WO2015124767A1 - Composé peptidique bifonctionnel pour adhérence cellulaire - Google Patents

Composé peptidique bifonctionnel pour adhérence cellulaire Download PDF

Info

Publication number
WO2015124767A1
WO2015124767A1 PCT/EP2015/053724 EP2015053724W WO2015124767A1 WO 2015124767 A1 WO2015124767 A1 WO 2015124767A1 EP 2015053724 W EP2015053724 W EP 2015053724W WO 2015124767 A1 WO2015124767 A1 WO 2015124767A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
sequence
seq
biomaterial
cell
Prior art date
Application number
PCT/EP2015/053724
Other languages
English (en)
Inventor
Frederic Cuisinier
Ivan PANAYOTOV
Csilla Gergely
Christian Larroque
Elias Estephan
Original Assignee
Ctre Hosp Universitaire De Montpellier
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ctre Hosp Universitaire De Montpellier filed Critical Ctre Hosp Universitaire De Montpellier
Publication of WO2015124767A1 publication Critical patent/WO2015124767A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/04—Metals or alloys
    • A61L27/06—Titanium or titanium alloys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/08—Materials for coatings
    • A61L29/085—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/25—Peptides having up to 20 amino acids in a defined sequence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention first relates to a bi-functional peptidic compound characterized in that it comprises a metal-binding peptide (MBP) domain and a cell- specific peptide (CSP) domain, said MBP and CSP domains being linked by a linker.
  • a bi-functional peptidic compound according to the invention is able to bind to the metallic surface of a biomaterial compound to gingival cells, thereby enhancing the cell- adhesion of said biomaterial compound.
  • the present invention also relates to a composition comprising said bi-functional peptidic compound, and to a biomaterial compound associated with said bi-functional peptidic compound, it relates in particular to a dental implant functionalized by the adsorption on its surface of a bi-functional peptide according to the invention.
  • Implantable biomaterials may be functionalized by surface modification in order to improve their properties.
  • WO 2010 094212 discloses plasma treated and controllable release antimicrobial peptide coated titanium alloy for surgical implantation. The surface forms antimicrobial layers on the alloy capable of resisting microbial adhesion and proliferation, while allowing mammalian cell adhesion and proliferation when the alloy is implanted to human body.
  • JP 2009007339 discloses an adhesive for dental implant, reinforcing boundability of the dental implants with biological tissues, especially soft tissues such as gingival epithelium.
  • the adhesive for dental implants comprises a ceil adhesive artificial peptide (P). Further, a siiane coupling agent and/or a titanium coupling agent is preferably contained in the adhesive.
  • US 7897163 discloses a bone graft material and a scaffold for tissue engineering applications having an osteogenesis-promoting peptide immobilized on their surface.
  • the shielding membrane and implant comprise ceil adhesion derived peptides including ROD amino acids.
  • RGD bio-active peptide sequence to promote cell adhesion on different surfaces. It is found in most extracellular matrix proteins such as fibronectin, laminin, and vitronectin influencing cell adhesion, mobility, proliferation, and cell survival and has been shown to bind approximately half of the 24 known human integrins.
  • the LbL film deposition method consists of the alternate adsorption of oppositely charged polyelectrolytes that auto-assemble, leading to the formation of polyelectrolyte multilayer (PEM) films.
  • PEM polyelectrolyte multilayer
  • EP 2 385 057 discloses a peptide having the amino acid sequence SVSVGMKPSPRP (SEQ ID N° l) and its binding to Silicon substrates. Estephan et al. (2012 ) also disclose a peptide hav ing the sequence SEQ ID N° l and the large variety of inorganic and organic substrates bound by this peptide.
  • WO 2006/1 16737 discloses a peptide having the sequence SWELL. YPLRANL (SEQ ID N°4) and its ability to disrupt cell lines adhesion. Devemy and Blaschuk (2009) disclose an antagonist of E- and N-cadherin having the sequence SEQ ID N°4 and its ability to disrupt intercellular adhesive complexes.
  • Ti titanium
  • Ti alloys T1 6 AI 4 V
  • the epithelial sealing at the abutment has been identified as the critical factor to prevent periimplant inflammation.
  • basal lamina ⁇ 200 nm
  • hemidesmosomes formation.
  • the adhesion must be stable and resistant to various external factors like mechanical constraints and oral bacterial pathogens.
  • the present invention provides a new bi-functional metal-cell specific peptidic compound, and in a particular a metal-cell specific peptide (MCSP) and a method to functionalize the surface of a biomaterial compound, wherein said bi-functional peptidic compound comprises a metal binding peptide (MBP) domain linked to a cell-specific peptide (CSP) domain to increase the cell adhesion of said functionalized biomaterial compound.
  • MBP metal binding peptide
  • CSP cell-specific peptide
  • a functionalization method according to the invention is easy-to-use, and the bi- functional peptidic compounds can be synthetized in desired quantities and modified according to the envisaged application by standard methods.
  • the invention goes beyond the state of the art in sensitivity and detection limit due to the ordered array of molecules assured by the peptides.
  • a bi-functionalization process according to the present invention is able to overcome the problem related to the unwanted polymerization and denaturation of capturing molecules when amine-activation is performed via glutaraldehyde chemistry.
  • a process according to the invention is able to confer biocompatibility and stability to a functionalized biomaterial, and may reduce its toxicity.
  • the present invention discloses four artificial MCSP among which was selected one best candidate for implant surface functionalization.
  • the present invention will become more fully understood from the detailed description given herein and from the accompanying drawings, which are given by way of illustration only and do not limit the intended scope of the invention.
  • amino acid is herein represented according to the IUPAC amino-acid abbreviation, such as follows:
  • the present invention first relates to a peptidic compound characterized in that it comprises: a) a metal-binding peptide (MBP) domain able to bind to a titanium surface, said MBP domain comprising an amino acid sequence selected from the group consisting of:
  • CSP cell-specific peptide
  • metal-binding peptide By “metal-binding peptide”, “metal-binding peptide domain” or MBP domain, it is intended a peptide comprising an amino acid sequence which is able to bind to an inorganic substrate, and preferably to a metallic surface.
  • the ability of a MBP domain of a peptide according to the invention to bind to a titanium surface is characterized by the affinity of said MBP domain for a titanium surface, and preferably a high affinity of said MBP domain for a titanium surface.
  • a person skilled in the art of determining the affinity of a peptide for another compound will be able to choose the appropriate method and conditions to determine the ability of a MBP domain of the invention to bind to a titanium surface.
  • in vitro affinity measurement methods including ELISA and competitive inhibition, biopanning, microscopy, including fluorescence microscopy and atomic force microscopy, spectroscopy, including Fourier transform infrared spectroscopy, spectrometry, including MALDI-TOF mass spectrometry.
