WO2011145077A2 - Branched polypeptides and their use in promoting adhesion of cells to solid surfaces - Google Patents
Branched polypeptides and their use in promoting adhesion of cells to solid surfaces Download PDFInfo
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- WO2011145077A2 WO2011145077A2 PCT/IB2011/052207 IB2011052207W WO2011145077A2 WO 2011145077 A2 WO2011145077 A2 WO 2011145077A2 IB 2011052207 W IB2011052207 W IB 2011052207W WO 2011145077 A2 WO2011145077 A2 WO 2011145077A2
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- Prior art keywords
- cells
- sak
- branched
- polypeptide conjugate
- branched polypeptide
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- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 108010000222 polyserine Proteins 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 210000004129 prosencephalon Anatomy 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011894 semi-preparative HPLC Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 108010052768 tyrosyl-isoleucyl-glycyl-seryl-arginine Proteins 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
- C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
Definitions
- the invention lies in the field of branched polypeptides.
- novel branched peptide polymers and their use in serum-free attachment of cells to solid surfaces.
- the herein-disclosed novel polypeptides are especially useful for serum- free attachment of stem cells to solid surfaces.
- Adherent cells need appropriate surfaces for attachment.
- the attachment of cells to artificial surfaces gained special attention with the development of bioimplants including dental, joint, bone and vascular prostheses, and with the interest in culturing stem cells as potential tools of therapeutic cell replacement.
- sera have been included in the culture medium.
- Serum is known to contain various adhesion-molecules, such as fibronectin, collagens, laminins, vitronectin, etc. (Anderson NL and Anderson NG, Mol Cell Proteomics, 2002, 11 :845-67), which make the culturing surface more similar to the extracellular environment and help the attachment of most cell types.
- Application of serum provides active soluble components, including hormones, growth factors, and cytokines.
- the composition of sera is not fully clarified. Moreover, it changes from sample to sample depending on the physiological / developmental stage of the source animals and also on handling the sample.
- Serum-free, chemically defined media are available for a wide variety of cells (http://www.zet.or.at/publikationen/Studien/fcs_update_l_2004.htm).
- these tissue - and cell-specific defined media impair cell attachments.
- the need for specific attachment-promoting surfaces under serum-free conditions resulted in the development of various coating layers for tissue culture dishes or clinical implants.
- These coating materials include e.g. complex extracellular matrix preparations, amnion membrane, purified/recombinant extracellular matrix proteins, physico-chemical modification of solid surfaces and synthetic peptides.
- synthetic peptides are the following: Laminin-derived peptides e.g.
- Extracellular proteins or basement membrane preparations resemble a more or less native adhesion environment, and seem to provide serum- free attachment possibilities for a number of cell types.
- Synthetic peptide -fragments of extracellular matrix proteins represent a relatively cheap, reproducible and chemically defined group of artificial adhesion molecules.
- Various polymer and co-polymer scaffold molecules had been modified with various RGD-peptides and are generally acknowledged as promising coating materials for promoting cell adhesion (see above Hersel et al).
- the RGD tripeptide sequence is a common motif in various extracellular matrix (ECM) proteins (like fibronectin, vitronectin, etc.) and is responsible for the binding of many ECM proteins to cell surface receptors (Piersbacher MD and Ruoslahti E, Nature, 1984, 309:30-3).
- Cyclic RGD containing pentapeptides such as c(RGDfV) were originally described as potent inhibitors of cell adhesion to vitronectin and laminin fragment PI (Aumailley et al, FEBS Lett, 1991, 291(l):50-4).
- the cyclic RGD pentapeptides exert various effects on cultured cells including rapid aggregation and reduced proliferation (Market K et al., Int J Dev Neurosci. 2004, 12(8), 689-90). In vivo, they were reported to reduce tumor growth presumably by hindering vascularisation through cell-attachment blocking activity (Stupp R and Ruegg C. J Clin Oncol. 2007, 25(13): 1637-8).
- the pentapeptides do not form adhesive molecular coatings on solid surfaces, as their structure is distorted upon binding to solid surfaces, but when embedded into a protein matrix, they seem to support the attachment and long-term serum- free survival of a number of mammalian cells (Marko et al, Bioconj. Chem 2008, 19 (9), 1757-1766).
- the invention relates to a branched polypeptide conjugate of formula I poly ⁇ Lys(c[Arg-Gly-Asp-D-Phe-Cys(CH 2 CO)]i-Ser j -DL-Ala m ) ⁇ (I)
- m is 1 to 7
- j is 0.7 to 1
- i is 0.01 to 0.5.
- m is 2 to 5
- j is 0.9 to 1.0 and i is 0.05 to 0.3, preferably m is about 2.7 and i is about 0.1.
- the degree of polymerization (DP) of the branched polypeptide conjugate of the invention is preferable below 500. More preferably DP is from about 200 to about 400. Even more preferably DP is from about 250 to about 350 and most preferably DP is about 300.
- the degree of polymerization can be measured by different processes well known to a person skilled in the art [see e.g. Schachman, H. K. Ultracentrifugation, diffusion, and viscosimetry. In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press: New York, 1957; Vol. 4].
- the degree of polymerization of polypeptides was herein actually determined by applying a relative method based on the viscosities of their aqueous solution. Flow time of aqueous solution of polypeptides at a concentration range of 1-0.5w% was measured using a capillary viscometer (Ostwald-type) at a given temperature and specific viscosity was calculated.
- the branched polypeptide conjugates of the invention can be used in therapy and in diagnosis.
- the diagnostic applications of the polypeptide conjugates of the invention include, inter alia, the attachment of cells of interest to solid surfaces to facilitate the performance of diagnostic assays performed with the attached cells.
- the therapeutical use of said polypeptide conjugates includes manufacturing implants coated with said polypeptide conjugates, thus promoting adhesion of cells to the implant.
- Such bioimplants may be dental, joint, bone and vascular prostheses and scaffold materials serving for e.g. growing artificial organs.
- the branched polypeptide conjugates of the invention may also be used for coating any solid material, for example plastic, polystyrene, glass, Si0 2 , Ti0 2 and the like, as well as combination of solid materials which may have any form.
- the branched polypeptide conjugates of the invention may be used for coating tissue culture dishes or various implant surfaces, if tissue cells of the host are expected to settle onto the surface.
- said branched polypeptide conjugates can be used for promoting adhesion of cells to solid surfaces and implants.
- Said implants are preferably dental, joint, bone or vascular implants or scaffold materials serving for growing artificial tissues or organs.
- the branched polypeptide conjugates of the invention are especially useful for enhancing the adhesion of stem cells.
- said branched polypeptide conjugates according to the invention can be used to coat a solid surface, preferably said solid surface is comprised in a tissue culture vessel.
- the branched polypeptide conjugate of the invention can be used in bioindustrial-range cell growth under defined, xeno-material-free conditions for protein production by tissue cells and in large-scale cultivation of cells for biomedical applications.
- solid surfaces in a tissue culture vessel, a cellular bioreactor or a fermentor are coated with the branched polypeptide conjugate according to the invention.
- the invention relates to implants comprising a surface, preferably a solid surface coated with the branched polypeptide conjugate according to the invention and the invention relates to the use of such an implant in therapy.
- Fig. 1 shows a schematic representation of a branched polymer according to the invention.
- Fig. 2 shows the initial attachment and spreading of A431 aorta smooth muscle -derived cells to SAK (left panels) and to SAK-c( GDfC) (right panels) coated polystyrene surfaces.
