Scientific paper Determination of the Interactions Between Zn2+ and Water Soluble Polymer Ligands with Potential Use in Controlled Drug Delivery Nada Verdel,1* Andrej Krzan,2 Mojca Bencina2 and Majda Zigon2 1 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia 2 National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: nadaverdel@gmail.com Received: 28-01-2013 Abstract Biodegradable copolymers of aspartic and lactic acids were synthesized for potential use in controlled drug release. The proportion of aspartic acid moieties in the copolymers was 0.9 and 0.1, the molecules were partially branched and had absolute molar masses over 100,000 g/mol. The drug could be attached to the copolymer via metal (particularly zinc) ions, so a method to estimate the interactions between zinc ions and the water-soluble polymers by fluorescence spectroscopy was developed. The stability constants of binding of zinc and the concentrations of zinc bound to polymer were determined. The results confirm that zinc ions at pH 6 preferentially bind to side groups of aspartic acid units of the copolymers. Keywords: Polyamino acid, controlled drug delivery, biodegradable carrier, fluorescence spectroscopy, zinc, polymer coordination compound. 1. Introduction This study focused on biodegradable polymers that can potentially be used for pharmaceutical purposes in controlled drug release. Polymers as drug carriers are used in two types of delivery systems, colloidal carriers and polymer-drug conjugates. In colloidal formulations, the polymer encapsulates drug within micro- or nanopar-ticles.1 In polymer-drug conjugates, the drug is covalently bound to the polymer.2 For such purposes, the drug could be attached to the polymer via dative covalent bonds or coordinate bonds with metal ions like zinc, and for the polymer components lactic and aspartic acid moieties could be combined. Poly(aspartic acid), PA, is a typical hydrophilic biodegradable polymer with aspartic acid monomer units. The most common method for the synthesis of PA involves the polycondensation of aspartic acid into polysucci-nimide, followed by its hydrolysis to sodium polyasparta-te. The hydrolyzed form, sodium polyaspartate, has hydrophilic carboxylate side groups that can interact with metal ions.3 It degrades quickly under physiological and biological conditions. However, it is insoluble in organic solvents and does not have thermoplastic properties, which makes it difficult to obtain PA films or mouldings; it can only be processed as an aqueous solution or in a hygroscopic powder form.4 On the other hand, polylactide or poly(lactic acid), PLA, is one of the most intensively studied biodegradable thermoplastic polymers and contains lactic acid monomer units. PLA is hydrophobic and soluble in organic solvents. It is currently used in a number of biomedical applications, such as sutures or fibres,5 stents, implants for bone fixation6 and drug delivery devices.7 It is also being evaluated as a material for tissue engineering.8 One of the shortcomings of this polymer is that there is no pendant functional group on the backbone. One of the possibilities to combine the advantages of both PLA and PA derivatives is to make amphiphilic copolymers.4 PA can bind metal ions and is water-solub-le,9 whereas PLA has good mechanical properties and dissolves in organic solvents. By changing the ratio of aspar-tic and lactic acid units we can therefore obtain amphiphi-lic copolymers to which a drug could be attached via zinc ion bridges. For many years zinc has been used to treat epithelial disorders, ranging from wound healing to diarrhea and ulcerative colon disease.10 It is found in all body tissues, with ~85% of the whole body zinc in muscle and bone, and another 11% found in the skin and liver.11 Typically, humans appear to have the capacity to regulate whole body zinc content over a ten-fold change in intake.12 Zinc ions bind to copolymers of aspartic and lactic acids to form polymer coordination compounds. In polymer coordination compounds metal ions interact with li-gand groups situated on the polymer chains. The kind and strength of the metal ion to ligand interaction can be described by stability