheres

[0236] A 100 L solution containing gentamicin-loaded spheres was drop-casted on 11 cm.sup.2 glass substrates. Then, the samples were left to dry, dipped in TDW to remove excess drug and peptide, and dried again.

Gentamicin Release from Surfaces

[0237] 11 cm.sup.2 glass substrates coated with gentamicin-loaded spheres were placed in 3 mL of PBS (10 mM, pH=7.4) and incubated for 24 hours. After incubation, 10 L were collected from the PBS and placed on an agar plate streaked with E. coli. The plates were incubated at 37 C. overnight. After incubation, the zone of the inhibition diameters were measured, and the amount of antibiotic release was calculated using a calibration chart.

Preparation of GOx-Loaded Spheres

[0238] GOx-loaded spheres were prepared by diluting a peptide stock solution (100 mg/mL) to a concentration of 1 mg/mL in Tris buffer containing GOx at a concentration of 100 g/mL.

Preparation of Surfaces Coated with GOx-Loaded Spheres

[0239] A 150 L of solution containing GOx-loaded spheres was drop-casted on 11 cm.sup.2 glass substrates. Then, the samples were left to dry, dipped in TDW to remove excess enzyme and peptide, and dried again.

Bacterial Growth

[0240] E. coli (ATCC 25922) were grown in TSB medium at 37 C., for 6 hours, in loosely capped tubes with agitation (120 rpm), to late logarithmic phase. Then, the bacteria were centrifuged and washed 3 times with PBS, re-suspended, and diluted to 10.sup.5 CFU/mL with TSB.

Antifouling Activity of the Peptide Assemblies

[0241] Three mL of the culture were transferred to each petri dish, and the substrates were placed horizontally in the plate, and incubated in a humidified incubator at 37 C. for either 24 hours or 3 days. One additional mL of TSB was added to each plate after two days to ensure a sufficient supply of nutrients.

[0242] After incubation, the substrates were gently rinsed with 3 mL of PBS, and transferred into test tubes with 5 mL of PBS. Then, the test tubes were sonicated for 1 minute to detach bacteria from the substrates, and vortexed for 15 seconds. The number of viable bacteria was determined by plating the samples in 10-fold serial dilutions on LB agar plates.

Antibacterial Dual Activity of the Surfaces

[0243] 150 L of the bacterial culture were gently placed on each substrate, and the substrates were placed horizontally in the plate and incubated in a humidified incubator at 37 C. for 1 hour.

[0244] After incubation, the substrates were gently rinsed with 1 mL of PBS, and transferred into test tubes with 3 mL of PBS. Then, the test tubes were sonicated for 1 minute to detach bacteria from the substrates, and vortexed for 15 seconds. The number of viable bacteria was determined by plating the samples in 10-fold serial dilutions on LB agar plates.