- Phase contrast pictures were taken at 15 min (upper panels) and at 45 min after seeding. Note the spherical shape of non- attached cells (left panels) on SAK surfaces, and the numerous spreading cells (arrows on the right panels) on SAK-c(RGDfC).
- Fig. 3 shows that attached cells 1.5 hour after seeding indicated an increased initial adhesion to SAK- c(RGDfC) coating (determined by photometry).
- the adhesivity was density-dependent and reflected the differences in adhesion-preference among different types of cells.
- the data points were calculated as percentages of optical density values obtained from cultures on bare polystyrene surfaces (100%) and represents averages and standard deviations of data from 4 to 8 identically treated cultures.
- Fig. 4 Coating polystyrene tissue culture surfaces with SAK-c( GDfC) (C) resulted in significantly increased adhesion of astrocytes regardless of the presence of serum. In serum-free conditions, increased attachment was seen even at 0.0025 g/cm 2 (C 0.0025) density. At higher (>0.25 g/cm 2 ) surface densities, SAK-c(RGDfC) increased the attachmenti in the presence of 5% fetal bovine serum, as well. Cells (10 5 cells/cm 2 ) were seeded for 2 hours.
- the attached cell mass was fixed with 4% paraformaldehyde (w/v in phosphate buffered saline), stained with methylene blue and measured by photometry. 100% (solid line) + stdev (dashed lines) show the cell mass attached on bare polystyrene surfaces.
- Fig. 5 shows that SAK-c(RGDfC) even in low (0.025 g/cm 2 ) surface density supported the serum-free survival of fetal mouse forebrain-derived neural stem cells. Attached cell mass was determined by methylene blue staining of paraformaldehyde-fixed cultures grown for 48 hours in serum-free defined medium on bare polystyrene (control) or on surfaces coated with 2.5, 0.25 or 0.025 g/cm 2 SAK-c(RGDfC) (SAK), AK- c(RGDfC) (AK) or poly-L-lysine (PLL).
- a cyclic arginyl-glycyl-aspartyl-D-phenylalanyl-cysteine (c(RGDfC)) pentapeptide moiety was bound to a poly-lysine/serine-oligo-alanine ⁇ poly[Lys(Ser j -DL-Ala m ], where j is from 0.9 to 1.0 and m is from 1 to 7; SAK ⁇ polypeptide backbone with controlled branching pattern.
- the alanine units in the side chain are preferably in their DL form, however, D and/or L alanine may also be used.
- the SAK-c(RGDfC) polymer conjugate according to the invention was easily bound to various solid surfaces (Table 1) either by linking covalently to surface NH 2 groups (APTES-treated glass surfaces) or by simple ionic/polar interactions.
- the established layer carried outward- looking serine -oligo-alanine side chains with a single cyclic pentapeptide moiety at many termini.
- the cyclic RGD pentapeptide is characterized by a sequence (RGDOZ) wherein Z may be any amino acid capable of conjugating with the terminus of the side chain; through a thiol group, such as Cys, Hey or other non- natural amino acid containing thiol group, through an amino group, such as Lys, Orn or other non-natural amino acid containing amino group in the side chain or through a carboxyl group, such as Glu, Asp or other non-natural amino acid containing -COOH in the side chain.
- RGD pentapeptide is (RGDfC) of the following formula:
- novel peptide polymers can be prepared with processes well-known in the art of peptide syntheses, especially by using different protecting groups to obtain a specifically designed branched structure.
- the first step of the synthesis is the preparation of the polylysine backbone. This reaction is well-known in the art, wherein the ⁇ -amino group of lysine is protected, e.g. by benzyloxycarbonyl group (Z); polymerization may be accomplished e.g. by the N-carboxyanhydride method in the presence of an initiator, e.g. diethylamine (DEA).
- DEA diethylamine
- the polymerization grade of the reaction may be influenced by the molar ratio of the Lys(Z)-NCA and DEA; a molar ratio of 50 to 1 provides a polymer with about 250 to 350 Lys units (measured by viscosimetry).
- the polymerization is carried out preferably for 5 days followed by terminating the reaction by adding 1M HC1 solution, separating and deprotecting the dried insoluble polymer using 4M HBr/acetic acid solution. Dialysis of polylysine against d.i. water is the most preferred approach for separation of the appropriate size of polymers from smaller oligomers.
- the branched structure is formed by polymerizing the side chains to the polylysine backbone.
- a number of activated alanine molecules e.g. Ala-NCA
- DL-Ala is used considering the inhibition of ordered structure formation such as a-helix structure that decreases the solubility of the polymer.
- the resulted polypeptide is marked as poly[Lys(DL-Ala m )] (AK), where m is generally from 1 to 7 and preferably from 2 to 5.
- the present inventors found that attaching serine amino acid residue to the side chains of branched polypeptide increased the solubility thereof.
- a preferred Ser derivative for coupling is Z-Ser-OPcp.
- HOBt catalyzed active ester coupling method is preferred for the attachment of Ser [Mezo G. et al. Carrier design: Synthesis and conformational studies of poly[L-lysine] based branched polypeptides with hydroxyl groups. Biopolymers 42, 719-730 (1997)].
- the benzyloxycarbonyl protecting group is cleaved similarly to the method applied in the synthesis of polylysine mentioned above followed by dialysis as a preferred purification procedure.
- Conjugation of the cyclic pentapeptides is carried out also by using methods known in the art for coupling amino acids and peptides.
- An example for the conjugation is the thioether bond formation through the SH group of a cyclic pentapeptide with the chloroacetylated branched polymer.
- Coupling of the cyclic peptide to SAK polymer by thioether linkage is preferred because of its easy and selective formation and high stability under circumstances of chemical synthesis, purification and biological measurements.
- the SAK polymer was chloroacetylated using ClAc-OPcp (Mezo G et al. Bioconjugate Chem. 2003, 14, 1260-1269), while cysteine residue was incorporated into the sequence of the cyclic GD peptide.
- glicine as C-terminal amino acid residue was chosen.
- acetate counter ion was changed for hydrochloride by using pyridinium hydrochloride.
- the "head-to-tail" cyclisation was achieved in a diluted solution of DMF in the presence of BOP and HOBt coupling reagents and DIEA base to serve slightly alkaline condition.
- the deprotection of side chain protecting groups of the cyclic peptide was performed with modified reagent-K cleavage mixture (Marko K. et al. Bioconjugate Chemistry 2008, 19 (9), 1757-1766).
- cyclic peptide was converted into thiol-protected form as well by carboxamidation of the Cys side chain (cyclo[RGDfC(Cmc)]). Carboxamidation of the thiol group was carried out in Tris buffer under slightly alkaline conditions using iodoacetamid. The cyclic peptides were characterized by RP-HPLC and ESI- MS (results are not shown).
- the known composition of the adhesive material, its cost-effective production, and easy application, together with the avoidance of any non-clarified additives provide novel routes for attaching cultured cells for animal and human therapeutic purposes.
- the polymer of the invention can be used for coating tissue culture dishes or various implant surfaces, if tissue cells of the host are expected to settle onto the surface.
- SAK- c(RGDfC) coated tissue culture surfaces can easily be prepared and used for serum-free attachment and further growth of the cells in defined media.
- Linear side chain protected precursor peptide H-Asp(O i Bu)-D-Phe-Cys(Trt)-Arg(Pbf)-Gly-OH was synthesized manually by solid phase peptide synthetic method according to Fmoc Bu strategy using 2-Cl-trityl chloride resin as a support [Barlos, K., Chatzi, O., Gatos, D., Stavropoulos, G. (1991) 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage. Int. J. Pept. Prot. Res. 37, 513-520].