constants. Stability constants of polymer coordination compounds can be calculated with the help of many theoretical models. For our system, the most suitable model is described by Flory's concept of infinitely large polymer chains.13,14 In this paper we report a method of determining the interactions between polymer ligands and Zn2+ in aqueous solutions by fluorescence spectroscopy. To determine the concentration of free zinc, a fluorescent indicator that enables minimal interference with the equilibrium of zinc, was used. As zinc carriers copolymers of aspartic and lactic acids were prepared and the properties of the copoly-mers for potential use as carriers in chemically controlled drug release were characterized. 2. Experimental 2. 1. Materials L-aspartic acid (> 98%), L-lactic acid (> 85%), L-lactide - the cyclic dimer of lactic acid (98%), N,N-di-methylformamide (p. a., 99%) and lithium bromide, LiBr, (> 99%), purchased from Aldrich, N,N-dimethylacetamide, DMAc, (99%), purchased from Merck and zinc chloride (p. a., 98+%), purchased from Fluka, were used as received. Benzoylated dialysis tubing with pore size of < 1,200 g/mol was purchased from Sigma Aldrich. Sodium poly((a,P)-D,L-aspartate) (40 w.%) was purchased from Aldrich, with FTIR: v = 2600-3600 (N-H and O-H), 1598 (C=O, car-boxylate), 1648 (amide I), 1240 (amide III), 670 (amide V), 2867 and 2930 (C-H and CH2) and 3086 cm-1 (-HC=, ma-leamic acid) and 1H NMR (in D2O): 5 = 4.7 (a-CH), 4.5 (P-CH), 2.76 (P-CH2) and 2.65, 2.55 ppm (a,P-CH2). The degree of ionization according to Saudek15 was 1. The fluorescent dye Fluozin-1, N-(2-methoxyphenyl)iminodiacetic acid indicator, "cell impermeant", purchased from Molecular Probes, was dissolved in twice deionized Milli-Q water and kept protected from light at -20 °C. 8 g of N-(carba-moylmetyl)iminodiacetic acid buffer (ADA) (Kd(Zn2+) = 127 nmol/L at an equimolar ratio of Zn2+ and ADA, 20 °C, pH 7 and ionic strength 0.1 mol/L), purchased from Sigma, was dissolved in 50 mL 1 N NaOH. 2. 2. Syntheses Poly(succinimide-co-lactic acid), PSL, copolymers were synthesized in a 100-mL glass flask that was deoxy-genated by continuous degassing and back-filling with argon, except during the third step of synthesis when a vacuum pump was applied (Table 1). The reaction mixture consisted of aspartic acid and lactic acid or lactide without addition of catalysts or solvents. ALa copolymers were synthesized by reaction of aspartic and lactic acids, and ALt copolymers with aspartic acid and lactide. The reactions proceeded similarly to the procedure with aspartic acid and lactide reported by Shinoda et al.4 under the reaction conditions summarized in Table 1. Upon heating the reaction mixtures changed into viscous liquids, which were very difficult to stir. At the end of the syntheses the reaction flask was taken out of the oil bath and cooled to 45 °C. The products were purified by dissolution in the minimal quantity of DMF and the filtrate was poured into distilled water to precipitate the polymer. The yellowish white polymer powder was dried for 12 hours at 80 °C in an oven. FTIR (KBr): v = 1750 (C=O, ester), 1790, 1720 (C=O, succinimide), 1660-1630 (amide I), 1550-1530 (amide II), 1290-1260 (amide III), 640 (amide V), 3700-3000 (N-H and O-H), 2990-2945 (C-H and CH2, stretching vibrations), 1130 (C-O, ester) and 1400 cm-1 (CH2, deformational vibrations). In the next step, amphiphilic poly(sodium a,P-as-partate-co-lactic acid) (PAL) copolymers were prepared by hydrolyses of cyclic succinimide rings to more hydrop-hilic aspartic acid units bearing carboxylate groups. The hydrolysis were performed with 1 M NaOH added drop-wise. The products were purified by three days of dialysis with exchanging distilled water twice per day where compounds with < 1,200 g/mol were separated from compounds having molecular weight over 2,000 g/mol. FTIR (KBr): v = 1600 (COO-, carboxylate), 3400 (N-H and NH2), 1240 (amide III) and 670 cm-1 (amide V). Table 1: Reaction conditions for the synthesis of copolymers of aspartic (A) and lactic acid (La) or lactide (Lt) sample N A La Lt A/Lafeed temperature (°C) / time (h) I. step II. step III. step IV. step melting cooling vacuum (~ 4 mbar) further reaction AM 4 16 17 / 0.95 180 / 2.5 180-40 / 0.5 40 / 0.3 180 / 2.5 AL 3 42 / 245 0.17 180 / 2.5 180-160 / 0.5 160 / 18 / N = number of parallels, A/Mfeed = feed molar ratio 2. 