  • a MBP domain according to the invention is also able to bind to metallic surface other than titanium.
  • a metallic surface other than titanium, able to be bound by a MBP domain according to the invention may be chosen among one of the following: a titanium alloy, gold, carbon, cobalt, platinum, steel, surgical stainless steel, silicone, chrome and a chrome alloy.
  • cell-specific peptide By "cell-specific peptide”, “cell-specific peptide domain” or CSP domain, it is intended a peptide comprising an amino acid sequence which is able to bind or to adhere to cells, preferably to mammalian cells, said mammalian cells being chosen in the group consisting of mice cells, rat cells and human cells, preferably human cells.
  • a CSP domain of a bifunctional peptide according to the invention is able to bind to epithelial cells, and more particularly to keratinocytes or to endothelial cells.
  • a CSP domain according to the invention is able to bind to E-cadherin, to N-cadherin, to a domain thereof or to a combination thereof.
  • a CSP-domain according to the invention is chosen in the group consisting of E-cadherin antagonists, N-cadherin antagonists, or E- and N-cadherin antagonists.
  • amino acid sequence and “sequence” will be employed indifferently in the present specification.
  • amino acid and “amino acid residue” will also be employed indifferently.
  • the amino acid sequences are read from the N-Terminal extremity to the C-Terminal extremity of said amino acid sequence of the polypeptide. Therefore the amino acid in position 1, i.e. the first amino acid, of a sequence is the amino acid at the N-terminal extremity of the sequence.
  • peptide means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “polypeptide” or "protein”.
  • peptidic compound designates a compound comprising a peptide, or comprising two peptide domains linked by a linker, wherein said linker has a peptidic or a non-peptidic nature. In a particular embodiment of the invention, two peptide domains are linked by a peptidic linker and said peptidic compound is a peptide.
  • identity means that two amino acid sequences are identical (i.e. on an amino acid basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, in which the amino acid sequence to be compared can contain additions or deletions with respect to the reference sequence for optimal alignment between those two sequences.
  • the percentage of sequence identity is calculated by determining the number of positions at which the identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size) and multiplying the result by 100 to yield the percentage of sequence identity.
  • the percentage of sequence identity of an amino acid sequence can also be calculated using BLAST software with the default or user defined parameter.
  • the invention also relates to a functional derivative of a bi-functional peptidic compound according to the invention, comprising one or more modifications that do not substantially affect the activity of the bi-functional peptidic compound such as previously defined.
  • Modifications include addition and/or deletion and/or substitution of one or more amino acids, and/or replacement of one or more amino acid side chain by a different chemical moiety, and/or protection of the N-terminus, the C-terminus, or one or more of the side chains by a protecting group, and/or introduction of double bonds, and/or cyclization and/or modification of stereospecificity in the amino acids chain.
  • An amino acid may be substituted by an equivalent amino acid without modifying the peptide structure.
  • leucine, valine and isoleucine may be substituted, or aspartic acid and glutamic acid, or glutamine and asparagine, or asparagine by lysine.
  • the substituting amino acids are not limited to those naturally occurring in proteins, such as L-a-amino acids, or their D-isomers.
  • the peptides can be substituted with a variety of moieties such as amino acid mimetics well known to those of skill in the art.
  • An amino acid mimetic as used here is a moiety other than a naturally occurring amino acid that conformationally and functionally serves as a substitute for an amino acid in a polypeptide of the present invention.
  • the individual amino acids can be incorporated in the peptide by a peptide bond or peptide bond mimetic.
  • a peptide bond mimetic of the invention includes peptide backbone modifications well known to those skilled in the art.
  • the present invention relates to a bi-functional peptidic compound characterized in that it comprises:
  • MBP metal-binding peptide domain able to bind to a titanium surface
  • said MBP-domain comprising an amino acid sequence selected from the group consisting of: SVSVGMKPSPRP (MBP-1, SEQ ID N°l), a sequence having at least 80% identity with the sequence SEQ ID N°l , WDPPTLKRPVSP (MBP-2, SEQ ID N°2) and a sequence having at least 80%> identity with the sequence SEQ ID N°2, and
  • CSP cell-specific peptide domain
  • PPFLMLLKGSTRFC CSP-1, SEQ ID N°3
  • SWELYYPLRANL CSP-2, SEQ ID N°4
  • SEQ ID N°4 a cell-specific peptide domain comprising an amino acid sequence selected from the group consisting of: PPFLMLLKGSTRFC (CSP-1, SEQ ID N°3), a sequence having at least 80% identity with sequence SEQ ID N°3, SWELYYPLRANL (CSP-2, SEQ ID N°4) and a sequence having at least 80%> identity with the sequence SEQ ID N°4,
  • a bi-functional peptidic compound according to the invention comprises a MBP domain comprising a sequence having at least 80% identity, preferably at least 90%> identity with sequence SEQ ID N°l, or having the sequence SEQ ID N°1.
  • a bi-functional peptidic compound according to the invention comprises a MBP domain comprising a sequence having at least 80% identity, preferably at least 90%> identity with sequence SEQ ID N°2, or having the sequence SEQ ID N°2.
  • the present invention relates to a bi-functional peptidic compound according to the invention comprising a CSP domain comprising an amino acid sequence having at least 80% identity, preferably 90%> identity, with sequence SEQ ID N°3, or having the sequence SEQ ID N°3.
  • the present invention relates to a bi-functional peptidic compound according to the invention comprising a CSP domain comprising an amino acid sequence having at least 80%> identity, preferably 90%> identity, with sequence SEQ ID N°4, or having the sequence SEQ ID N°4.
  • a peptide according to the invention comprises a MPB domain and a CSP domain, wherein said MBP and CSP are linked by a peptide linker.
  • a bi-functional peptide according to this embodiment of the invention is defined as a metal-cell specific peptide (MCSP).
  • a peptide according to the invention comprises from 20 to 50 amino acids, preferably from 20 to 40 amino acids, preferably from 25 to 35 amino acids, preferably from 25 to 30 amino acids, even more preferably consists of 27 amino acids.
  • the invention relates to a peptidic compound comprising a metal-binding peptide and a cell-specific peptide, linked by a linker, which is adapted for the functionalization of a biomaterial.
  • the invention relates to a peptidic compound comprising a MBP-domain and a CSP-domain, linked by a linker, wherein said peptide is modified by any standard biochemical procedure well known by a person skilled in the art, in order to improve its biochemical characteristics, in particular its stability.