- the attachment of C-terminal amino acid to the resin was as follows: i) prior to use, the resin was dried over solid KOH in a dessicator overnight; ii) swelling of the resin in dichloromethane (DCM) for half an hour, then washing it with ⁇ -dimethylformamide (DMF); iii) coupling of the C-terminal amino acid to the resin using 2.5 equivalents of Fmoc-Gly-OH dissolved in DMF and 2.5 equivalents of A -ethyldiisopropylamine (DIEA) (1+1.5 equiv adding in two portions) for 1 hour, then capping with methanol (0.8 mL/g resin) for 10 minutes; iv) washing with DMF (5x0.5 min); v) removal of Fmoc protecting group in three steps: 5% piperidine in DMF for 10 minutes, 30% piperidine in DMF for 15 minutes, 50% piperidine in DMF for 30 minutes; vi) DMF washing (6x1 min).
- DCM dich
- the Fmoc-Gly resin loading was determined by Gude's method resulted in 0.6 mmol/g resin capacity [Gude, M., Ryf, J., White, P.D. (2002) An accurate method for the quantitation of Fmoc-derivatized solid phase supports. LIPS 9, 203-206].
- the peptide -resin Prior to the cleavage of side chain protected peptide from the resin, the peptide -resin was washed with DMF, DCM, methanol and diethyl-ether, then dried in vacuo over P 2 O 5 .
- linear side chain protected peptide was not necessary because its high purity (over 90%) was detected by RP-HPLC.
- the linear peptide derivative was dissolved in methanol in the presence of 10 equivalents of pyridinium hydrochloride and the solvent was evaporated afterwards.
- the dried linear peptide was dissolved in DMF (1 mg/niL), then 3-3 equivalents of (benzotriazol-1- yloxy)tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) and HOBt coupling agents in the presence of 6 equivalents of DIEA was added to the solution.
- the pH was kept under slightly alkaline condition (pH 7.5-8) during the reaction time.
- the reaction was followed by RP-HPLC. After 24 hours the reaction was completed, then the solvent was evaporated in vacuo and the crude product was precipitated with 5% NaHCOs (aqueous solution), filtered off and washed with d.i. water until the filtrate was neutral.
- the cleavage of side chain protecting groups of the dried cyclic peptide was performed by using modified reagent-K cleavage mixture: trifluoroacetic acid (9.75 mL), water (0.25 mL), phenol (0.75 g), thioanisole (0.25 mL), ethane- 1,2-dithiol (0.25 mL), triisopropyl silane (0.125 mL) at room temperature for 3.5 hour. After cleavage, the crude product was precipitated with cold diethyl ether, washed with ether three times, filtered off and dried in vacuo over ⁇ 2 ⁇ 5 . The crude product was purified by semi-preparative RP-HPLC. The pure cyclic peptide was characterized by analytical RP-HPLC and ESI-MS (description can be seen below).
- SAK-(cyclofArg-Gly-Asp-D-Phe-CysJ) by conjugation of cyclic peptide to chloroacetylated branched chain polymeric polypeptide
- Branched chain polymeric polypeptide (SAK) was prepared according to the method previously described [Mezo, G., Kajtar, J., Nagy, L, Szekerke, M, Hudecz, F. (1997) Carrier design: Synthesis and conformational studies of poly(L -lysine) based branched polypeptides with hydroxyl groups in the side chains. Biopolymers 42, 719-730]. Chloroacetylation of SAK polimer was performed according to Mezo et al.
- Conjugation reaction of multivalent chloroacetyl-SAK polymer and cyclo[ GDfC] resulting in a thioether linkage was carried out in a 0.1M Tris buffer (pH 8.0) solution.
- the cysteine containing cyclic peptide (2 equivalents altogether to the CI content of the polymer) was added in solid form to the solution of chloroacetylated polypeptide (1 mg/mL) time by time during 3h.
- the reaction was terminated after 24 hour by addition an excess of cysteine to block the unreacted chloroacetyl groups.
- the crude product was dialyzed against d.i.
- Electrospray ionization mass spectrometry was performed with a Bruker Daltonics Esquire 3000 Plus (Bremen, Germany) mass spectrometer, operating in continuous sample injection at 4 L/min flow rate. Samples were dissolved in 50% acetonitrile -water mixture. Mass spectra were recorded in positive mode in the m/z 200-1500 range.
- amino acid composition of peptides was determined by amino acid analysis using SYKAM S-433 amino acid analyzer (SYKAM GmbH, Eresing, Germany) equipped for the determination of amino acids with post- column reaction with ninhydrin. Prior to analysis the samples were hydrolysed in 6 M HC1 in sealed and evacuated tubes at 110 °C for 24 hours.
- the degree of polymerization was about 300 for the lysine that means in average 300 lysine residues were coupled together. On the side chains in average there were 2.7 DL-Ala residues and the length of the side chains varied from 1 to 7 racemic alanine residue. Roughly every side chain contained one serine residue. The cyclopeptide bound approximately to 10% of the side chains.
- Example 1 The product of Example 1, SAK-c(RGDfC) was easily bound to various solid surfaces either by linking covalently to the surface amino groups (APTES-treated glass surfaces) or by simple ionic/polar interactions.
- the produced solid materials coated with SAK-c(RGDfC) for serum-free cell cultivation are listed in Table I.
- the SAK-c(RGDfC) polymer was dissolved in water and introduced to the surface for 20 minutes. After aspirating the solution, the surfaces were let to dry at room temperature. Dried surfaces were ready to use or could be stored at 4°C for months.
- the surface density of the pentapeptide moieties were varied by binding SAK-c(RGDfC) from standard volumes of solutions with different (0.002 - 20 g/ml) concentrations.
- the calculated surface densities varied from 0.00025 to 2.5 ⁇ g SAK-c(RGDfC) /cm 2 area of the coated solid surface.
- Example 2 In cell cultivation experiments, the solid materials described in Example 2 were used.
- Serum-free attachment to the SAK-c(RGDfC) coated surfaces was tested on cell types listed in Table II.
- Cell attachment was quantified by adhesion assays, modified after Oliver et al (Oliver MH et al, J Cell Sci, 92, 513- 518 (1989)) or by determining the living cell mass by MTT -reduction test (Mossmann T (1983) J. Immunol Methods 65:55-58) after washing down non-attached cells.
- the SAK-c(RGDfC) surface provided highly adhesive surfaces for a number of cell types without serum or any exogenous, non-defined biological substances. Except neurons, which did not adhere to it, SAK-c(RGDfC) promoted initial attachment, supported spreading (Fig. 2) and long-term growth of all cells listed in Table II.
- Table II shows the cell types used in the cell cultivation experiments:
- Fig. 3 the photometric determination of attached cells 1.5 hour after seeding indicated an increased initial adhesion to SAK-c(RGDfC) coating.
- the adhesivity was density-dependent and reflected the differences in adhesion-preference among different types of cells.
- the data points were calculated as percentages of optical density values obtained from cultures on bare polystyrene surfaces (100%) and represents averages and standard deviations of data from 4 to 8 identically treated cultures.
- SAK-c(RGDfC) For most cell types, 0,025 - 2.5 g/cm 2 SAK-c(RGDfC) provided ideal attachment, while the control peptides (SAK, -linear RGDfV, c(RADfC), AK, or PLL) did not support the adhesion.
- the SAK-c( GDfC) surface resulted a two to five-fold survival in comparison to bare polystyrene or to SAK, AK or PLL coated (2.5 g/cm 2 ) surfaces.
