Bioconjugate Chem. 2008, 19, 617–625
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Synthesis of Multifunctional Polyvinylsaccharide Containing Controllable Amounts of Biospecific Ligands V. Korzhikov,† S. Roeker,‡ E. Vlakh,† C. Kasper,‡ and T. Tennikova*,† Institute of Macromolecular Compounds, Russian Academy of Science, Bolshoy pr. 31, 199004, St. Petersburg, Russia, and Institut für Technische Chemie der Universität Hannover, Callinstr. 3, 30167 Hannover, Germany. Received October 16, 2007; Revised Manuscript Received December 20, 2007
In the present study, the attempt to synthesize a multibiofunctional polymeric vector to be used for construction of composite scaffolds for bone tissue engineering has been undertaken. The polymers based on 2-deoxy-2methacrylamido-D-glucose were functionalized by a growth factor (BMP-2), GRGDSP peptide, and poly(L-lysine) using aldehyde chemistry. The covalent modification process was quantitatively studied, and a polymer conjugate containing all these ligands was formed. In addition, the impacts of coupled ligands toward the adsorption of polymers on the commercial mineral macroporous matrix Sponceram used in cell culture applications were studied.
1. INTRODUCTION For a long time, biomaterials used for surgical purposes were generally intended to remain inert, thus, unaffected by the reactions with surrounding tissues (1–7). While these approaches were useful in many applications, numerous investigators have been currently addressing the means by which biological recognition might be imported (3, 8–15). Therefore, it may be possible to utilize such biologically active materials to control cellular interactions and cell function. For example, the new approach for construction of three-dimensional (3-D) composite scaffolds for bone tissue engineering (16) supposes the use of biofunctional polymers. In this case, a suitable mineral support should be adsorptively covered by a biocompatible but nondegradable macromolecular compound bearing covalently coupled special ligands responsible for cell adhesion, growth, and differentiation. In this manner, the general idea of this work is the functionalization of the matrix surface in order to intensify in Vitro or/and in ViVo tissue formation. The biomolecules of interest are growth factors (17–19) and adhesive peptides (20–23). The former are polypeptides involved in the regulation of a variety of cellular activities such as growth and differentiation. Bone morphogenetic proteins (BMP) belonging to the transforming growth factor-β (TGF-β) family and regulating main cellular functions can be proposed as the most probable candidates for our goals. The latter are the so-called RGD peptides. They represent integrin-binding mediators and contain the Arg-Gly-Asp sequence in their chemical structure. Modification of the scaffold surface with RGD peptides may enhance cell adhesion to the matrix and activate integrin signaling pathways. Additionally, different oligo- and polycations, such as poly(L-lysine), could be used to simplify a nonspecific electrostatic cell adhesion. There are few basic requirements that should be met while designing the polymer for the purpose described in ref (24). First, the polymer has to be biocompatible. Second, it has to possess the required molecular mass (MM). This means that, on one hand, the polymer has to be of sufficient MM to exhibit * Correspondence to: Prof. Dr. Chem. Sci. Tennikova T. B. Bolshoy pr. 31, 199004, St. Petersburg, Russia, tel: (812) 323-10-50, fax: (812) 323-68-69, e-mail:
[email protected]. † Russian Academy of Science. ‡ Institut für Technische Chemie der Universität Hannover.
a tendency to be adsorbed on the mineral matrix, and on the other hand, the polymer MM should not hinder its removal from the body (usually, MM ) (20–30) × 103). Finally, the polymer has to have sufficient adsorption capability with regard to the mineral supports and contain reactive groups for ligands covalent conjugating. For these purposes, the use of a new type of polymer known as polyvinylsaccharides (PVS) (25–31), which are carbon backbone polymers containing carbohydrate residues as side groups, looks very promising. The presence of saccharide groups in such polymers gives the possibility of forming a structure that may induce a significant increase of specific interactions (32–39) in cells with a scaffold surface. As the reactions of bioligand covalent coupling should proceed at mild conditions without yielding toxic byproducts and considering the fact that proteins and peptides contain amino groups, aldehyde chemistry was chosen for this purpose. In our previous studies, the new hydrophilic polymers based on 2-deoxy-N-methacrylamido-D-glucose (MAG) with a controllable amount of aldehyde groups and the evidence for those to be adsorbed on several mineral supports were obtained (40). Nevertheless, the important issue in our studies is the supervised functionalization of the mineral matrix surface, which could be achieved by quantitatively controllable coupling of special ligands to the polymer prior to its adsorption on a solid matrix. Thus, the biological response of the system might be governed. Moreover, the combination of several bioligands with different functions on one polymer chain is of special interest. In the present work, the detailed study of the obtained polymer modification with the bone morphogenetic protein (BMP-2) model, namely, RNase, RGD-peptide, and poly(L-lysine), as well as the synthesis of a polymeric conjugate containing all these ligands on one polymer chain were carried out. The impact of the introduced ligands on the adsorption parameters of polymers was evaluated. In addition, in order to assay the applicability of synthesized conjugates in bone tissue engineering, the cytotoxicity of the initial and modified polymers was examined.
