Classifications

• • 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
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F122/00—Homopolymers of compounds having one or more unsaturated aliphatic radicals each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides or nitriles thereof
  • C08F122/36—Amides or imides
  • C08F122/38—Amides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa

Definitions

• the present invention relates to a bioactive amino acid sequence and its use in a functional peptide, in particular to promote cell adhesion, cell growth and/or cell differentiation.
• Bioactive or biologically active peptide (or amino acid) sequences are known in the art. Bioactive sequences may be derived from any of a diverse range of naturally occurring proteins and peptides including ECM components, cell adhesion molecules, cell surface receptors, growth factors, cytokines, chemokines, etc.
• the -RGD- sequence is a prototypic cell recognition sequence found in fibronectin and well known to be recognized by integrins and to mediate cell attachment. The integrin-mediated cell attachment influences and regulates cell migration, growth, differentiation, and apoptosis.
• the purpose of the present invention is to provide a bioactive amino acid sequence that has particularly advantageous properties to promote cell adhesion, cell growth and/or cell differentiation.
• the present invention therefore relates to the use of the amino acid sequence Har-Gly-Asp (hRGD) as a bioactive sequence in a functional peptide to promote cell adhesion, cell growth and/or cell differentiation.
• hRGD amino acid sequence Har-Gly-Asp
• this amino acid sequence exhibits mainly improved cell adhesion, and cell growth.
• the present amino acid sequence shows a longer half-life time, in particular via a stronger resistance towards enzymatic degradation.
• the present amino acid sequence also shows higher affinity towards specific cell lines.
• biological sequence or “biologically active sequence” is intended an amino acid sequence which has a specific biological function, here the promotion of cell adhesion (or cell attachment), cell growth and/or cell differentiation (or induction of a cellular phenotype).
• An example cell differentiation is the transformation of pluri or omni potent cells, for example stem cells, into dedicated cell types, such as bone cells, muscle cells, insulin secreting cells etc.
• amino acid As used herein, the term "amino acid” (Xaa) is intended to denote any compound comprising at least one RiR 2 group, preferably H 2 group, and at least one carboxyl group.
• the amino acids of this invention can be natural amino acids or non-natural amino acids, naturally occurring or synthetic.
• the natural amino acids, with exception of glycine, contain a chiral carbon atom. Unless otherwise specifically indicated, the compounds containing natural amino acids with the L-configuration are preferred.
• the aminoacids can be selected from, for example ⁇ -alanine (PAla), ⁇ -aminobutyric acid (GABA), 5- aminovaleric acid, glycine (Gly or G ), phenylglycine, arginine (Arg or R), homoarginine (Har or hR), alanine (Ala or A), valine (Val or V), norvaline, leucine (Leu or L), norleucine (Nle), isoleucine (He or I), serine (Ser or S), isoserine, homoserine (Hse), threonine (Thr or T), allothreonine, methionine (Met or M), ethionine, glutamic acid (Glu or E), aspartic acid (Asp or D), asparagine (Asn or N), cysteine (Cys or C), cystine, phenylalanine, tyrosine (Tyr or Y), tryptophan (
• peptide comprises peptides and peptide analogous.
• Peptide analogous comprise natural amino acids and non-natural amino acids. They can also comprise modifications such as glycosylations. All amino acids can be either the L- or D- isomer.
• the peptides or peptide analogues can also comprise amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
• the peptides may also be formed from amino acids analogues that have modified R groups or modified peptide backbones.
• Peptide analogues usually include at least one bond in the peptide sequence which is different from an amide bond, such as urethane, urea, ester or thioester bond.
• Peptides or peptide analogues according to the present invention can be linear, cyclic or branched and are preferably linear.
• “functional peptide moiety” is intended a peptide moiety comprising a bioactive sequence, thus a peptide moiety exhibiting a biological activity.
• the amino acid sequence Har-Gly-Asp (hRGD) can represent in itself the bioactive sequence of a functional peptide moiety, promoting cell adhesion, cell growth and/or cell differentiation.