  • a peptide according to the invention comprises a metal-binding peptide and a cell-specific peptide, linked by a peptide linker, wherein said MBP domain has the sequence SEQ ID N°l, or an amino acid sequence having at least 80%> identity, preferably at least 90%> identity with sequence SEQ ID N°l, and said
  • CSP domain has the sequence SEQ ID N°4, or an amino acid sequence having at least
  • a peptide according to the invention comprises a metal-binding peptide and a cell-specific peptide, linked by a peptide linker, wherein said MBP domain has the sequence SEQ ID N°l and said CSP domain has the sequence
  • a peptide according to the invention comprises a MBP domain and a CSP domain, linked by a peptide linker, wherein said peptide linker comprises, or consists of,a short amino-acid sequence of 1 , 2, 3, 4 or 5 amino acids residues in length.
  • the linker is designed to facilitate the chemical immobilization of the MBP domain to the inorganic substrate and the binding of the CSP domain to the cells.
  • a bifunctional peptide according to the invention comprises, or consists of, 1, 2, 3, 4 or 5 amino acids chosen among any amino acids.
  • the peptide linker is a flexible linker.
  • the peptide linker is a rigid linker.
  • the peptide linker comprises at least one Gly, Ser, Arg, Leu, Cys, and/or Lys amino acids.
  • the peptide linker comprises Gly and Ser amino acids, or is defined as a glycine-rich linker or a serine -rich linker.
  • a peptide according to the invention comprises a peptide linker which exclusively comprises Gly amino acids.
  • the linker in a bifunctional peptide according to the invention, consists of three Gly amino acids.
  • a peptide according to the invention comprises a metal-binding peptide and a cell-specific peptide, linked by a peptide linker, wherein said peptide comprises an amino acid sequence selected from the group consisting of:
  • a bi-functional peptide according to the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID N°5, SEQ ID N°6, SEQ ID N°7 and SEQ ID N°8.
  • a bi-functional peptide according to the invention has an amino acid sequence selected from the group consisting of: SEQ ID N°5, SEQ ID N°6, SEQ ID N°7, and SEQ ID N°8.
  • a peptide according to the invention has the sequence SEQ ID N°6.
  • the present invention relates to a composition for the functionalization of a biomaterial compound, said composition comprising at least a bi- functional peptide according to the invention.
  • a composition according to the invention is adapted for the functionalization of a biomaterial intended to be implanted or administered to a host, in particular to a mammal host and more particularly to an animal or a human host.
  • a composition according to the invention consists preferably in a liquid phase comprising a sterile buffer.
  • the buffer is chosen among buffers well known by a person skilled in the art, and in particular Dulbecco's Phosphate Buffered Saline (DPBS - Sigma Aldrich) without calcium chloride and magnesium chloride.
  • This buffer has a neutral pH and the following chemical composition: Potassium Phosphate Monobasic 0.20 g/L; Potassium Chloride 0.20 g/L; Sodium Chloride 8.00 g/L; Sodium Phosphate Dibasic [Anhydrous] 1.15 g/L.
  • the buffer was chosen according the instruction for dilution of lyophilizated sterile peptides, like the most available solution for cell culture application.
  • a composition according to the invention comprises at least one a peptide comprising a metal-binding peptide and a cell-specific peptide, linked by a peptide linker wherein the peptide concentration is comprised between about 0.001 mM and about 1 mM, more preferably between about 0.01 mM and about 0,2 mM, and more preferably is of about 0,1 mM, wherein "about” designates a 10% variation of said concentration value.
  • the present invention also relates to an inorganic substrate functionalized by adsorption onto its surface of a peptide according to the invention.
  • the present invention also relates to an inorganic substrate functionalized by adsorption onto its surface of a peptide according to the invention, wherein said inorganic substrate is a biomaterial compound.
  • biomaterial compound it is intended a material compatible with biological tissues and organisms.
  • a biomaterial compound functionalized by adsorption onto its surface of at least one peptide according to the invention is chosen in the group consisting of: a microparticle, a nanoparticle, a tube, a nanotube, or a biomaterial incorporating thereof.
  • a microparticle, a nanoparticle, a tube, a nanotube, which surface has been functionalized with a peptide according to the invention may be part of a self-assembled structure or of a complex material.
  • a biomaterial compound according to the invention is a medical device, preferably a medical device for implantation.
  • a biomaterial compound or a medical device according to the invention is a part of a multi-component material.
  • a biomaterial compound according to the invention is a medical device chosen in the group consisting of: an implant, a dental implant, an implant for maxilo-facial surgery, a biosensor, a balloon, a stent, a shunt, a catheter, a myocardial plug, a pacemaker lead, a dialysis access graft, a heart valve, a pump, a prosthesis, an electronic device, an optic device, or a functional part thereof.
  • a biomaterial compound according to the invention is chosen in the group consisting of: a dental implant, the abutment screw of a two-parts dental implant or the cervical zone, or collar, of a monoblock implant.
  • the invention relates to a biomaterial compound functionalized by a peptide according to the invention, wherein said biomaterial compound is made of a material chosen in the group consisting of: titanium, a titanium alloy, surgical stainless steel, gold, carbon, cobalt, platinum, silicone, chrome and a chrome alloy.
  • the invention relates to a biomaterial compound functionalized by a peptide according to the invention, wherein the surface of said biomaterial compound is made of an inorganic material, said inorganic material being preferably chosen in the group consisting of: titanium, a titanium alloy, surgical stainless steel, gold, carbon, cobalt, platinum, silicone, chrome and a chrome alloy.
  • the invention relates to a biomaterial compound according to the invention, wherein the surface of said biomaterial compound is made of a titanium alloy, and preferably of T1 6 AI 4 V.
  • the invention relates to a biomaterial compound according to the invention, wherein the surface of said biomaterial compound is made of silicone (Si), and preferably of porous silicone.
  • the invention relates to a Si nano-particle or to a Si micro- particle, which surface has been functionalized with a peptide according to the invention, and which exhibit a better solubility, stability and molecular recognition properties than the non-functionalized Si nano-particle or to a Si micro-particle.
  • This functionalized Si nano-particle or to a Si micro-particle may be part of a self-assembled structure, or part of a complex material.
  • the present invention relates to a dental implant functionalized with a peptide comprising a sequence chosen in the group consisting of: the sequence SEQ ID N°6, a sequence having at least 80% identity with SEQ ID N°6, and a sequence having at least 90% identity with SEQ ID N°6.
  • the invention relates to a titanium dental implant, or to a T1 6 AI 4 V dental implant functionalized with a peptide comprising the sequence SEQ ID N°6.
  • the invention relates to a biomaterial compound associated with a peptide according to the invention, said biomaterial compound being characterized in that the cell adhesion force on epithelial cells of said biomaterial compound is comprised between about 0,1 and about 10 nN, preferably between about 0,5 and about 5 nN.