2. EXPERIMENTAL SECTION 2.1. Materials and Instruments. All buffer salts and other reagents of pro analysi (p.a.) quality were purchased from Fluka (Buchs, Switzerland) and Sigma (Taufkirchen, Germany). The
10.1021/bc700383w CCC: $40.75 2008 American Chemical Society Published on Web 03/04/2008
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dialysis bags Spectra/Pore (MWCO: 1000) used for polymer desalting were purchased from Spectra (USA). The separation of nonreacted low- and high-molecular ligands from polymer conjugates was performed in spin-columns (VIVASCIENCE, Sartorius Group, Göttingen, Germany)smembrane MWCO: 3000, 30 000. For adsorption studies, the macroporous monolithic Sponceram (Sp) (doped ZrO2 ceramic material; pore size 600 µm; surface area ∼1.4 m2/g) kindly donated by Zellwerk GmbH (Germany) was used as a mineral support. Poly(L-lysine) (pLL) of MM 15 000 and ribonuclease A (RNase) were the products of Sigma (Germany). Bone morphogenetic protein-2 (BMP-2) was a kind gift of Prof. Dr. W. Sebald (Institut für Physiologische Chemie, Universität Würzburg, Germany). For UV–vis measurements, the full-spectrum UV/vis spectrophotometer NANO-DROP ND-1000 (peQLab Biotechnologie GmbH, Germany) was used. The fluorescence measurements were performed using Multiscan Fluorimeter FLUOROSCAN ASCENT (Thermo Electron Corp., UK). 2.2. Methods. 2.2.1. Polymer Synthesis and Aldehyde Group Generation. The samples of water-soluble 2-deoxy-2methacrylamido-D-glucose (MAG) homopolymer (pMAG) and its copolymer with 1-vinyl-2-pyrrolidone (VP) and acrolein diethylacetal (DAAc) (p(MAG-co-VP-co-DAAc)) were used as functional vectors. 2.2.1.1. Synthesis of pMAG. The synthesis of MAG polymer wasperformedsimilarlytoapreviouslypublishedprocedure(31,40). MAG (6 g, 0.024 mol) was dissolved in 57 mL of DMF, and 0.3 g (5 wt % from MAG amount) of AIBN was added. The reaction mixture was purged with nitrogen. Polymerization was carried out in a sealed ampule at 60 °C for 24 h. The product was precipitated in diethyl ether. After filtration and vacuum drying, 5.7 g (95%) of polymer was obtained. The specific viscosity was measured in 0.1 M Na2SO4 at 25 °C and was found to be equal to 0.11 dL/g that corresponded to MMη ) 21 000 ([η]25 °C ) 8.29 × 10–4. M0.49). MMw(light-scattering) was equal to 25 800. 2.2.1.2. Synthesis of p(MAG-co-VP-co-DAAc). The samples of p(MAG-co-VP-co-DAAc) were synthesized by free radical polymerization of comonomers in the presence of AIBN in DMF (40) as described above. The comonomers ratio was [MAG]: [VP]:[DAAc] ) 15:45:40 mol %. The copolymerization proceeded for 72 h. The resulting polymer product was purified Via dialysis for 24 h against water and then lyophilized. MMw (light-scattering) was found to be equal to 13 500. 2.2.1.3. Oxidation of pMAG. The synthesis of dialdehyde derivatives of pMAG was carried out as follows (40): 0.5 g (0.002 mol) of pMAG was dissolved in 80 mL of water and cooled down to 5 °C. Then, the sodium periodate was added in an amount corresponding to the desirable amount of aldehyde groups to be obtained. Generally, [NaIO4]:[MAG] ) 0.5 and 0.7 mol were used to obtain 20 and 30 mol % of aldehyde groups. The reaction was allowed to proceed for 24 h in the dark at 5 °C. Then, the product was purified by dialysis against water (24 h) and dried by lyophilization. The appearance of aldehyde groups in oxidized polymer was registered via IR spectra measurements. There are two small peaks, 1755 and 1735 cm-1, at the foot of wide amide carbonyl band, which might be referred to the absorption of two aldehyde groups arising from R-glycol cleavage. 2.2.1.4. ActiVation of p(MAG-co-VP-co-DAAc). The activation, e.g., the removal of blocking acetal groups, of p(MAGco-VP-co-DAAc) was carried out in aqueous solution of hydrochloric acid containing 5 mg/mL of polymer at 50–60 OC for 2 h, pH 2.0. After the reaction completed, the solution of activated polymer was dialyzed for 12 h against water and