• the amino acid sequence Har-Gly-Asp can also be part of a longer bioactive sequence of a functional peptide moiety, promoting cell adhesion, cell growth and/or cell differentiation.
• the hRGD sequence may comprise additional amino acids covalently bound to its N-terminus ( l3 ⁇ 4).
• the bioactive sequence may for instance be selected from (Xaa) n -hRGD sequences wherein Xaa is any natural or unnatural amino acid and n is 1 to 10.
• the n Xaa amino acids may be the same or different. Suitable examples of such sequences are GhRGD, YhRGD, YGhRGD, GGGGhRGD, pAla-hRGD, GABA-hRGD and 6-aminovalericamide-hRGD.
• the hRGD sequence may comprise additional amino acids covalently bound to its C-terminus (COOH).
• the bioactive sequence may for example be selected from hRGD-(Xaa) m sequences wherein Xaa is any natural or unnatural amino acid and m is 1 to 10.
• the m Xaa amino acids may be the same or different.
• Suitable examples of such sequences are hRGDS, hRGDY, hRGDF, hRGDK, hRGDV, hRGDT hRGDWP, hRGDYK, hRGDFK, hRGDSP, hRGDSPK, hRGDSY, hRGD P, hRGDTP, and hRGDSP, in particular hRGDWP.
• the first and second aspects as described above may be combined, the hRGD sequence comprising additional amino acids covalently bound to both its N- and C-terminus, i.e. (Xaa) n -hRGD- (Xaa)m where Xaa is any natural or unnatural amino acid, n is 1 to 10, and m is 1 to 10.
• Such sequences may for instance be selected from the group consisting of GhRGD S, GhRGDY, GhRGDF, YGhRGD, GhRGD SY, GhRGD SP,
• the present invention therefore also relates to the use of a bioactive sequence in a functional peptide to promote cell adhesion, cell growth and/or cell differentiation, wherein the bioactive sequence is selected from the group consisting of GhRGD, YhRGD, YGhRGD, GGGGhRGD, pAla-hRGD, and GABA-hRGD, 6-aminovalericamide-hRGD, hRGDS, hRGDY, hRGDF, hRGDK, hRGDV, hRGDT, hRGDWP, hRGDFK, hRGDYK, hRGDSP, hRGDSPK, hRGDSY, hRGD P, hRGDTP, and hRGDSP, GhRGDS,
• GhRGDY GhRGDF
• GhRGDS Y GhRGDSP
• GhRGDSPK GhRGDSPK
• GhRGDTP GhRGDSPK, GhRGDSP, GhRGDK, GGGGhRGDS, GhRGDNP, and combinations thereof; in particular hRGDS, GhRGDS, GhRGDSY, Ala- hRGD, GABA-hRGD, 6-aminovalericamide-hRGD, and hRGDWP; more particularly Ala-hRGD, GABA-hRGD, 6-aminovalericamide-hRGD, and hRGDWP; most preferably hRGDWP.
• the hRGD sequence can also be linked to mercaptopropionic acid (Mpr) on its N-terminus.
• Mpr mercaptopropionic acid
• Such sequences can read Mpr-(Xaa) n -hRGD-(Xaa) m wherein Xaa is any natural or unnatural amino acid selected independently from one another, n is 0 to 10, and m is 0 to 10.
• two mercaptopropionic acid moieties may be covalently bound together, in particular via a disulfur bond.
• Such sequences typically read (Xaa) m -DGhR-(Xaa) n -Mpr-Mpr-(Xaa) n '-hRGD-(Xaa) m ' wherein (Xaa) is any natural or unnatural amino acid selected independently from one another, n and n' range independently from 0 to 10, and m and m' range independently from 0 to 10.
• the functional peptide moiety can correspond to the bioactive sequence comprising the hRGD sequence.
• the functional peptide moiety can also comprise additional amino acids, further to the bioactive sequence comprising the hRGD sequence.