3. Characterization Methods Fourier transform infrared spectroscopy, FTIR, was carried out with a Perkin-Elmer 1725X FTIR spectrometer. Typical FTIR spectra were obtained using KBr pellets or FTIR spectra in aqueous solutions using AgCl plates, resolution 4-cm-1, range 400-4500 cm1, 20 °C and with 10 scans for dry or 300 scans for aqueous samples. Nuclear magnetic resonance, NMR, spectra were recorded on a VXR 300 Varian spectrometer. The solvent for PSL was deuterated dimethyl sulfoxide, DMSO-d6 and the solvent for PAL was D2O with addition of a standard, (CH3)3SiCH2CH2CH2SO3Na, DSS at a temperature of 25 °C and a pulse angle of 90°. Acquisition parameters for 1H NMR spectra were 300 MHz, 35 scans and 5-s relaxation time, and for 13C NMR spectra 75 MHz, 30,000 scans and 2-s relaxation time. Differential scanning calorimetry, DSC, was conducted on a Perkin Elmer DSC-7 in the temperature range from -60 to 200 °C at a heating and cooling rate of 20 and 200 °C/min, respectively. Size exclusion chromatography with a detector for multiple angle light scattering, SEC-MALS, was conducted on a Dawn-DSP detector, laser photometer (He-Ne laser, 633 nm), Optilab-DSP differential refractometer (Wyatt Technology Co.), Perkin Elmer Series 200 pump and a PL Gel Mixed D 5 pm column with pre-column (Polymer Laboratories, Ltd.), with N,N-dimethyl aceta-mide (DMAc) containing lithium bromide (0.1 M) as mobile phase. The nominal flow rate of eluents was 0.8 mL/min. The mass of the samples injected onto the column was typically 0.1-0.2 mg. Data acquisition and evaluation utilized Astra 4.73.04 software (Wyatt Technology). The method is described in detail in Gricar et al..16,17 2. 4. Interactions Between Polymer Ligands and Zn2+ The interactions between polymer ligands and metal ions can be deduced by different analytical methods. By means of chemical methods like elemental analysis the coordination number and atomic structure of polymer coordination compounds can be determined.14 By physi-cochemical methods like potentiometric titration the stability constants and coordination numbers of metal ions can be deduced, whereas by conductometric titration the density of charge distribution, distribution of free metal ions, transport of total charged particles and the conductivity of polyelectrolyte solutions can be determined.18 By spectroscopic methods like FTIR spectroscopy the molecular coordination structures, the position of coordination bonds on linear and crosslinked polymer chains, the special features of conformations, the shapes of functional groups and hydration layers can be defined. And by means of NMR spectroscopy the positions of metal ions on polymer ligands can be deduced.14 Above mentioned methods mostly demand a purified polymer coordination compound with no free metal ions in the solution. However, the equilibrium of free and bound Zn2+ in aqueous solutions of the polymer ligands prevents complete removal of free Zn2+. The extent of the interactions between polymer ligands (L) and Zn2+ was therefore determined by fluorescent spectroscopy where the addition of an appropriate fluorescence indicating dye permits minimal interference with the equilibrium. For this, the indicator FluoZin-1 was used and the resulting fluorescence intensity indicated the concentration of free Zn2+ in the aqueous solution from which the concentration of bound Zn2+ was calculated according to the equation [Zn2+]t = [Zn2+]f + [Zn2+]b. FluoZin-1 is based on N-(2-methoxyphenyl)imino-diacetate chelator with stability constant for binding of Zn2+, Kd(Zn2+) = 8 pmol/L. FluoZin-1 is designed for detection of Zn2+ in the 0.05-50 pmol/L concentration range and exhibits a fluorescence intensity dependent on the concentration of free Zn2+ with no accompanying spectral shift. According to the manufacturer's specification, the dissociation constant of FluoZin-1 is dependent on pH, temperature, ionic strength and viscosity of the solvent, the presence of other ions and compounds, photobleac-hing