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lyophilized. In the IR-spectra of activated p(MAG-co-VP-coDAAc), the evident swell (in comparison with pMAG spectra) at the foot of the amide carbonyl band is observed that might be referred with confidence to the absorption of aldehyde groups. 2.2.2. Analytical Methods. 2.2.2.1. Quantification of Aldehyde Groups (Schiff’S Test). The molar amount of aldehyde groups was determined by a reaction of polymers with fuchsinsulfite reagent (Shiff’s reagent). For this purpose, the calibration curve was plotted using glutaric dialdehyde as a standard, and the extinction coefficient of CHO-groups was calculated. The analytical procedure was carried out as follows: to a sample of 0.5 mL of aldehyde containing substance, 2.5 mL of Shiff reagent was added, and the absorbance of colored complex was measured at 550 nm in 40 min. 2.2.2.2. Iodine test. The molar amount of VP in p(MAGco-VP-co-DAAc) and the concentration of this copolymer at adsorption studies was determined using iodine test. For that, 3 mL of 3 mM iodine in 0.2 M sodium acetate buffer, pH 4.6, was added to 0.5 mL of polymer solution. The absorbance of the colored complex was measured immediately at 460 nm. 2.2.2.3. UV Measurements. In addition to the Schiff’s test, the direct measurements at 265–270 nm were used to quantify the amount of the polymers and their conjugates with high- and low-molecular mass ligands. 2.2.3. Polymer Modification. All covalent coupling reactions were allowed to proceed at room temperature for 2 h with stirring (400–700 rpm). After polymer functionalization, all modified samples were treated with sodium borohydride, then purified by spin-column dialysis (membrane MWCO: 3000, 30 000) and stored in a refrigerator at 4 °C. 2.2.3.1. Coupling of Low-Molecular Mass Ligand (GRGDSP– Peptide). In this case, the molar proportions of polymer CHO groups and NH2 groups of peptide were used. 1, 2, 5, and 10 equiv (in the case of p(MAG-co-VP-co-Ac) modification) and 1 and 2 equiv (in the case of oxidized pMAG modification) of GRGDSP-peptide, calculated according to the molar content of polymer CHO groups, were dissolved in 0.5 mL 0.01 M sodium borate buffer, pH 10.0, and added to the same volume of activated polymer containing 1 equiv of aldehyde groups (aqueous solution, pH 10.0). To prove quantitatively the covalent coupling of peptide to the polymer, a fluorescent test was carried out. For that, 5 equiv of dansylcadaverine were dissolved in 2 mL of 7 M urea solution containing 7.5% MeOH and added to 1 equiv of polymer-peptide conjugate dissolved in 2 mL of sodium borate buffer, pH 10.0. Then, 10 equiv of water-soluble 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) dissolved in 1 mL of urea was introduced. The reaction proceeded for 40 min at room temperature. Low-molecular mass components of the reaction mixture were separated by spin-column dialysis, and the determination of fluorescent emission of bound to the peptide marker was carried out at 485 nm (corresponding to excitation wavelength of 355 nm). The preliminary calibration curve for free dansylcadaverine allowed quantitative determination of peptide amount bound to the polymer. 2.2.3.2. Coupling of Macromolecular Ligands. In the case of coupling of proteins and pLL, both containing numerous amino groups, for the reaction mixture preparation the mass proportions were used. SDS-PAGE characterization of modified products was carried out using 16% gels prepared according to the standard procedure (41). Silver staining was used for oxidized pMAG, proteins, and conjugate zone detection (16, 41). (a) Poly(L-Lysine). To study the coupling of pLL to the polymers, the following model experiment was performed. First, the labeling of pLL with FITC was done. For this, 6 µL of fresh FITC aqueous solution, 1 mg/mL, was added to 1 mg/
Multifunctional Polyvinylsaccharide Containing Biospecific Ligands