• the functional peptide moiety comprises at least one or more hRGD sequences, such as two, three, four, five, six, seven, eight, nine or ten hRGD sequences, preferably at least one or more hRGDWP sequences, such as two, three, four, five, six, seven, eight, nine or ten hRGDWP sequences.
• the functional peptide moiety may comprise additional amino acids before, after or between bioactive sequences as defined above.
• hRGDWP sequences and their derivatives provide the advantage of mimicking cell adhesion proteins in the extracellular matrix and subsequently can bind integrin proteins on the cell surface.
• the functional peptide moiety comprising the bioactive amino acid sequence Har-Gly-Asp (hRGD) is covalently bound to a self-assembling peptide moiety.
• self-assembling peptide is intended a peptide able to self-assemble, i.e. a peptide defining a domain that folds into a specifically defined
• Self-assembling amino acid sequences are known in the art and, according to the present invention, peptide sequences capable of assembling into a ⁇ -sheet, a coiled coil a-helix structure, a peptide triple helix structure, or combinations thereof are preferred.
• a peptide moiety that is capable of self-assembly into a coiled coil structure is, for example, a peptide amino acid sequence providing an a-helical coiled coil structure. This is a tertiary structure which depends on the
• the peptide moiety of this embodiment comprises a variety of hydrophobic and polar residues, and is usually composed of at least 10 amino acids.
• the helix peptide moiety is designed to have all the polar residues on one face of the helix and all the hydrophobic residues on the other side of the helix.
• This helix can form part of two or more helix chains and form a coiled coil structure.
• the helices are associated together through hydrophobic interaction and form a coiled coil.
• the sequence of the peptide moiety can for example be a leucine zipper sequence.
• Peptide moieties capable of self-assembling into a ⁇ -sheet provide ⁇ -sheet stabilized by inter-molecular hydrogen bonding perpendicular to the peptide chain.
• the self-assembling occurs through hydrogen bond interactions between beta strands.
• the beta strand is a stretch of polypeptide chain with a backbone in an almost fully extended conformation.
• the ⁇ -sheet structure can be formed either from parallel or anti-parallel ⁇ -strands.
• An example of a ⁇ -sheet according to this embodiment is a peptide moiety that is able to self-assemble in an amyloid-like structure.
• Peptide moieties capable of self-assembling into a ⁇ -sheet comprise typically at least 5 or 6 amino acids.
• the peptide moiety that is able to self-assemble can form a hydrogel when the peptide is provided in suitable conditions.
• Hydrogels are three- dimensional networks of hydrophilic compounds, usually polymers, which have the ability to imbibe a large quantity of water and biological fluids. The network may be formed through either chemical crosslinking (covalent, ionic) or physical crosslinking (entanglements, crystallites, hydrogen bonds).
• hydrogels are three-dimensional structures capable of comprising at least 20wt% water in relation to the weight of the gel. Absorption of water by a hydrogel gel results in a significant increase of its dimensions, i.e. a significant swelling.
• the peptide moiety that is able to self-assemble into a ⁇ -sheet is an octapeptide moiety comprising alternating hydrophobic and charged amino acids.
• Hydrophobic amino acids are often selected from the group consisting of Phenylalanine (Phe or F), Tryptophan (Trp or W), Tyrosine (Tyr or Y),
• Isoleucine He or I
• Alanine Al or A
• Leucine Leu or L
• Valine Val or V
• Norleucine Nle
• Phenylalanine Phe or F
• Tryptophan Trp or W
• Tyrosine Tyr or Y
• Isoleucine He or I
• Norleucine Nle
• Charged amino acids are usually selected from the group consisting of Arginine (Arg or R), Aspartic acid (Asp or D), Glutamic acid (Glu or E), Lysine (Lys or K), and Histidine (His or H); particularly from Arginine (Arg or R), Aspartic acid (Asp or D), Glutamic acid (Glu or E), and Lysine (Lys or K).