  • the cell adhesion force on keratinocytes of said biomaterial compound is comprised between about 0,1 and about 10 nN, preferably between about 0,5 and about 5 nN, wherein "about” designates a 10%> variation of said value.
  • the present invention also relates to a method for the functionalization of a peptide according to the invention with a biomaterial compound, said method comprising a step of contacting said biomaterial compound with a composition comprising a at least one peptide according to the invention.
  • Functionalization of a biomaterial compound with a peptide according to the invention is obtained by adsorption of said peptide onto the surface of said biomaterial.
  • a method for the functionalization of the surface of a biomaterial compound according to the invention may comprise a step of contacting said biomaterial compound according to the invention by any method known by a person skilled in the art, such as, for example spraying, coating or printing a peptide on the surface.
  • This functionalization can be performed for example by dipping said biomaterial in a composition according to the invention for a time sufficient to ensure the adsorption of the peptide onto the surface of said biomaterial, such as for example 2 hours, then washed.
  • Biomaterial peptide functionalization may also be performed by any method known by a person skilled to contact said biomaterial with a phase comprising a peptide according to the invention.
  • the present invention also relates to the use of a peptide according to the invention for the functionalization of a biomaterial compound.
  • the present invention also relates to the use of a composition according to the invention for the functionalization of a biomaterial compound.
  • the present invention relates to the use of a peptide comprising the amino acid sequence SEQ ID N°6, or the use of a composition a peptide comprising the amino acid sequence SEQ ID N°6, for the functionalization of a biomaterial compound.
  • the present invention relates to the use of a peptide comprising the amino acid sequence SEQ ID N°6 for the functionalization of a dental implant, or a part of a dental implant.
  • the present invention relates to a kit for the functionalization of a biomedical compound, said kit comprising: a peptide according to the invention or a composition according to the invention, and at least one compound chosen in the group consisting of: buffers, positive control and instructions for use.
  • Figures 1A to ID represent the MALDl-TOF spectra of MBP-1 on titanium and on titanium-alloy Ti 6 Al 4 V ( Figures 1A and IB) and the MALDl-TOF spectra of MBP-2 on titanium and on titanium-alloy Ti 6 Al 4 V ( Figures 1C and ID).
  • Figures 2A and 2B represent the force spectroscopy of metal binding peptides affinity.
  • Figure 2A represents the typical adhesion force curve measured with AFM, wherein the force (expressed in nN) is a function of indentation (in microm).
  • the black curve represents the retracting curve (pulling).
  • the inset shows a schematic representation of the peptide-functionalized tip, wherein the biotinylated peptide binds to streptavidin which in turns binds to biotin at the surface of the tip.
  • Figure 2B represents the measured adhesion force, in pN, of MBP-1 (black box) and MBP-2 (white box) on Ti 6 Al 4 V (left part of the figure) and on Ti (right part of the figure).
  • Figure 3 represents a force-indentation curve showing the typical unbinding of a single keratinocyte from the Ti surface, as measured by AFM, wherein the force (expressed in nN) is a function of indentation.
  • the black curve represents the retracting curve (pulling). Arrows represent some adhesion events preceded by a force plateau.
  • the inset shows an image of a keratinocyte cell adsorbed on the lip-less AFM cantilever.
  • Figures 4A to 4D represent a single-cell force spectroscopy analysis.
  • Figure 4A represents, from left to right, the single cell adhesion forces on bare and functionalized (MCSP-1, MCSP-2, MCSP-3 and MCSP-4) Ti surfaces, measured with the same cell.
  • Figure 4B represents the single cell adhesion forces on bare and functionalized (MCSP- 1, MCSP-2, MCSP-3 and MCSP-4) Ti 6 Al 4 V surfaces, measured with the same cell.
  • Figure 4C represents the influence of the substrate to single cell adhesion forces on non- functionalized and functionalized (MCSP-2 and MCSP-4) Ti and Ti 6 Al 4 V surfaces.
  • Figure 4D represents the influence of the substrate to single cell adhesion forces on non- functionalized and functionalized (MCSP-1 and MCSP-3) Ti and Ti 6 Al 4 V surfaces.
  • Figure 5 represents variation of surface roughness on metal surfaces, on titanium (clear bars) and Ti 6 Al 4 V (dark bars), the RMS is respectively represented, from left to right, for bare surface, functionalized surface with MCSP-1, MCSP-2, MCSP-3 or MCSP-4.
  • Figure 6 represents the results of single cell force spectroscopy analysis before and after BSA, wherein adhesion force (nN) is indicated for the following surfaces, from left to right: Ti, Ti 6 Al 4 V, Ti/MCSP-2, Ti 6 Al 4 V /MCSP-2, Ti/MCSP-2/BSA, Ti 6 Al 4 V /MCSP-2/BSA.
  • Figure 7 is a histogram representation of cell adhesion on functionalized surfaces in a para-nitrophenil phosphate (pNPP) cellular test.
  • the relative adhesion expressed in arbitrary units, is represented for Ti (white bars) and Ti 6 Al 4 V (black bars) surface, wherein, from left to right, the surface are bare or functionalized with, from left to right, MCSP-1, MCSP-2, MCSP-3 or MCSP-4.
  • Figures 8 A-D represent Epi-fluorescent microscopy of oral keratinocyte cells labeled with cell-specific peptides; fluorescent microscopy analysis of oral keratinocyte cells labeled with SWELYYPLRANL amino-acid sequence; EABA (Endogenous Actine Biotine Activity) suppressed cells without peptides;
  • Figure 8B represents black and white (BTW) image of the green channel of the fluorescent image;
  • Figure 8C represents grey color distribution in each particle in the BTW image;
  • Figure 8D represents histograms of the mean grey color distribution in BTW image.
  • Figures 9 A-D represent Epi-fluorescent microscopy of oral keratinocyte cells labeled with cell-specific peptides; fluorescent microscopy analysis of oral keratinocyte cells labeled with EWMIHYDSALTS amino-acid sequence;
  • Figure 9B represents black and white (BTW) image of the green channel of the fluorescent image;
  • Figure 9C represents grey color distribution in each particle in the BTW image;
  • Figure 9D represents: histograms of the mean grey color distribution in BTW image.
  • Figures 10 A-D represent Epi-fluorescent microscopy of oral keratinocyte cells labeled with cell-specific peptides; fluorescent microscopy analysis of oral keratinocyte cells labeled with the peptide having the SWTWHFPESPPP amino-acid sequence;
  • Figure 10 B represent a black and white (BTW) image of the green channel of the fluorescent image;
  • Figure 10 C represents a grey color distribution in each particle in the BTW image;
  • Figure 10 D represents histogram of the mean grey color distribution in BTW image.
  • Figures 11 A-D represent Epi-fluorescent microscopy of oral keratinocyte cells labeled with cell-specific peptides; fluorescent microscopy analysis of EABA suppressed cells in the absence of peptides;
  • Figure 11 B represents black and wait (BTW) image of the green channel of the fluorescent image;