and the type of spectrofluorometer. Hence, it is important that all these factors are controlled or kept constant throughout the measurements. The measurements by fluorescence spectroscopy were performed on a Perkin Elmer LS50 spectrofluorometer under the following conditions: emission range 500-550, excitation 490, automatic standard employment of photo-multiplier, excitation slit 15 and emission slit 2.5 nm. Before a measurement was performed, the sample was thermostatted in the spectrofluorometer for 10 seconds at 20 °C. A 1 cm square quartz cuvette (0.5 mL) was used. The concentration of the indicator in the cuvette was 11.0 pmol/L and the ionic strength of the solution 0.1 mol/L. At the end of each experiment calibration was made by measurement of Fmin, a blank, and Fmax, the indicator saturation. Relative intensities of fluorescence, F*, were calculated according to the manufacturer's specifications via Equation (1): F* = (F-F . ) / (F -F) ^ mm' V max ' (1) F denotes the fluorescence intensity of the sample. Each series of measurements were calibrated with the calibration curve (F* = f([Zn2+]f)), where [Zn2+]f denotes the concentration of free Zn2+. The slope of the calibration curve of ZnCl2 solutions, k, and F* of solutions of ZnCl2 + polymer ligands, L, were used to calculate [Zn2+]f in solutions of ZnL by Equation (2): [Zn2+]f = k ■ F* (2) In spite of keeping the pH, temperature, ionic strength of the solvent and settings of the spectrofluoro- meter constant, the slope of the calibration curve changed daily. The reason, among others, was the alteration of air humidity. Therefore the instrument was calibrated daily with a new calibration curve. Photobleaching of the indicator was checked by measuring F of the solutions containing ZnCl2 + ADA and altering the duration of indicator exposure to daylight and any radiation in the spectrofluorometer before measurement. The statistical errors for six and four replicate measurements of F of solutions of ZnCl2 + ADA were calculated. Errors in F, F. and Fmax measurements, dF*/F*, we' min max ' ' re calculated via Equation (3); where dF - standard deviation of F, dF . - deviation of F ., dFmax - deviation of min min max F : dF*/F* = (dF + dFmm) / (F-Fmm) + (dF+ dF) / (F- F) (3) The interactions between Zn2+ and polymer ligands were determined by measuring [Zn2+]f in aqueous solutions of ZnL with PA, PAL ALa (90% A) and PAL Alt (10% A) at pH 6. The ZnL solutions were prepared by simply adding ZnCl2 ([Zn2+]t) to the aqueous solutions of polymer ligands without further purification. By this, the exact [Zn2+]t was known. The measurements were carried out at constant [Zn2+]t of 20, 40, 60 and 80 pmol/L on changing the total concentration of polymer ligand, [L]t. The concentrations of PA, PAL ALa and PAL ALt were calculated by knowing the precise volume and weight of the solution and the molecular weight of the average (co)monomer unit, Mn-unit; Mn-unit values were deduced by NMR spectroscopy. Two types of plots, describing linear dependence, were used: • Type 1: [PA]t = f(ln[Zn2+]f), [ALa] = f(ln[Zn2+]f) and [ALt]t = f(ln[Zn2+]f) at constant [Zn2+]t, where "t" and "f" denote total and free, respectively, (Figure 3), • Type 2: [Zn2+]b/[Zn2+]f = f([PA]t), [Zn2+]b/[Zn2+]f = f([ALa]t) and [Zn2+]b/[Zn2+]f = f([ALt]t); Figure 4. The linear relationships in plots of Type 1, [L]t = f(ln[Zn2+]f), describe the links between bound and total Zn2+ from which we could draw plots of Type 2. Plots of Type 2 are Flory's equations for unidentate ligands, independent of their position on the polymer chain, [Zn2+]b/[Zn2+]f = Kl ■ [L]t - KL ■ [L]b, where KL denotes the stability constant of the binding of Zn2+ to L and [L]b the concentration of bound polymer ligands.19 3. Results and Discussion 3. 