• the octapeptide moiety might for instance be selected from the group consisting of FEFKFEFK, FEFEFKFK, FDFKFDFK, FDFDFKFK, FEFRFEFR, FEFEFRFR, YDYKYDYK, YDYDYKYK, YEYRYEYR, YEYKYEYK, YEYEYKYK, WEWKWEWK, WEWEWKWK, WDWKWDWK,
• WDWDWKWK WDWDWKWK.
• amino sequences are FEFKFEFK or FEFEFKFK.
• the functional peptide moiety comprising the bioactive amino acid sequence Har-Gly-Asp is covalently bound to a polymer, in particular to a biocompatible polymer, more particularly to a thermo-responsive polymer.
• biocompatible polymer is intended a polymer which is compatible with living organisms. Suitable examples are polyesters like polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), polyhydroxyalkanoates like poly(hydroxyl butyrate-co-valerate) (PHBV), polycaprolactones, polyacrylamides like polyhydroxyacrylamide (pHPMA), polyanhydrides, polyimide, polyanhydride-co-imide, and polysaccharides like chitosan.
• PLA polylactic acid
• PGA polyglycolic acid
• PLGA poly(lactic-co-glycolic acid)
• PHBV polyhydroxyalkanoates
• PHBV poly(hydroxyl butyrate-co-valerate)
• pHPMA polyhydroxyacrylamide
• polyanhydrides polyimide
• polyanhydride-co-imide polysaccharides like chitosan.
• thermo-responsive polymer is intended a polymer which undergoes a physical change, such as conformational change, when exposed to external thermal stimuli such as an increase, or decrease, in temperature.
• the ability of thermo-responsive polymers to undergo physical changes in response to thermal stimuli classifies these polymers in the art in the category of smart materials. Contrary to the behavior of most polymers in aqueous solutions, thermal- responsive polymers become less soluble, or more hydrophobic, in water at elevated temperatures.
• the temperature providing the above phase transition from soluble to insoluble is designated in the art as the lower critical solution temperature (LCST), generally determined in deionized water at a neutral pH.
• LCST lower critical solution temperature
• thermo-responsive polymer is poly(N- isopropyl aery 1 amide) or PNIPAAm which LCST has been determined to be approximately 32 °C.
• Other especially suitable thermo-responsive polymers are PNIPAAm copolymers.
• PNIPAAm copolymers are copolymers comprising NIPAAm and at least one other monomer, the other monomer being selected from hydrophilic or hydrophobic monomers.
• Suitable hydrophilic monomers are for instance as N-methacryloyl-tris(hydroxymethyl)methylamide, hydroxyethyl acrylamide, hydroxypropyl methacrylamide (HPMA), N-aciylamido-1- deoxysorbitol, hydroxyl-ethylmethacrylate, hydroxypropyl actry late,
• HPMA hydroxypropylmethacrylamide
• Suitable hydrophobic monomers are derived from acrylamide monomers in which the amine nitrogen of the amide group is substituted with one or more alkyl residues, for example N-isopropylacrylamide, N,N-dimethylacrylamide, N,N- diethyl(meth)acrylamide, N-methyl methacrylamide, N-ethylmethacrylamide, N- propyl acrylamide, N-butylacrylamide, N-octyl (meth)acrylamide, N- dodecylmethacrylamide, N-octadecylacrylamide, propyl(meth)acrylate, decyl(meth)acrylate, stearyl(meth)acrylate, octyl-triphenylmethylacrylamide, butyl-triphenylmethylacrylamide, octadedcyl-triphenylmethylacrylamide, phenyl-triphenylmethylacrylamide, benzyl-triphenylmethylacrylamide,
• Thermo-responsive polymers can be used for the preparation of hydrogels and more particularly for the preparation of smart hydrogels, i.e. environmental sensitive hydrogels. These smart hydrogels can undergo a reversible volume change in response to environmental stimuli such as pressure, pH, temperature or ionic strength making them especially suitable to be used in biomedical and pharmaceutical fields, in particular in the field of cell and tissue culturing.
• the functional peptide moiety comprising the bioactive amino acid sequence Har-Gly-Asp (hRGD) is preferably covalently bound to the polymer through a linkage, preferably selected from the group consisting of thioether, amino, amido, ester and ether linkage.