  • Figure 11 C represents grey color distribution in each particle in the BTW image;
  • Figure 11 D represents histograms of the mean grey color distribution in BTW image.
  • Example 1 Selection and surface affinity characterization of metal binding peptides (MBP)
  • the medium was changed every two days until cells were used. For all experiments cells from 7 to 9 population number were used. After 90%> of cell confluence, the cells were detached with 0.05%> Trypsin-EDTA (Gibco®, Invitrogen, Carlsbad, CA, USA) for 5 min.
  • Phage display selection of surface binding peptides An Ml 3 bacteriophage library (PhD- 12 PD Peptide Library KitTM) supplied by New England Biolabs (Beverly, USA) in phosphate -buffered saline solution containing 0.1% TWEEN-20 (PBST; Sigma Aldrich) was exposed to Ti and T1 6 AI 4 V samples. After rocking for 1 h at room temperature, the Ti and Ti-alloy surfaces were thoroughly washed with PBST to rinse off unbound phages. Bound phages were then eluted from the surface under acidic conditions (glycine -HC1 pH 2.2, 10 min), which disrupt the interaction between the displayed peptide and the target.
  • acidic conditions glycine -HC1 pH 2.2, 10 min
  • target wells were changed to prevent elution of phages bound to the plastic walls.
  • Tris-HCL pH 9.1
  • the eluted phages were infected into the bacterial host strain Escherichia coli ER2738 and thereby amplified.
  • monoclonal phage populations were selected and analyzed individually. Finally, ten phages were selected and amplified from each sample, followed by the extraction of their DNA that will define the genetic code of the expressed peptide.
  • Samples were analyzed using a 4800 Plus MALDI-tandem time-of-flight system (MALDI-TOF/TOF) Proteomics Analyzer (Applied Biosystems, Foster City, USA) in positive reflector ion mode using a 20-kV acceleration voltage.
  • the YAG laser was operated at a 200-Hz firing rate with a wavelength of 355 nm.
  • Mass spectrometry spectra were acquired for each measure using 1500 laser shots. All acquired spectra of samples were processed using 4000 Series ExplorerTM software (Applied Biosystems, Foster City, USA) in default mode.
  • the peptide was identified by searching in the Swiss-Prot database using Protein Pilot TM 2.0 software (Applied Biosystems, Foster City, USA) or Protein Prospector.
  • the ExPASy database was used to calculate the monoisotopic theoretical mass of the peptide.
  • AFM measurements were carried out with an Asylum MFP-3D head and controller (Asylum Research, Santa Barbara, CA), mounted on an Olympus inverted microscope. Height images were recorded in tapping mode and in liquid at room temperature. Typically, 512 x 512 point scans were taken at a scan rate of 1 Hz per line. Both trace and retrace images were recorded and compared. Force Measurements by Atomic Force Microscopy. Relative binding strengths of peptides onto Ti and T1 6 AI 4 V surfaces were measured in contact mode and in a liquid medium (PBS pH 7.4) with a functionalized tip. Force measurements were taken at constant loading rates (vertical piezo-velocity of 1 ⁇ /s).
  • the spring constant of the tip was calibrated in the presence of PBS solution by the thermal fluctuation method and found to be about 18pN/nm.
  • the ultrasoft AFM cantilever tips Biolever-Olympus
  • tip functionalization was performed: the AFM cantilever tips were incubated in 1 ⁇ g/ml biotinylated bovine serum albumin (BSA; Sigma-Aldrich) solution in PBST, pH 7.0, at room temperature overnight, and then the tip was incubated for 30 min in 100 ⁇ g mL-1 streptavidin in PBST, and finally in BSA (1%) for 1 h to block the nonspecific binding sites.
  • BSA biotinylated bovine serum albumin
  • MCSP-2 functionalized surfaces were incubated in bovine serum albumin solution (lmg/ml in PBS) for 10 min. Single cell force spectroscopy measurement on a BSA treated surfaces was accomplished as previously described.
  • Confluent cells were washed with PBS and detached with 0.05% Trypsin-EDTA for 5 min at 37 C°. Cells were then resuspended in supplemented KSFM, and 5X105 cells per well was incubated with the un-functionalized and functionalized Ti and T1 6 AI 4 V surfaces. The samples were incubated at 37 C° under a 5% C02 humidified atmosphere for four hours.
  • the cells were washed three times with PBS and lysed with 500 ⁇ , of the acid phosphatase lysing buffer (0.1 M sodium acetate, 0.1% Triton X-100, pH 5.5), supplemented with 1 mg/mL of pNPP (para-ntropheny lphosphate, Sigma- Aldrich). After 1 h incubation at 37 C°, the reaction was stopped by the addition of 50 ⁇ ⁇ of 1 N NaOH (Sigma-Aldrich) for 30 min at room temperature. The yellow colorimetric reaction was measured by a microtiter plate reader (EL-800 Universal Microplate Reader, BioTec Instruments INC Vermont, US.) at 405 nm. To determinate the concentration of adherent cells, a a linear relationship was used between the percentage of adhering cells and the light absorption yellow-coloured solution due to para-nitrophenyl phosphate coloration.
  • the acid phosphatase lysing buffer 0.1 M sodium acetate, 0.1% Triton
  • HPAWALGKLNVE (SEQ ID N°14) 12,5 % 0 %
  • MBP-1 peptide has a great potential as a linker to functionalize metallic surfaces if specificity is not a key factor.
  • 6 bacteriophage clones (every clone have has been amplified separately and a bank of 6 bacteriophages was then created by mixing the clones) were used for the fourth biopanning round.
  • the WDPPTLKRPVSP sequence (MBP-2) was expressed by 40% of the phages.
  • MBP-1 and MBP-2 were further chosen as the metal binding part of the bi-functional peptides.
  • Table 4 The obtained value of mass-to-charge ratio (m/z) for MBP-1 was 1239.6 in agreement with the theoretical mass (table 3) and on both metals was identified a small peak at m/z of 1255.6 corresponding to the oxidized form of the peptide.
  • Table 5 presents the physico-chemical characteristics of the peptides.
  • the sequence of the MBP-1 peptide presents a hydrophobic first half and a hydrophilic second half.
  • the adhesion resists also to high ionic strength solvent indicating that electrostatic interactions are not critical.
  • the obtained value of mass-to-charge ratio (m/z) for MBP-2 was 1391.6 and correspond to the theoretical [M + H] + mass ( Figures 1C and ID).
  • the MBP-2 peptide contains two positively charged (K, R) amino acids and one negatively charged (D) amino acid, hence his total charge is twofold lower than the charge of MBP-1 (table 3). This may explain its lower electrostatic interaction with the negatively charged metal surfaces indicated by its rinsing with 1M NaCl.
  • the mass-spectrometry spectra of MBP-2 resisting to acetonitrile rinsing reveal two peaks at m/z 1406 and m/z 1423.6 suggesting the presence of oxygen ions in a peptide structure.
  • MBP-1 and MBP-2 affinity to the Ti metals force spectroscopy study was conducted by AFM. Monitoring unbinding processes of adsorbed molecules under external stress leads to quantification of adhesion forces.
  • the adhesion forces measured between MBPs and Ti and T1 6 AI 4 V surfaces are presented in Figures 2A and 2B.
  • MBP-1 adhesion force on T1 6 AI 4 V and Ti surfaces are 67.07 ⁇ 1.34 pN and 65.42 ⁇ 2.48 pN, respectively.
  • MBP-2 adhesion force on both surfaces are 109.76 ⁇ 2.62 pN and 134.61 ⁇ 2.65 pN.
  • MBP-2 peptide reveals statistically higher adhesion forces to both metals.
  • Adhesion of MBP-2 on T1 6 AI 4 V is almost twofold stronger than the adhesion force of MBP-1.
  • the strength of osteopontine and ⁇ 3 integrin bonds is 50 ⁇ 2 pN
  • ICAM-1 and Anti-ICAM-1 antibody bond is 100 ⁇ 50 pN30