mL solution of pLL in 0.01 M sodium borate buffer, pH 10.0. The reaction proceeded for 1 h, and the product was then purified from nonreacted FITC by spin-column dialysis (membrane MWCO: 3000). After that, the calibration curve from fluorescence measurement for the detection of pLL labeled with FITC was plotted. In the second stage of this experiment, the reaction of pLL-FITC with activated polymers at different mass proportions was carried out. 1 mass equiv of pLL-FITC conjugate dissolved in 0.01 M sodium borate buffer, pH 10.0, was added to 1 mL solution containing 5, 10, 20, or 40 mass equiv of activated polymer (borate buffer, pH 10.0). The reaction was allowed to proceed for 2 h with slight stirring (550 rpm). The resulting polymer conjugate was purified from nonreacted pLL-FITC via spin-column dialysis (membrane MWCO: 30 000), and the amount of Lys units coupled to the polymer was evaluated by fluorescence measurement. (b) Ribonuclease A and BMP-2. RNase A was chosen as a cheap BMP-2 model because of their similar isoelectric points and close molecular masses. Quantitative studies of RNase coupling to aldehyde-bearing polymers were carried out using the same model experiment as was made in the case of pLL. 10 µL of FITC aqueous solution, 1 mg/mL, was added to 2 mg/mL solution of protein in 0.01 M sodium borate buffer, pH 10.0. The labeling reaction was allowed to proceed for 1 h, and the product was purified by spin-column dialysis (membrane MWCO: 3000). For polymer modification, 1 mass equiv of protein-FITC conjugate dissolved in 0.01 M sodium borate buffer, pH 10.0, was added to 1 mL solution containing 2, 5, 10, and 20 mass equiv of activated polymer (borate buffer, pH 10.0). After the spin-column dialysis (membrane MWCO: 30 000), the fluorescence of polymer-(protein-FITC) conjugates was measured, and the molar amount of protein coupled was evaluated using the previously plotted calibration curve. For construction of composite matrices for cell culture experiments, about 400 mass excess of aldehyde-bearing polymer per amount of BMP-2 was used. 2.2.2.3. Synthesis of Triple Conjugate. The construction of multifunctional polymer conjugate containing RNase, pLL, and RGD-peptide was performed via “step-by-step” addition of ligands to the polymer solution and using the quantitative methods described above. All reactions were carried out in 0.01 M sodium borate buffer, pH 10.0. On the final step, the excess of sodium borohydride was added in order to reduce Schiff’s bases and possibly unnreacted aldehyde groups. (a) Coupling of RNase-FITC to Oxidized pMAG. In the first step, 1 mL of oxidized polyMAG solution, 2 mg/mL, was added to 0.1 mL of RNase-FITC solution, 1 mg/mL ([pMAG]: [RNase] ) 20:1 mass proportion). The reaction proceeded with slight stirring for 1 h. The product was purified by spin-column dialysis (membrane MWCO: 30 000) and two 0.1 mL aliquots were taken for fluorescence measurements. (b) Coupling of pLL–FITC to pMAG–(RNase–FITC) Conjugate. In the second step, 1 mg/mL, 1 mL of polyMAG–(RNase– FITC) conjugate solution, 1.6 mg/mL, was added to 0.08 mL of pLL–FITC solution ([pMAG]:[pLL] ) 20:1 mass proportion). In all cases, the concentration of polymer was considered to be the concentration of the whole conjugate. The reaction, purification, and fluorescence measurements were realized in the same way as in the case of RNase. (c,d) Coupling of RNase-FITC and pLL-FITC to p(MAGco-VP-co-Ac). The modification of p(MAG-VP-Ac) with RNase and pLL was carried out in the same way as was done with oxidized pMAG. The only difference consisted of a larger excess of polymer used in each step, namely, [p(MAG-co-VP-co-Ac)]: [ligand])30:1 mass proportion. (e) Coupling of RGD-Peptide. 0.2 mL of RGD-peptide solution, 1 mg/mL, was added to both conjugates of polymers
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with RNase-FITC and pLL-FITC. The reaction proceeded for 1.5 h with slight stirring. The product was purified by spincolumn dialysis, and the residual solution passed through membrane (MWCO: 3000) was measured at 226 nm. Thus, the amount of nonreacted peptide was evaluated, and the molar quantity of peptide coupled to the polymer was calculated using the previously plotted calibration curve. 2.2.4. Adsorption Studies. In all cases, the study of adsorption kinetics and isotherms building were performed at static conditions. Despite the experiments on covalent modification of aldehyde-bearing polymers by amino-containing ligands being carried out in 0.01 M sodium borate buffer, pH 10.0, it is essential for adsorption studies to be performed at physiological conditions, e.g., at pH 7.0. Therefore, 0.01 M sodium borate buffer, pH 10.0, adjusted to pH 7.0 by boric acid (called borate solution, pH 7.0) was used in all experiments. 