• linking groups can be directly, i.e. a covalent bond between the atoms of the polymer and the atoms of the functional peptide, or indirectly, i.e., through a linking group.
• Suitable examples of linking groups are among others linear or branched alkanes, especially polymethylene group comprising 1 to 10 carbon atoms.
• Other examples of linking groups are for instance polyether groups, such as polyethylene glycol (PEG).
• the functional peptide moiety comprising the hRGD sequence is covalently bound to both a self-assembling peptide and a polymer, in particular to both a self-assembling peptide and a biocompatible polymer, more particularly to both a self-assembling peptide and a thermo-responsive polymer.
• the present invention also relates to the use of the amino acid sequence Har-Gly-Asp (hRGD) as a bioactive sequence in a functional peptide to promote cell adhesion, cell growth and/or cell
• hydrogel differentiation in the preparation of hydrogels, preferably hydrogels for cell culture.
• hydrogels comprising the hRGD sequence, especially hydrogels wherein the hRGD sequence is part of the hydrogel scaffold.
• hydrogel scaffold is meant the material leading to the hydrogel structure, i.e. a physical entity comprising a polymer or a self- assembling peptide that will give rise to the hydrogel structure.
• the hRGD sequence as defined above preferably promotes cell adhesion, cell growth and/or cell differentiation.
• the hydrogels of the present invention further comprise a self-assembling peptide moiety and/or a polymer as defined above.
• the hRGD sequence is covalently bound to at least one of the self-assembling peptide moiety or polymer as defined above, optionally via a linkage and/or an additional peptide sequence.
• the present invention therefore also relates to a hydrogel comprising the hRGD sequence and a self-assembling peptide moiety and/or a polymer, in particular a self-assembling peptide moiety and/or a biocompatible polymer, more particularly a self-assembling peptide moiety and/or a thermo-responsive polymer.
• the present hydrogels are especially suitable for cell and tissue culture, providing, for example, improved cell adhesion and cell growth.
• the present hydrogel can be used for example for culturing cells, preferably fibroblast cells, chondrocyte cells or stem cells, or for tissue engineering.
• the present hydrogels may be used in 2- or 3-dimensional cell culture systems.
• Figure 1 optical micrographs of hydrogels based on octapeptide, hRGD and RGD modified octapeptides after 1, 3 and 7 days.
• Figure 2 fluorescence optical micrographs of hydrogels based on octapeptide, hRGD and RGD modified octapeptides after 1, 3 and 11 days.
• Figure 3 optical micrographs of hydrogels based on octapeptide, hRGD and RGD modified octapeptides after 1 day.
• Figure 4 fluorescence optical micrographs of hydrogels based on octapeptide, hRGD and RGD modified octapeptides after 1 and 3 days.
• Figure 5 cell number after 0, 1, 2, 3, 5, 7, 10 and 14 days in hydrogels based on octapeptide, RGD and hRGD modified octapeptides.
• (h)RGD means that RGD, hRGD or a mixture thereof can be used.
• Octapeptide Phe-Glu-Phe-Lys-Phe-Glu-Phe-Lys can be synthesized as disclosed in A. Maslovskis et al., Macromol. Symp., 296, 248-253 (2010), on a ChemTech ACT 90 peptide synthesizer using N-methyl-2- pyrrolidone (NMP) as solvent, and standard solid phase peptide protocols.
• NMP N-methyl-2- pyrrolidone
• Octapeptide can be also synthesized in a liquid phase approach (strategies
• RGD-octapeptide 80/20 octapeptide / (h)RGD-octopeptide molar ratio
• DMEM Gibco, Invitogen cell culture medium
• RGD-octapeptide 80/20 octapeptide / (h)RGD-octopeptide molar ratio
• 1 ml of a mixture of distilled water and lx Dulbecco's Phosphate Buffered Saline (70/30 ratio) were dissolved in 1 ml of a mixture of distilled water and lx Dulbecco's Phosphate Buffered Saline (70/30 ratio) at 90°C for 3 hours.