- Example 3 clearly supports that the integrin ligand cyclic RGDfC pentapeptide if attached covalently to a branched poly-L-lysine/serine-oligo-alanine backbone, proved to promote attachment of a number of different cells to solid surfaces. For attachment, serum or any biological additives could be omitted.
- SAK polypeptide according to the invention has a better solubility than the previously available AK polypeptide.
- a further advantage of the SAK polypeptide according to the invention is that it is less susceptible to ageing than a similar AK polypeptide. The difference between the solubilities of the different polypeptides is even more evident if 6 months old polypeptides are compared.
- the SAK polypeptide of the invention was surprisingly found to be superior to the previously disclosed AK polypeptide regarding its solubility and this better solubility was maintained for a longer period of time thus the SAK polypeptide is clearly less susceptible to ageing.
Abstract
Disclosed are novel poly{Lys(c[Arg-Gly-Asp-D-Phe-Cys(CH2CO)]i-Serj-DL-Alam)} branched peptide polymers wherein m is 1 to 7, j is 0.7 to 1.0 and i is 0.01 to 0.5. The use of said polypeptides in serum-free attachment of cells to solid surfaces is also disclosed. The herein-disclosed novel polypeptides are especially useful for serum- free attachment of stem cells to solid surfaces.
Description
BRANCHED POLYPEPTIDES AND THEIR USE IN PROMOTING ADHESION OF CELLS TO SOLID
SURFACES
Field of the invention
The invention lies in the field of branched polypeptides. Disclosed are novel branched peptide polymers and their use in serum-free attachment of cells to solid surfaces. The herein-disclosed novel polypeptides are especially useful for serum- free attachment of stem cells to solid surfaces.
Background of the invention
Adherent cells need appropriate surfaces for attachment. The attachment of cells to artificial surfaces gained special attention with the development of bioimplants including dental, joint, bone and vascular prostheses, and with the interest in culturing stem cells as potential tools of therapeutic cell replacement.
In traditional cell culture techniques, sera (of various origins) have been included in the culture medium. Serum is known to contain various adhesion-molecules, such as fibronectin, collagens, laminins, vitronectin, etc. (Anderson NL and Anderson NG, Mol Cell Proteomics, 2002, 11 :845-67), which make the culturing surface more similar to the extracellular environment and help the attachment of most cell types. Application of serum provides active soluble components, including hormones, growth factors, and cytokines. The composition of sera, however, is not fully clarified. Moreover, it changes from sample to sample depending on the physiological / developmental stage of the source animals and also on handling the sample. The complexity of active components hinders the identification of the actual needs of cells, therefore the elaboration of defined cell culture conditions and the appropriate scaffold materials. Furthermore many coating materials have been derived from animal sources and contain non-identified components. The presence of potential anaphylactic factors and a large number of biologically active - but not defined compounds - prevent the use of sera in cultivating cells for human cell therapy.
Serum-free, chemically defined media are available for a wide variety of cells (http://www.zet.or.at/publikationen/Studien/fcs_update_l_2004.htm). In exchange for their benefits, these tissue - and cell-specific defined media impair cell attachments. The need for specific attachment-promoting surfaces under serum-free conditions resulted in the development of various coating layers for tissue culture dishes or clinical implants. These coating materials include e.g. complex extracellular matrix preparations, amnion membrane, purified/recombinant extracellular matrix proteins, physico-chemical modification of solid surfaces and synthetic peptides. Examples of synthetic peptides are the following: Laminin-derived peptides e.g. SE ILESINE/Lipotec (http://www.centerchem.com/PDFs/Serilesine.pdf), IKVAV (Tashiro K et al, J. Biol. Chem., 264: 16174-16182), YIGSR and LGTIPG (http://bio.takara.co.jp/BIO_EN/ catalog_d.asp?C_ID=C0565); as well as RGD-modified peptides (see e.g. Hersel et al, Biomaterials, 2003, 24(24):4385-415).
Extracellular proteins or basement membrane preparations resemble a more or less native adhesion environment, and seem to provide serum- free attachment possibilities for a number of cell types. The high price and their biological origin, however, hinder their use for human clinical applications.
Synthetic peptide -fragments of extracellular matrix proteins represent a relatively cheap, reproducible and chemically defined group of artificial adhesion molecules. Various polymer and co-polymer scaffold molecules had been modified with various RGD-peptides and are generally acknowledged as promising coating materials for promoting cell adhesion (see above Hersel et al).
The RGD tripeptide sequence is a common motif in various extracellular matrix (ECM) proteins (like fibronectin, vitronectin, etc.) and is responsible for the binding of many ECM proteins to cell surface receptors (Piersbacher MD and Ruoslahti E, Nature, 1984, 309:30-3). Cyclic RGD containing pentapeptides, such as c(RGDfV) were originally described as potent inhibitors of cell adhesion to vitronectin and laminin fragment PI (Aumailley et al, FEBS Lett, 1991, 291(l):50-4). Cyclic (RGDfV) (Pfaff M et al, J Biol Chem, 1994, 269(32):20233-8), and (RGDfC) peptides have the highest affinity and selectivity towards ανβ3 (and ανβ5, ανβ6) integrins (Goodman et al, J Med Chem., 2002, 45(5): 1045-51) but bind to other integrins (such as 04^3 and α5βι) with lower affinity, as well (Pfaff M et al, J Biol Chem, 1994, 269(32):20233-8).
In solution, the cyclic RGD pentapeptides exert various effects on cultured cells including rapid aggregation and reduced proliferation (Market K et al., Int J Dev Neurosci. 2004, 12(8), 689-90). In vivo, they were reported to reduce tumor growth presumably by hindering vascularisation through cell-attachment blocking activity (Stupp R and Ruegg C. J Clin Oncol. 2007, 25(13): 1637-8).
In their native form, the pentapeptides do not form adhesive molecular coatings on solid surfaces, as their structure is distorted upon binding to solid surfaces, but when embedded into a protein matrix, they seem to support the attachment and long-term serum- free survival of a number of mammalian cells (Marko et al, Bioconj. Chem 2008, 19 (9), 1757-1766).
Marko et al. (see above) described a poly[Lys(DL-Alam)] (AK) branched polypeptide, carrying multiple copies of cyclic(arginyl-glycyl-aspartyl-D-phenylalanyl-cysteine) [cyclic RGD] pentapeptide on the N-terminal positions of the oligoalanine branches (termed AK-cyclo [RGDfC]) and shown that AK-cyclo[RGDfC] is able to coat various solid surfaces and enhances the adhesion of many cell types. However, the relatively low solubility of the peptide conjugate in water restricts its usability, furthermore the above conjugate is significantly susceptible to aging.
Brief description of the invention
To solve the above problem present inventors developed a novel family of branched polypeptide conjugates, based on a SAK (poly-lysine, oligo-D/L-alanine-serine) polymer, carrying cyclic RGD pentapeptides on terminal positions of serine in the branches. These polymers according to the invention promote adhesion, spreading, long- term survival and proliferation of a large number of anchorage -dependent cell types and at the same time are more soluble than the previously described AK polypeptides and are not susceptible to ageing.
Therefore according to a first aspect the invention relates to a branched polypeptide conjugate of formula I poly{Lys(c[Arg-Gly-Asp-D-Phe-Cys(CH2CO)]i-Serj-DL-Alam)} (I)
wherein m is 1 to 7, j is 0.7 to 1 and i is 0.01 to 0.5.
According to a preferred embodiment of the invention m is 2 to 5, j is 0.9 to 1.0 and i is 0.05 to 0.3, preferably m is about 2.7 and i is about 0.1.