  • cadherin- mediated cell-cell interaction has a minimal binding force in the range of 50 pN.
  • the highly specific and strongest non-covalent bond is the biotin-streptavidin interaction ranging from 250 to 320 pN according to the experimental conditions. Therefore, it can be concluded that the interactions of MBP-1 and MBP-2 with the two metal surfaces are in the range of antigen-antibody forces.
  • MCSP Metal-cell specific peptides
  • SCFS Single cell adhesion spectroscopy
  • the inset shows an image of a keratinocyte cell adsorbed on the tip-less AFM cantilever. Images of the cantilever were always recorded before and after each set of measurements and compared. No significant difference was observed between them. Scale bar represents 18 ⁇ .
  • the relatively short contact time of 5 s between cell and substrate means that no information is recorded about how the cell responds to the surface on a longer time scale, thus excluding the effects of, for example of changes in protein expression and limiting our study to the analysis of proteins and protein complexes that are already present at the cell membrane as well as to the study of mechanical properties of the cell.
  • the cell adhesion forces were registered towards the bare Ti surface and after its functionalization with the peptides (Figure 4A).
  • MCSP-2 and MCSP-4 functionalized surfaces presented higher cell adhesion force than MCSP-1 and MCSP-3 peptides.
  • Adhesion force values are significantly higher for MCSP-2 and MCSP-4 on both surfaces ( Figures 4A and 4B).
  • the MCSP-1 and MCSP-3 peptides have a common cell specific peptide CSP-2 and different MBP's, but they did not demonstrate the same comportment prior to cell adhesion force to metal surfaces.
  • the combination of MBP-1 and CPS-2 (cadherin binding peptide) in MCSP-2 appears to be the most successful peptide configuration for Ti functionalization to promote oral epithelial cell adhesion.
  • the measured cell adhesion forces are of the same order of magnitude as in previous works, however it should be stressed that measured forces are strongly depending on experimental conditions (contact time, loading rate).
  • Mesendodermal cells adhesion forces on substrates coated with fibronectin range from 0.198 - 0.405 nN, for contact times of 1- 5 s and maximal loading forces of 400 pN37.
  • Adhesion forces of Chinese hamster ovary (CHO) cells against collagen matrix, using a maximum loading force of InN and short contact times (5-10 s) are around several hundred pico-newtons.
  • Single cell adhesion forces between murin calvaria pre -osteoblast cells and partially denatured collagen (free RGD - motif) are close to 1 nN after a contact time of 5 s using a loading force of InN.
  • CHO Chinese hamster ovary
  • AFM height images recorded in tapping mode demonstrates the following: Bare Ti surface Rms: 5.4 nm, Ti/ MCSP-2 surface Rms: 21.3 nm, Bare Ti 6 Al 4 V surface Rms: 6.02nm and Ti 6 Al 4 V /MCSP-2 surface Rms: 13.8 nm.
  • the RMS of bare Ti 6 Al 4 V surface increased from 6.0 nm to 13.8 nm after absorption of MCSP-2.
  • RMS increased also for MCSP-3 functionalized Ti surface.
  • AFM height images of metal surfaces after functionalization with MCSP-2 (Figure 4C for Ti; Figure 4D for ⁇ 6 ⁇ 14) demonstrated similar peptide deposition patterns with agglomerates of about 200 nm. Increasing of surface roughness after MCSP-2- functionalization could explain its higher adhesion performances over other peptides.
  • BSA bovine serum albumin
  • the number of adherent living cells was compared on functionalized and non- functionalized Ti metal surfaces, after four-hour cell incubation, by means of a colorimetric para-nitrophenil phosphate cell viability test (pNPP).
  • pNPP colorimetric para-nitrophenil phosphate cell viability test
  • This methodology can be also applied for testing the cell-adhesion or more precisely the mechanical resistance of adherent cell to rinsing procedure, on different surfaces. After eliminating non-adherent cells with rinsing, cells attached to the surface were subjected to the acid phosphatase viability test.
  • These pNPP tests complemented the SCFS measurements where was tested the force between one cell and five different positions on the surface, to obtain information about a great number of adherent cells on the whole surface of the implant, four hours after incubation.
  • CAR cell adhesion recognition
  • the (CAR) sequence was presented in WO2006/116737 and is defined by the following formula: W-X-L/I/V/F/M/A, wherein X is an amino acid selected from E, T, Y, Q, M, F, D, or L and wherein L/I/V/F/M/A is Leu or He or Val or Phe or Met or Ala.
  • This sequence according to the above formula is selected from the group consisting of WEL, WTL, WYI, WEF, WQM, WEV, WTV, WYV, WQV, WTM, WQF, WTF, WMI, WDF, WTI, WFI, WQL, WEI, and WLA.
  • the peptide SWTWHFPESPPP was described like less soluble than SWELYYPLRANL in aqueous solutions (Devemy and Blaschuk, 2009) and in the same time the place of (CAR) sequence in SWTWHFPESPPP was changed.
  • the (-WHF-) sequence was not presented like cell adhesion recognition sequence, although its responds to the presented formula.
  • the three peptides were applied with a) cell line of non-tumoral, immortalized oral keratinocyte cells, TERT-2 OKF-6 (BWH Cell Culture and Microscopy Core, USA). Cells lines described in WO 2006/116737 (MCF-7 and SKOV-3) are carcinoma cell lines.
  • MCF-7 cells are a carcinoma cells isolated from human breast adenocarcinoma with estrogen and cytokeratin sensitivities.
  • SKOV-3 cells are a human ovary cancer cells with epithelial-like morphology.
  • oral keratinocyte cells TERT-2 OKF-6 are immortalized human epithelial cells with origin from oral mucosa (Dickson et al.).
  • Our aim was to find an adequate amino acid sequence for specific recognition of oral keratmocyte cell surface. Subsequently this sequence will be used in the construction of bi-functional peptides to link keratmocyte cells and titanium.
  • Biotin product number: B4639
  • FITC labeled Avidin product number: A2050
  • Sigma Aldrich Sigma Aldrich, St. Louis, USA
  • Cells from non- tumoural, immortalized oral keratmocyte cell line, TERT-2 OKF-6 (BWH Cell Culture and Microscopy Core, USA) were used for all experiment.
  • the protocol contains the following steps: 1) PBS incubation and wash x 10 min; 2) Avidin incubation x 20 min; 3) PBS incubation and wash x 5 min; 4) Biotin incubation x 20 min; 5) PBS incubation and wash x 5 min.
  • concentrations of Avidin ranging from 0.1-0.01% and biotin ranging from 0.01-0.001% were adequate for use in EABA suppression.
  • After calibration we used a PBS solution for the Avidin with concentration 200 ⁇ g/ml and for the biotin 20 ⁇ g/ml. 6) Incubation with biotinilated peptide x 30 min in concentration 500 ⁇ g/ml.
  • Epi-fluorescent microscopy was realized with an Eclipse TE2000-E (Nikon) Microscope.
  • Image analysis The analysis of fluorescent images was realized by Image J sofwere (NIH, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/). The aim was to measure the degree of fluorescence by measuring the degree of the grey color into the images. Using the function "color - split channels” we isolated the green channel of each image. Each green channel image was transformed in black and wait 8 bit image using the function threshold - BTW (image - adjust - threshold - BTW). The histograms for the grey color distribution was created with the function "analyze -histogram". To illustrate the grey color distribution of each particle into the image we use the function "analyze - image plot". Results and discussion:

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Transplantation (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Dermatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Vascular Medicine (AREA)
  • Biochemistry (AREA)
  • Surgery (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Inorganic Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne en premier lieu un peptide bifonctionnel caractérisé en ce qu'il comprend un domaine de peptide de liaison à un métal (MBP) et un domaine de peptide spécifique à une cellule (CSP), lesdits domaines de MBP et de CSP étant liés par un lieur. Un peptide bifonctionnel selon la présente invention est apte à se lier à la fois au métal d'un composé biomatériau et à des cellules, améliorant ainsi l'adhérence cellulaire dudit composé de biomatériau. La présente invention concerne en outre une composition comprenant ledit peptide bifonctionnel. L'invention porte par ailleurs sur un composé biomatériau associé audit peptide bifonctionnel, en particulier un implant dentaire fonctionnalisé par l'adsorption sur sa surface d'un peptide bifonctionnel de l'invention.
PCT/EP2015/053724 2014-02-21 2015-02-23 Composé peptidique bifonctionnel pour adhérence cellulaire WO2015124767A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP14305242.1 2014-02-21
EP14305242 2014-02-21

Publications (1)

Publication Number Publication Date
WO2015124767A1 true WO2015124767A1 (fr) 2015-08-27

Family

ID=50236128

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/053724 WO2015124767A1 (fr) 2014-02-21 2015-02-23 Composé peptidique bifonctionnel pour adhérence cellulaire

Country Status (1)

Country Link
WO (1) WO2015124767A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2627455C1 (ru) * 2016-02-01 2017-08-08 Федеральное государственное автономное образовательное учреждение высшего образования "Белгородский государственный национальный исследовательский университет" (НИУ "БелГУ") Способ изготовления биомеханического сенсора для измерения сил адгезии в системе "клетка-клетка"

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006116737A2 (fr) 2005-04-28 2006-11-02 Mcgill University Composes et methodes permettant de moduler les processus medies par la cadherine
JP2009007339A (ja) 2007-05-30 2009-01-15 Sanyo Chem Ind Ltd 歯科用インプラント用接着剤
WO2010094212A2 (fr) 2009-02-19 2010-08-26 Versitech Limited Surface antibactérienne et son procédé d'obtention
US7897163B2 (en) 2004-03-19 2011-03-01 Seoul National University Industry Foundation Bone graft and scaffolding materials immobilized with osteogenesis enhancing peptides on the surface
EP2385057A1 (fr) 2010-05-05 2011-11-09 Centre National de la Recherche Scientifique Dérivés de peptides pour la biofonctionnalisation de substrats de silicium et leurs applications

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7897163B2 (en) 2004-03-19 2011-03-01 Seoul National University Industry Foundation Bone graft and scaffolding materials immobilized with osteogenesis enhancing peptides on the surface
WO2006116737A2 (fr) 2005-04-28 2006-11-02 Mcgill University Composes et methodes permettant de moduler les processus medies par la cadherine
JP2009007339A (ja) 2007-05-30 2009-01-15 Sanyo Chem Ind Ltd 歯科用インプラント用接着剤
WO2010094212A2 (fr) 2009-02-19 2010-08-26 Versitech Limited Surface antibactérienne et son procédé d'obtention
EP2385057A1 (fr) 2010-05-05 2011-11-09 Centre National de la Recherche Scientifique Dérivés de peptides pour la biofonctionnalisation de substrats de silicium et leurs applications