• 500 ⁇ of sample were placed in each cell culture well plates with 40 ⁇ of 0.5M NaOH solution and stirred.
• 100 ⁇ of cells suspended in medium were added to the wells and stirred in.
• 100 ⁇ of cell culture medium were then added on top of the gels in the wells and the well plate were placed in the incubator.
• 125 ⁇ of NaOH 2N were added to 5.1 ml of Dulbecco's Modified Eagle Medium (DMEM).
• DMEM Dulbecco's Modified Eagle Medium
• the solution was vigorously shaken and rapidly added to 715 ⁇ of a dimethylsulfoxide (DMSO) solution containing the a mixture octapeptide/(h)RGDWP-octapeptide (80/20 w/w)(concentration 200 mg peptide/ml DMSO).
• DMSO dimethylsulfoxide
• the immediate formed gel was shaken for 3 min.
• the obtained gel was washed on a 5 ⁇ membrane four times by 15 ml Dulbecco's Modified Eagle Medium (DMEM).
• the washed gel was directly used for 2D cell culture or then transferred into a vial, slightly diluted by DMEM (5-10% of the gel volume), and shaken for few minutes.
• the suspended gel could then be transferred into culture flasks and be inoculated with cells for 3D cell culture.
• hydrogels were compared: a hydrogel based on self-assembling octapeptide FEFKFEFK, a hydrogel based on a RGD modified octapeptide (Mpr-RGDWP -FEFKFEFK), and a hydrogel based on a hRGD modified octapeptide (Mpr-hRGDWP -FEFKFEFK) .
• the cell morphology and cell attachment were observed using optical microscopy.
• Figure 1 shows optical microscopy pictures of hydrogels based on octapeptide, hRGD and RGD modified octapeptides, taken after 1, 3 and 7 days.
• FEFKFEFK hydrogel have a rounded morphology up to day 7 in culture. Very few dead cells were recorded. In contrast, images revealed that after day 1 and day 3 in culture, the HDF's seeded on hRGD modified hydrogels showed a highly stretched morphology compared to the ones seeded on RGD modified gels. These results correlated well with the optical microscopy images presented in example 7.1.1. Medium change after alternating days provided sufficient nutrients to facilitate cell growth; consequently very few dead cells were detected.
• FIDF Human Dermal Fibroblasts
• FIG. 3 shows optical microscopy pictures of hydrogels based on octapeptide, hRGD and RGD modified octapeptides, taken after 1 day.
• Figure 4 shows fluorescence optical microscopy pictures of hydrogels based on octapeptide, hRGD and RGD modified octapeptides, taken after 1 and 3 days.
• the fluorescence micrographs show that majority of the cells encapsulated within the FEFKFEFK hydrogel demonstrated a rounded morphology up to day 3 in culture. Dead cells were observed at day 1. At day 3 the cell viability improved and therefore, few dead cells were detected. In contrast, it can be seen from the images that between day 1 and day 3, FIDF's embedded within the hRGD modified hydrogels demonstrated a highly stretched morphology compared to the FIDF's embedded within the RGD modified hydrogels.
• FIG. 5 shows the cell number after respectively 0, 1, 2, 3, 5, 7, 10 and 14 days in hydrogels based on octapeptide, RGD and hRGD modified octapeptides.

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• 2011
  • 2011-10-06 WO PCT/EP2011/067479 patent/WO2012045822A1/en not_active Ceased
  • 2011-10-06 EP EP11764580.4A patent/EP2625191A1/en not_active Withdrawn
  • 2011-10-06 JP JP2013532197A patent/JP2013538864A/ja active Pending
  • 2011-10-06 WO PCT/EP2011/067481 patent/WO2012045824A1/en not_active Ceased
  • 2011-10-06 EP EP11764581.2A patent/EP2625192A1/en not_active Withdrawn
  • 2011-10-06 US US13/877,915 patent/US20130210147A1/en not_active Abandoned
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