The degree of polymerization (DP) of the branched polypeptide conjugate of the invention is preferable below 500. More preferably DP is from about 200 to about 400. Even more preferably DP is from about 250 to about 350 and most preferably DP is about 300.
The degree of polymerization can be measured by different processes well known to a person skilled in the art [see e.g. Schachman, H. K. Ultracentrifugation, diffusion, and viscosimetry. In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press: New York, 1957; Vol. 4]. The degree of polymerization of polypeptides was herein actually determined by applying a relative method based on the viscosities of their
aqueous solution. Flow time of aqueous solution of polypeptides at a concentration range of 1-0.5w% was measured using a capillary viscometer (Ostwald-type) at a given temperature and specific viscosity was calculated. Similar measurements for chemically identical polypeptides with known degree of polymerization (Alamanda Polymers, Inc, Huntsville, AL, USA) used as standards were performed. A correlation curve was obtained from these data relating the degree of polymerization to the specific viscosity of the aqueous polypeptide solution. Using this correlation curve, the degree of polymerization of any further polypeptide with the same chemical composition can be determined measuring the viscosity of its aqueous solution at the given temperature.
The branched polypeptide conjugates of the invention can be used in therapy and in diagnosis. The diagnostic applications of the polypeptide conjugates of the invention include, inter alia, the attachment of cells of interest to solid surfaces to facilitate the performance of diagnostic assays performed with the attached cells.
The therapeutical use of said polypeptide conjugates includes manufacturing implants coated with said polypeptide conjugates, thus promoting adhesion of cells to the implant. Such bioimplants, without limitation, may be dental, joint, bone and vascular prostheses and scaffold materials serving for e.g. growing artificial organs. The branched polypeptide conjugates of the invention may also be used for coating any solid material, for example plastic, polystyrene, glass, Si02, Ti02 and the like, as well as combination of solid materials which may have any form. The branched polypeptide conjugates of the invention may be used for coating tissue culture dishes or various implant surfaces, if tissue cells of the host are expected to settle onto the surface.
According to the invention, said branched polypeptide conjugates can be used for promoting adhesion of cells to solid surfaces and implants. Said implants are preferably dental, joint, bone or vascular implants or scaffold materials serving for growing artificial tissues or organs.
The branched polypeptide conjugates of the invention are especially useful for enhancing the adhesion of stem cells.
Furthermore, said branched polypeptide conjugates according to the invention can be used to coat a solid surface, preferably said solid surface is comprised in a tissue culture vessel.
According to another aspect of the invention the branched polypeptide conjugate of the invention can be used in bioindustrial-range cell growth under defined, xeno-material-free conditions for protein production by tissue cells and in large-scale cultivation of cells for biomedical applications. In these applications solid surfaces in a tissue culture vessel, a cellular bioreactor or a fermentor are coated with the branched polypeptide conjugate according to the invention.
Furthermore, the invention relates to implants comprising a surface, preferably a solid surface coated with the branched polypeptide conjugate according to the invention and the invention relates to the use of such an implant in therapy.
Brief description of the drawings
Fig. 1 shows a schematic representation of a branched polymer according to the invention.
Fig. 2 shows the initial attachment and spreading of A431 aorta smooth muscle -derived cells to SAK (left panels) and to SAK-c( GDfC) (right panels) coated polystyrene surfaces. Phase contrast pictures were taken at 15 min (upper panels) and at 45 min after seeding. Note the spherical shape of non- attached cells (left panels) on SAK surfaces, and the numerous spreading cells (arrows on the right panels) on SAK-c(RGDfC).
Fig. 3 shows that attached cells 1.5 hour after seeding indicated an increased initial adhesion to SAK- c(RGDfC) coating (determined by photometry). The adhesivity was density-dependent and reflected the
differences in adhesion-preference among different types of cells. The data points were calculated as percentages of optical density values obtained from cultures on bare polystyrene surfaces (100%) and represents averages and standard deviations of data from 4 to 8 identically treated cultures.
Fig. 4: Coating polystyrene tissue culture surfaces with SAK-c( GDfC) (C) resulted in significantly increased adhesion of astrocytes regardless of the presence of serum. In serum-free conditions, increased attachment was seen even at 0.0025 g/cm2 (C 0.0025) density. At higher (>0.25 g/cm2) surface densities, SAK-c(RGDfC) increased the attachmenti in the presence of 5% fetal bovine serum, as well. Cells (105cells/cm2) were seeded for 2 hours. After removing non-attached cells, the attached cell mass was fixed with 4% paraformaldehyde (w/v in phosphate buffered saline), stained with methylene blue and measured by photometry. 100% (solid line) + stdev (dashed lines) show the cell mass attached on bare polystyrene surfaces.
Fig. 5 shows that SAK-c(RGDfC) even in low (0.025 g/cm2) surface density supported the serum-free survival of fetal mouse forebrain-derived neural stem cells. Attached cell mass was determined by methylene blue staining of paraformaldehyde-fixed cultures grown for 48 hours in serum-free defined medium on bare polystyrene (control) or on surfaces coated with 2.5, 0.25 or 0.025 g/cm2 SAK-c(RGDfC) (SAK), AK- c(RGDfC) (AK) or poly-L-lysine (PLL).
Detailed description of the invention
According to the invention, a cyclic arginyl-glycyl-aspartyl-D-phenylalanyl-cysteine (c(RGDfC)) pentapeptide moiety was bound to a poly-lysine/serine-oligo-alanine {poly[Lys(Serj-DL-Alam], where j is from 0.9 to 1.0 and m is from 1 to 7; SAK} polypeptide backbone with controlled branching pattern. According to a preferred embodiment of the invention, the alanine units in the side chain are preferably in their DL form, however, D and/or L alanine may also be used. The SAK-c(RGDfC) polymer conjugate according to the invention was easily bound to various solid surfaces (Table 1) either by linking covalently to surface NH2 groups (APTES-treated glass surfaces) or by simple ionic/polar interactions. The established layer carried outward- looking serine -oligo-alanine side chains with a single cyclic pentapeptide moiety at many termini.
The cyclic RGD pentapeptide is characterized by a sequence (RGDOZ) wherein Z may be any amino acid capable of conjugating with the terminus of the side chain; through a thiol group, such as Cys, Hey or other non- natural amino acid containing thiol group, through an amino group, such as Lys, Orn or other non-natural amino acid containing amino group in the side chain or through a carboxyl group, such as Glu, Asp or other non-natural amino acid containing -COOH in the side chain. Preferably, the RGD pentapeptide is (RGDfC) of the following formula:
which is attached to the chloroacetylated N-terminal amino acid of the side chain(s) through the -SH group of the cysteine moiety via thioether bond.
In position O D-isomer of Phe and Tyr as well as their N-methyl derivatives can be applied.
The novel peptide polymers can be prepared with processes well-known in the art of peptide syntheses, especially by using different protecting groups to obtain a specifically designed branched structure. The first step of the synthesis is the preparation of the polylysine backbone. This reaction is well-known in the art, wherein the ε-amino group of lysine is protected, e.g. by benzyloxycarbonyl group (Z); polymerization may be accomplished e.g. by the N-carboxyanhydride method in the presence of an initiator, e.g. diethylamine (DEA). The polymerization grade of the reaction may be influenced by the molar ratio of the Lys(Z)-NCA and DEA; a molar ratio of 50 to 1 provides a polymer with about 250 to 350 Lys units (measured by viscosimetry). The polymerization is carried out preferably for 5 days followed by terminating the reaction by adding 1M HC1 solution, separating and deprotecting the dried insoluble polymer using 4M HBr/acetic acid solution. Dialysis of polylysine against d.i. water is the most preferred approach for separation of the appropriate size of polymers from smaller oligomers.