2.2.4.1. Adsorption Kinetics and Desorption Studies. 1.5 mL of polymer (or conjugate) solution with concentration 1 mg/ mL in borate solution, pH 7.0, was added to 50 mg of mineral support. The adsorption proceeded with slight stirring (300–400 rpm) at 25 °C for 3 h. At regular intervals, the content was centrifuged, and the amount of adsorbed polymer was estimated by measuring its concentration in the supernatant using the methods described above (reaction with Shiff’s reagent, iodine test, and UV measurements). The control of possible polymer desorption was carried out by incubation of mineral supports in 0.01 M PBS 7.0 over 14 days using the same quantitative methods to evaluate the desorbed amount of polymer derivatives. 2.2.4.2. Isotherm Building. For this purpose, several samples containing 50 mg of mineral support each were used. The concentrations of polymers (conjugates) solutions in borate solution, pH 7.0, varied from 0.25 to 3.5 mg/mL. The samples of Sponceram were incubated for 2 h (the time corresponding to the plateau on the kinetics curves) at 25 °C in polymer solution, and the amount of adsorbed polymer at fixed concentration was determined as mentioned above. 2.2.4.3. Statistics. For each point, both for kinetics measurements and isotherm building, the set of three equivalent experiments (n ) 3), with a pure Sponceram as a control, were performed and three UV–vis measurements were done for each sample. The presented results are given as a mean values and the average deviation is 5%. 2.2.4.4. Adsorption of Polymers and Conjugates for Cell Culture Experiments. For cell culture experiments, the adsorption of initial polymers was performed in 15 mL tubes containing 20 pieces of Sponceram 15–20 mg each, and 5 mL of sterilized (0.2 µm filter) polymer solution in sodium borate buffer, pH adjusted to 7.0, with concentration 0.5 mg/mL was added to the tubes. The tubes were slightly stirred (300 rpm) or rotated (1 rpm) at least overnight. Then, the solution of the polymer was removed, and the pieces of Sponceram were washed twice with phosphate buffering saline (PBS), pH 7.0. Subsequently, the composite materials were incubated with cell culture medium for 2 h at room temperature or overnight at 4 °C. The conjugates adsorption was carried out in a similar way, except the 2 mL tubes and 1.5 mL of 1.0 mg/mL or 0.5 mg/mL solutions were used. 2.2.5. Cell Culture Experiments. 2.2.5.1. CultiVation of MC3T3-E1 Cells. MC3T3-E1 cells were stored in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal bovine serum and antibiotics. For assays in 96-well plates, 5000 cells per well were seeded. For cell seeding onto Sponceram, 15–20 mg pieces of Sponceram were shaken (350 rpm) with 20 µL of a cell suspension (1 mio cells/mL) for 30 min at 37 °C, 5% CO2. The Sponceram pieces were sterilized by auto-
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Table 1. Modification of pMAG (30 mol % CHO groups, MM 20 000) and p(MAG-co-VP-co-Ac) (7 mol % CHO groups, MM 13 500) with GRGDSP-Peptidea [CHO]polym:[NH2]GRGDSP mol ratio
[CHO] group conversion, mol % pMAG
1 0.5
75 94 p(MAG-co-VP-co-Ac)
1 0.5 0.2 0.1
40 50 63 93
Reaction conditions: 0.01 M sodium borate buffer, pH 10.0, 25 °C, 2 h stirring (550 rpm). Purification: spin-column dialysis (MWCO: 3000). The aldehyde groups conversion (the amount of peptide bound to the polymer) was determined by fluorescent test with use of dansylcadaverine as a fluorescent label (λex.) 355 nm, λem.) 485 nm). a
claving and incubated with cell culture medium for at least 2 h before cell seeding. 2.2.5.2. MTT Assay. MTT assays were performed with n ) 5 and one negative control. The matrices were placed into new wells to measure exclusively the viability of the cells adherent to the Sponceram but not those on the bottom of the well. Cells/ matrices were incubated with 100 µL of cell culture medium and 10 µL MTT (5 mg/mL in PBS) for 4 h at 37 °C, 5% CO2. Formazan crystals were dissolved overnight with 10% SDS in 0.01 M HCl. The formazan absorption was measured at 570 nm subtracted by the 630 nm absorption. 2.2.5.3. Statistical Analysis. A p value of