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
BOUDOU T. ET AL., ADV. MATER., vol. 22, 2010, pages 441 - 467
DEVEMY; BLASCHUK, PEPTIDES, vol. 30, 2009, pages 1539 - 1547
DICKSON, M. A ET AL., MOLECULAR AND CELLULAR BIOLOGY, vol. 20, no. 4, 2000, pages 1436 - 47
ELIAS ESTEPHAN ET AL: "SVSVGMKPSPRP: a broad range adhesion peptide Background", BIOMED TECH, 1 January 2012 (2012-01-01), pages 481 - 489, XP055126046, Retrieved from the Internet <URL:http://www.degruyter.com/dg/viewarticle.fullcontentlink:pdfeventlink/$002fj$002fbmte.2012.57.issue-6$002fbmt-2011-0109$002fbmt-2011-0109.pdf?t:ac=j$002fbmte.2012.57.issue-6$002fbmt-2011-0109$002fbmt-2011-0109.xml> [retrieved on 20140630], DOI: 10.1515/bmt-2011-0109 *
ESTEPHAN E. ET AL., BIOMED TECH (BERL, vol. 57, no. 6, December 2012 (2012-12-01), pages 481 - 9
GUILLAUME VIDAL ET AL: "Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides", ACTA BIOMATERIALIA, vol. 9, no. 1, 1 January 2013 (2013-01-01), pages 4935 - 4943, XP055126102, ISSN: 1742-7061, DOI: 10.1016/j.actbio.2012.09.003 *
H. YAZICI ET AL: "Biological response on a titanium implant-grade surface functionalized with modular peptides", ACTA BIOMATERIALIA, vol. 9, no. 2, 1 February 2013 (2013-02-01), pages 5341 - 5352, XP055126091, ISSN: 1742-7061, DOI: 10.1016/j.actbio.2012.11.004 *
IVAN PANAYOTOV ET AL: "Bi-functionnal Pepides to Promote Epithelial Sealing on Ti and Ti6Al4V", BIOENGINEERING CONFERENCE (NEBEC), 2013 39TH ANNUAL NORTHEAST, IEEE, 5 April 2013 (2013-04-05), pages 319 - 320, XP032447991, ISBN: 978-1-4673-4928-4, DOI: 10.1109/NEBEC.2013.170 *
MEYER S. ET AL., ADV. MATER., vol. 19, 2007, pages 2492 - 2498
MEYER S. ET AL., BIOMACROMOLECULES, vol. 12, 2011, pages 533 - 539
MEYER S. ET AL., BIOMATERIALS, vol. 30, 2009, pages 277 - 286
MEYER S. ET AL., CHEMMEDCHEM, vol. 3, 2008, pages 1645 - 1648
MEYERS S R ET AL: "Endothelialization of titanium surfaces", ADVANCED MATERIALS, WILEY - V C H VERLAG GMBH & CO. KGAA, DE, vol. 19, no. 18, 2 August 2007 (2007-08-02), pages 2492 - 2498, XP002528796, ISSN: 0935-9648, DOI: 10.1002/ADMA.200700029 *
WOOD, G. S. ET AL., THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY: OFFICIAL JOURNAL OF THE HISTOCHEMISTRY SOCIETY, vol. 29, no. 10, 1981, pages 1196 - 204
YAZICI H. ET AL., ACTA BIOMATER., vol. 9, no. 2, February 2013 (2013-02-01), pages 5341 - 52

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2627455C1 (ru) * 2016-02-01 2017-08-08 Федеральное государственное автономное образовательное учреждение высшего образования "Белгородский государственный национальный исследовательский университет" (НИУ "БелГУ") Способ изготовления биомеханического сенсора для измерения сил адгезии в системе "клетка-клетка"

Similar Documents

Publication Publication Date Title
Khatayevich et al. Biofunctionalization of materials for implants using engineered peptides
Chen et al. Surface biofunctionalization by covalent co-immobilization of oligopeptides
AU2002367566B2 (en) Interfacial biomaterials
US7972615B2 (en) Peptide compositions for coating metal medical devices with vancomycin
Khoo et al. Staphylococcus aureus resistance on titanium coated with multivalent PEGylated-peptides
NZ560649A (en) Angiogenic heparin binding peptide amphiphiles, peptide amphiphiles, self-assembled composition and related methods of use
CN103096932A (zh) 细胞穿透肽及其用途
JP6856618B2 (ja) 接着性ペプチド
US20110129611A1 (en) Decorating hydroxyapatite biomaterials with modular biologically active molecules
Fischer et al. Controlling cell adhesion to surfaces via associating bioactive triblock proteins
AU2007205023A1 (en) Compositions and methods for promoting attachment of cells of endothelial cell lineage to medical devices
KR102053895B1 (ko) 세포의 부착, 증식과 분화를 촉진하는 신규 펩티드 fnin3 및 이의 용도
US7531505B2 (en) Compositions and methods for promoting attachment of cells of endothelial cell lineage to medical devices
JP2018527404A (ja) 環状ｒｇｄ細胞結合モチーフ及びその使用
Herranz-Diez et al. Bioactive compounds immobilized on Ti and TiNbHf: AFM-based investigations of biofunctionalization efficiency and cell adhesion
US10851132B2 (en) Protein based adhesive composition and method
WO2015124767A1 (fr) Composé peptidique bifonctionnel pour adhérence cellulaire
JP4406013B2 (ja) 細胞の接着・伸展を促進するペプチド、その断片及びその誘導体
EP3349811B1 (fr) Peptide pour le revetement de surfaces
KR102010871B1 (ko) 세포접착용 조성물 및 방법
Najjar et al. An L-to D-amino acid conversion in the cell penetrating peptide dfTAT influences proteolytic stability, endocytic uptake, and endosomal escape
KR101794482B1 (ko) 생체 또는 비생체 접착성 펩타이드 및 그 용도
CN101998860B (zh) 用于将糖肽抗生素递送至医学装置表面的组合物和方法
Zehbe et al. Anodic cell-protein deposition on inverse inkjet printed micro structured gold surfaces

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15710724

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15710724

Country of ref document: EP

Kind code of ref document: A1