Obviously, the preparation of the polylysine is not limited to the process described above and described in the examples more in detail. It is obvious for those skilled in the art that other methods may equivalently be used as well.
The branched structure is formed by polymerizing the side chains to the polylysine backbone. In the simplest embodiment, a number of activated alanine molecules (e.g. Ala-NCA) are incorporated to the side chains of polylysine. Preferably DL-Ala is used considering the inhibition of ordered structure formation such as a-helix structure that decreases the solubility of the polymer. The resulted polypeptide is marked as poly[Lys(DL-Alam)] (AK), where m is generally from 1 to 7 and preferably from 2 to 5.
The present inventors found that attaching serine amino acid residue to the side chains of branched polypeptide increased the solubility thereof.
A preferred Ser derivative for coupling is Z-Ser-OPcp. To achieve the complete substitution of the branches,
HOBt catalyzed active ester coupling method is preferred for the attachment of Ser [Mezo G. et al. Carrier design: Synthesis and conformational studies of poly[L-lysine] based branched polypeptides with hydroxyl groups. Biopolymers 42, 719-730 (1997)]. The benzyloxycarbonyl protecting group is cleaved similarly to the method applied in the synthesis of polylysine mentioned above followed by dialysis as a preferred purification procedure.
Conjugation of the cyclic pentapeptides is carried out also by using methods known in the art for coupling amino acids and peptides. An example for the conjugation is the thioether bond formation through the SH group of a cyclic pentapeptide with the chloroacetylated branched polymer.
Coupling of the cyclic peptide to SAK polymer by thioether linkage is preferred because of its easy and selective formation and high stability under circumstances of chemical synthesis, purification and biological measurements. For development of a thioether bond between the polymer carrier molecule and the cyclic peptide, the SAK polymer was chloroacetylated using ClAc-OPcp (Mezo G et al. Bioconjugate Chem. 2003, 14, 1260-1269), while cysteine residue was incorporated into the sequence of the cyclic GD peptide. To avoid racemisation in the "head-to -tail" cyclisation step, glicine as C-terminal amino acid residue was chosen. This way the sequence of the linear side chain protected precursor peptide built up on the solid phase was DfCRG. The peptide was prepared on 2-Cl-trityl chloride resin by Fmoc Bu strategy. The side chain protected linear precursor
peptide with free N- and C-terminus (H-D(0'Bu)fC(Trt)R(Pbf)G-OH) was produced by the cleavage using a mixture of DCM:MeOH:AcOH = 8: l: l(v/v/v). The fairly pure crude product was applied for the cyclisation without further purification. To avoid acylation with acetic acid, prior to cyclisation, acetate counter ion was changed for hydrochloride by using pyridinium hydrochloride. The "head-to-tail" cyclisation was achieved in a diluted solution of DMF in the presence of BOP and HOBt coupling reagents and DIEA base to serve slightly alkaline condition. The deprotection of side chain protecting groups of the cyclic peptide was performed with modified reagent-K cleavage mixture (Marko K. et al. Bioconjugate Chemistry 2008, 19 (9), 1757-1766). For comparative study the cyclic peptide was converted into thiol-protected form as well by carboxamidation of the Cys side chain (cyclo[RGDfC(Cmc)]). Carboxamidation of the thiol group was carried out in Tris buffer under slightly alkaline conditions using iodoacetamid. The cyclic peptides were characterized by RP-HPLC and ESI- MS (results are not shown).
Prior to conjugation the "activation" of SAK polymer was necessary. This was achieved by incorporation of a chloroacetyl group into some of the amino groups of the polypeptide using ClAc-OPcp. The incorporated chlorine atoms could be exchanged for a thiol containing cyclopeptide in a nucleophile substitution reaction under slightly alkaline conditions. To avoid significant dimerisation of the cyclic peptide by disulfide bond formation, the cysteine containing cyclic peptide was added in solid form to the solution of choloracetylated polypeptide time by time keeping low concentration of the cyclic peptide in the reaction mixture. After this conjugation reaction blocking of unreacted chloroacetyl groups was done by addition of an excess of cysteine to prevent alkylation during biological experiments.
The known composition of the adhesive material, its cost-effective production, and easy application, together with the avoidance of any non-clarified additives provide novel routes for attaching cultured cells for animal and human therapeutic purposes. The polymer of the invention can be used for coating tissue culture dishes or various implant surfaces, if tissue cells of the host are expected to settle onto the surface. For cell culturing, SAK- c(RGDfC) coated tissue culture surfaces can easily be prepared and used for serum-free attachment and further growth of the cells in defined media.
EXAMPLES
For the syntheses presented in the examples, all amino acid derivatives were purchased from Novabiochem (Laufelfingen, Switzerland) or Reanal (Budapest, Hungary). Scavengers, coupling agents and cleavage reagents were Fluka products (Buchs, Switzerland). Solvents for synthesis and 2-Cl-trityl chloride resin were obtained from Reanal. Acetonitrile for HPLC was delivered by Sigma- Aldrich Kft (Budapest, Hungary).
Example 1
Synthesis of SAK-c(RGDfC)
Synthesis of cyclo[Arg-Gly-Asp-D-Phe-Cys]
Linear side chain protected precursor peptide H-Asp(OiBu)-D-Phe-Cys(Trt)-Arg(Pbf)-Gly-OH was synthesized manually by solid phase peptide synthetic method according to Fmoc Bu strategy using 2-Cl-trityl chloride resin as a support [Barlos, K., Chatzi, O., Gatos, D., Stavropoulos, G. (1991) 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage. Int. J. Pept. Prot. Res. 37, 513-520]. Side chain protecting groups of A^-fluorenylmethyloxycarbonyl (Fmoc) protected amino acid derivatives used for the assemble of peptide chain were as follows: tert-butyl ester (O'Bu) for Asp, trityl (Trt) for Cys and 2,2,4,6,7- pentamethyl-dihydrobenzofurane-5-sulfonyl (Pbf) for Arg.
The attachment of C-terminal amino acid to the resin was as follows: i) prior to use, the resin was dried over solid KOH in a dessicator overnight; ii) swelling of the resin in dichloromethane (DCM) for half an hour, then washing it with ΛζΛ^-dimethylformamide (DMF); iii) coupling of the C-terminal amino acid to the resin using 2.5 equivalents of Fmoc-Gly-OH dissolved in DMF and 2.5 equivalents of A -ethyldiisopropylamine (DIEA) (1+1.5 equiv adding in two portions) for 1 hour, then capping with methanol (0.8 mL/g resin) for 10 minutes; iv) washing with DMF (5x0.5 min); v) removal of Fmoc protecting group in three steps: 5% piperidine in DMF for 10 minutes, 30% piperidine in DMF for 15 minutes, 50% piperidine in DMF for 30 minutes; vi) DMF washing (6x1 min). The Fmoc-Gly resin loading was determined by Gude's method resulted in 0.6 mmol/g resin capacity [Gude, M., Ryf, J., White, P.D. (2002) An accurate method for the quantitation of Fmoc-derivatized solid phase supports. LIPS 9, 203-206].
After this procedure the further amino acid derivatives were built in according to the next protocol: i) coupling of Fmoc-amino acid derivative with 1 -hydro xybenzotriazole (HOBt) and ΛζΛ^'-diisopropylcarbodiimide (DIC) (3 equivalents each) in DMF. The efficacy of the coupling reaction was monitored by ninhydrin assay [Kaiser, E., Colescott, R.L., Bossinger, CD., Cook, P.I. (1970) Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595-598]; ii) DMF washing (5x0.5 min); iii) deprotection in two steps with 30 % piperidine in DMF for 3+17 minutes, iv) DMF washing (6x1 min).
Prior to the cleavage of side chain protected peptide from the resin, the peptide -resin was washed with DMF, DCM, methanol and diethyl-ether, then dried in vacuo over P2O5. Cleavage of the side chain protected peptide from the resin was performed using a mixture of DCM:methanol: acetic acid=8: l : l twice for 2+2 hour at room temperature. After combination of the cleavage mixtures, the solution was concentrated in vacuo and the intermediate crude product was precipitated with cold ether, filtered, washed with ether three times afterwards. The solid material was dissolved in acetic acid prior to freeze drying. The purification of the linear side chain protected peptide was not necessary because its high purity (over 90%) was detected by RP-HPLC. For changing the acetate counter ion to chloride, the linear peptide derivative was dissolved in methanol in the presence of 10 equivalents of pyridinium hydrochloride and the solvent was evaporated afterwards.
The dried linear peptide was dissolved in DMF (1 mg/niL), then 3-3 equivalents of (benzotriazol-1- yloxy)tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) and HOBt coupling agents in the presence of 6 equivalents of DIEA was added to the solution. The pH was kept under slightly alkaline condition (pH 7.5-8) during the reaction time. The reaction was followed by RP-HPLC. After 24 hours the reaction was completed, then the solvent was evaporated in vacuo and the crude product was precipitated with 5% NaHCOs (aqueous solution), filtered off and washed with d.i. water until the filtrate was neutral. The cleavage of side chain protecting groups of the dried cyclic peptide was performed by using modified reagent-K cleavage mixture: trifluoroacetic acid (9.75 mL), water (0.25 mL), phenol (0.75 g), thioanisole (0.25 mL), ethane- 1,2-dithiol (0.25 mL), triisopropyl silane (0.125 mL) at room temperature for 3.5 hour. After cleavage, the crude product was precipitated with cold diethyl ether, washed with ether three times, filtered off and dried in vacuo over Ρ2θ5. The crude product was purified by semi-preparative RP-HPLC. The pure cyclic peptide was characterized by analytical RP-HPLC and ESI-MS (description can be seen below).
Synthesis of SAK-(cyclofArg-Gly-Asp-D-Phe-CysJ) by conjugation of cyclic peptide to chloroacetylated branched chain polymeric polypeptide
Branched chain polymeric polypeptide (SAK) was prepared according to the method previously described [Mezo, G., Kajtar, J., Nagy, L, Szekerke, M, Hudecz, F. (1997) Carrier design: Synthesis and conformational studies of poly(L -lysine) based branched polypeptides with hydroxyl groups in the side chains. Biopolymers 42, 719-730]. Chloroacetylation of SAK polimer was performed according to Mezo et al. [Mezo, G. et al. (2003) Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII. Bioconjug. Chem. 14, 1260-1269]. Briefly: for the preparation of polypeptide - cyclic peptide conjugate, SAK (DPn=309, Ser:Ala:Lys= 0.9:2.7: 1, Mw: 116900) was reacted with chloroacetic acid pentachlorophenyl ester in DMF/water solution (9/1, v/v): first 25 mg SAK was dissolved in 1ml water, then it was diluted with 4 ml DMF, after that 0.5 equiv chloroacetic acid pentachlorophenyl ester (ClAc-OPcp) calculated to the branches (8.2 mg) dissolved in 5 ml DMF was added. The mixture was reacted overnight followed by dialysis against 1 % acetic acid aqueous solution for two days. The precipitate (pentachloro phenol) was filtered off and the filtrate was freeze-dried. According to the organic chlorine determination the molar ratio of chlorine and lysine in polypeptide was 0.2: 1 that means, 20% of the amino groups of the branched polypeptide SAK was chloroacetylated.
Conjugation reaction of multivalent chloroacetyl-SAK polymer and cyclo[ GDfC] resulting in a thioether linkage was carried out in a 0.1M Tris buffer (pH 8.0) solution. To avoid significant dimerization of cyclic peptide via disulfide bond formation, the cysteine containing cyclic peptide (2 equivalents altogether to the CI content of the polymer) was added in solid form to the solution of chloroacetylated polypeptide (1 mg/mL) time by time during 3h. The reaction was terminated after 24 hour by addition an excess of cysteine to block the unreacted chloroacetyl groups. The crude product was dialyzed against d.i. water to remove the unreacted cyclopeptide and cyst(e)ine, then freeze-dried and analysed for amino acid composition. According to the amino acid analysis (Lys : Ala : Ser: Asp : Gly : Phe : Arg = 1.00 : 2.67 : 0.90 : 0.10 : 0.13 : 0.11 : 0.09) the ratio of Lys and amino acids derived from cyclic peptide was about 10: 1 meaning the one -tenth of the side chains of the branched polypeptide SAK was substituted by the cyclic peptide.
Analytical methods
Analytical RP-HPLC measurements for the determination of the purity of intermediate derivatives and cyclic peptides were carried out on Vydac 218TP ds column, 250x4.6 mm, particle size 5 μπι, pore size 300 A (Vydac, Hesperia, CA) using Shimadzu HPLC apparatus [two LC-6A pump, SPD-6AV UV-detector, SIL-6B auto injector, SCL-6B system controller; (Shimadzu Corporation, Kyoto, Japan)] with eluent A: 0.045% TFA in H20 and eluent B: 0.036% TFA in acetonitrile. Linear gradient of 5-95 % eluent B was developed with 1 mL/min flow rate, during 30 min. Peaks were detected at λ=220 nm.
Purification of cyclic peptides was performed by using Phenomenex Jupiter Cis column (250x 10 mm, 10 μτη, 300 A) on a Knauer laboratory assembled semipreparative HPLC system [Knauer Pump Type 120, Dynamic Mixing Chamber Analytical, Spectro Photometer K-2501 ; (Knauer, Berlin, Germany)]; with eluent A: 0.1% TFA in H20 and eluent B: 0.1% TFA in acetonitrile-H20 (80:20, v/v). An isocratic elution with 10 % of eluent B was applied from 0 to 5 minutes, then 5-28 minutes a gradient elution of 10-33 % of eluent B was used with 4 mL/min flow rate. Peaks were detected at λ=220 nm.
The identification of the products was achieved by mass spectrometry. Electrospray ionization mass spectrometry was performed with a Bruker Daltonics Esquire 3000 Plus (Bremen, Germany) mass spectrometer,
operating in continuous sample injection at 4 L/min flow rate. Samples were dissolved in 50% acetonitrile -water mixture. Mass spectra were recorded in positive mode in the m/z 200-1500 range.
The amino acid composition of peptides was determined by amino acid analysis using SYKAM S-433 amino acid analyzer (SYKAM GmbH, Eresing, Germany) equipped for the determination of amino acids with post- column reaction with ninhydrin. Prior to analysis the samples were hydrolysed in 6 M HC1 in sealed and evacuated tubes at 110 °C for 24 hours.
Determination of organic chlorine was carried out using modified Schoniger combustion method [Schoniger, W. (1955) Eine mikroanalytische Schnellbesimmung von Halogenen in organischen Substanzen. Microchimica Acta, 1, 123-129].
The exact composition of the produced SAK-c(RGDfC) was the following
poly{Lys(c[Arg-Gly-Asp-D-Phe-Cys(CH2CO)]0.i-Ser0.9-DL-Ala2.7)} .
The degree of polymerization was about 300 for the lysine that means in average 300 lysine residues were coupled together. On the side chains in average there were 2.7 DL-Ala residues and the length of the side chains varied from 1 to 7 racemic alanine residue. Roughly every side chain contained one serine residue. The cyclopeptide bound approximately to 10% of the side chains.
Example 2
Preparation of solid surfaces coated with SAK-c(RGDfC)
The product of Example 1, SAK-c(RGDfC) was easily bound to various solid surfaces either by linking covalently to the surface amino groups (APTES-treated glass surfaces) or by simple ionic/polar interactions. The produced solid materials coated with SAK-c(RGDfC) for serum-free cell cultivation are listed in Table I.
Table I
For coating, the SAK-c(RGDfC) polymer was dissolved in water and introduced to the surface for 20 minutes. After aspirating the solution, the surfaces were let to dry at room temperature. Dried surfaces were ready to use or could be stored at 4°C for months.
The surface density of the pentapeptide moieties were varied by binding SAK-c(RGDfC) from standard volumes of solutions with different (0.002 - 20 g/ml) concentrations. The calculated surface densities varied from 0.00025 to 2.5 μg SAK-c(RGDfC) /cm2 area of the coated solid surface.
Example 3
Cell cultivation
In cell cultivation experiments, the solid materials described in Example 2 were used.
For positive control of cell attachment, surfaces were coated with native extracellular matrix proteins, fibronectin or vitronectin. For negative control, bare polystyrene and glass surfaces were used or surfaces were
coated with SAK, or with that conjugated with linear pentapeptide (RGDfC) or with cyclic pentapeptide with a corrupted RGD motive [c(RADfC)]. For comparison poly-L-lysine coatings were also produced and tested.
Serum-free attachment to the SAK-c(RGDfC) coated surfaces was tested on cell types listed in Table II. Cell attachment was quantified by adhesion assays, modified after Oliver et al (Oliver MH et al, J Cell Sci, 92, 513- 518 (1989)) or by determining the living cell mass by MTT -reduction test (Mossmann T (1983) J. Immunol Methods 65:55-58) after washing down non-attached cells.
The SAK-c(RGDfC) surface provided highly adhesive surfaces for a number of cell types without serum or any exogenous, non-defined biological substances. Except neurons, which did not adhere to it, SAK-c(RGDfC) promoted initial attachment, supported spreading (Fig. 2) and long-term growth of all cells listed in Table II.
Table II shows the cell types used in the cell cultivation experiments:
Table II
All investigated cell types (except neurons) showed increased adhesivity to SAK-c(RGDfC) surface in comparison to surfaces coated either with fibronectin (FN), poly-L-lysine (PLL) or with the control peptides SAK, AK, AK -bound linear pentapeptide (AK-RGDfC), or AK-bound cyclic non-RGD reference peptide AK- c(RADfC) (see Fig. 3) .
According to Fig. 3 the photometric determination of attached cells 1.5 hour after seeding indicated an increased initial adhesion to SAK-c(RGDfC) coating. The adhesivity was density-dependent and reflected the differences in adhesion-preference among different types of cells. The data points were calculated as percentages of optical density values obtained from cultures on bare polystyrene surfaces (100%) and represents averages and standard deviations of data from 4 to 8 identically treated cultures.
For most cell types, 0,025 - 2.5 g/cm2 SAK-c(RGDfC) provided ideal attachment, while the control peptides (SAK, -linear RGDfV, c(RADfC), AK, or PLL) did not support the adhesion.
The assays presented on Fig. 3 were carried out under serum-free conditions, and all biological additives were omitted. The attachment to SAK-c(RGDfC) was not compromised by the absence of serum. Even the highly serum-dependent astroglial cells attached readily to the surface in serum-free conditions, which is shown on Fig. 4. The lack of serum or other biological additives did not impair the initial growth of some of the cells, either. As shown on Fig 5, in a 48-hour culture period, SAK-c(RGDfC) coating (from 0.025 to 2.5 g/cm2 densities) supported the survival of fetal mouse brain derived neural stem cells in the absence of serum. By methylene blue
mass determination or by MTT-reduction (Mossman et al see above) viability tests, the SAK-c( GDfC) surface resulted a two to five-fold survival in comparison to bare polystyrene or to SAK, AK or PLL coated (2.5 g/cm2) surfaces.
Example 3 clearly supports that the integrin ligand cyclic RGDfC pentapeptide if attached covalently to a branched poly-L-lysine/serine-oligo-alanine backbone, proved to promote attachment of a number of different cells to solid surfaces. For attachment, serum or any biological additives could be omitted.
Example 4
Comparative study of solubility and ageing
The solubility data of freshly prepared and 6 months old branched polypeptides with a polymerization rate
DPn<500 are presented in Table III below.
Table III
Qualitative comparison of solubility
The above data clearly support that the SAK polypeptide according to the invention has a better solubility than the previously available AK polypeptide. A further advantage of the SAK polypeptide according to the invention is that it is less susceptible to ageing than a similar AK polypeptide. The difference between the solubilities of the different polypeptides is even more evident if 6 months old polypeptides are compared.
Furthermore, it may be noted that in line with the state of the art, we found that by increasing the polymerization rate, the solubility of the polymers decreased (data not shown).
In conclusion, the SAK polypeptide of the invention was surprisingly found to be superior to the previously disclosed AK polypeptide regarding its solubility and this better solubility was maintained for a longer period of time thus the SAK polypeptide is clearly less susceptible to ageing.
Claims
1. A branched polypeptide conjugate of formula I
poly{Lys(c[Arg-Gly-Asp-D-Phe-Cys(CH2CO)]i-Serj-DL-Alam)} (I)
wherein m is 1 to 7, j is 0.7 to 1.0 and i is 0.01 to 0.5.
2. The branched polypeptide conjugate according to claim 1, wherein m is 2 to 5, j is 0.9 to 1.0 and i is 0.05 to 0.3.
3. The branched polypeptide conjugate according to claim 1 or 2, wherein m is 2.7, j is 0.9 and i is 0.1.
4. The branched polypeptide conjugate according to any of claims 1 to 3, wherein the degree of polymerization is below 500.
5. The branched polypeptide conjugate according to claim 4, wherein the degree of polymerization is from 200 to 400.
6. The branched polypeptide conjugate according to claim 5, wherein the degree of polymerization is about 300.
7. The branched polypeptide conjugate according to any of claims 1 to 6, for use in therapy.
8. The branched polypeptide conjugate according to any of claims 1 to 6, for use in diagnosis.
9. Use of a branched polypeptide conjugate according to any of claims 1 to 6 for promoting adhesion of cells to a solid surface.
10. Use of a branched polypeptide conjugate according to any of claims 1 to 6 for promoting adhesion of cells to an implant.
11. The use according to claims 9 or 10 wherein said cells are stem cells.
12. The use according to claim 10, wherein said implant is a dental, joint, bone or vascular implant or a scaffold material serving for growing artificial tissue or organ.
13. Use of a branched polypeptide conjugate according to any of claims 1 to 6 for coating a solid surface.
14. The use according to claim 13, wherein said solid surface is comprised in a tissue culture vessel, in a cellular bioreactor or in a fermentor.
15. An implant comprising a solid surface coated with the branched polypeptide according to any of claims 1 to 6.
16. The implant according to claim 15 for use in therapy.
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US10066204B2 (en) * | 2013-03-15 | 2018-09-04 | Wisconsin Alumni Research Foundation | Chemically labile peptide-presenting surfaces for cellular self-assembly |
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