1. Synthesis and Characterization Amphiphilic biodegradable copolymers with aspar-tic and lactic acid moieties were synthesized as described in the literature4, with some modifications in the preparation procedure. NMR and FTIR spectra show that under the conditions (summarized in Table 1) poly(succinimide-co-lactic acid) (PSL) copolymers were synthesized. By basic hydrolysis of PSL poly(sodium (a,P)-aspartate-co-lactic acid) (PAL) copolymers were prepared. Copolymers with a higher amount of aspartic acid, ALa, were synthesized from aspartic and lactic acid, and copolymers with a higher amount of lactic acid, ALt, from aspartic acid and lactide (Table 2). The PAL ALa copolymers were water-soluble, but insoluble in organic solvents, whereas PAL ALt were amphiphilic. Throughout the hydrolysis of PSL to PAL, the ratio of comonomer units and end groups are preserved.20 Ma-leimide end groups in PSL transform through hydrolysis to maleamic acids and due to the presence of double bonds between carbon atoms, form irregular structures in PSL as well as in PAL (Scheme 1). Succinimide end groups transform through hydrolysis to asparagine end groups and succinamic acid end groups, whereas car-boxy and hydroxy end groups remain unchanged after hydrolysis.21 Shortly, preservation of comonomer units, irregular structures and end groups throughout the hydrolysis were reasons the characterization of copoly-mers was discussed mainly via characterization of PSL (Table 2). poly(succinimide-co-lactic acid) branched structures maleiniide end units succinimide end units Scheme 1. Schematic structures of poly(succinimide-co-lactic acid) copolymers. Table 2. Average characteristics of synthesized copolymers of aspartic and lactic acids ALa and Alt, where the mole ratios between aspartic and lactic acid moieties in the feed stock and in the copolymer are A/Lafeed and A/LaL, the average molar masses of comonomer units are Mn-unit and the number of end comonomer units per copolymer chain and the percentage of end groups per copolymer are Nchain and NC, respectively FTIR (CO) NMR DSC L N (/) A/Lafeed (/) Nchain (/) (ester) (cm-1) (SI) (cm-1) A/LaLa (/) Aa (%) NCb(%) Mn-unit (g/m0l) Tg (°C) ALa 4 0.95 141 c 1787 1718 8 90 8±4 94 58 ± 5 d ALt 3 0.17 390 1759 1723 0.13 10 6 ± 2 75 15 ± 5 N = number of parallels,a = ± 10%, estimated from 13C NMR spectra of PSL in DMSO-d6, b - ± 10%, percentage of end groups in copolymers, de- termined from 1H NMR spectra of PSL in DMSO-rf6, c = band overlapped by other bands; SI = succinimide, d = results of DSC measurements after first heating, because after Tg the copolymers degrade. The structure of PSL copolymers was elucidated by 1H and 13C NMR spectroscopy (Scheme 1) and the signals were consistent with Shinoda's NMR study4 on the copolymers of aspartic and lactic acids, Matsubara's NMR studies22 on thermally prepared polysuccinimides and Es-partero's study23 on low molecular weight poly(lactic acid)s. Two major resonances in the 1H NMR spectra of PSL, corresponding to methyl (k) and methine groups (f, g, h), were observed at 1.6-1 ppm and 5.4-4 ppm, respectively (Scheme 1). The signal of water overlaps the signal of methylene protons. Therefore on addition of CF3COOH to the deuterized solvent containing dissolved PLS,4 the residual water peak displayed a higher chemical shift (approx. 8.8 ppm) and the characteristic multiplets at 3.8-2.2 ppm were assigned to the methylene protons (i) of succinimide. The methine protons of succinimide (i) at 5.4-4.5 ppm overlap with the PLA methine resonances (f). Resonances at 9.3-7.6 ppm indicate amide protons (-CONH-) (e) in branched laurylamide structures and a-, 0-succinimide open units. The small resonance at 7.05 ppm indicates the presence of maleimide end groups (d), that at 11.5 ppm succinimide end groups (c), and the resonances at 5.5 and 12-13 ppm -OH (a) and -COOH (b) lactic acid end groups, respectively. 13C NMR spectra (Figure 1) show signals of succinimide CO at 178-170 ppm, ester CO at 174-169 ppm and amide CO at 171-166 ppm. The lactic acid and succinimi-de CH were assigned to 72-67 ppm and 48-47 ppm, respectively. The succinimide CH2 groups absorbed at 35-33 ppm and CH3 lactic acid at 21-16 ppm. The 13C NMR spectra of ALa and ALt copolymers mainly differ in the CH and CO regions. ALa spectra show intense signals of succinimide CH and CO groups, whereas in ALt spectra, the signals of lactic acid CH and CO groups are predominant. The differences in the chemical shifts and intensities of signals in the NMR spectra indicate (in accordance with the FTIR spectra) different ratios of aspartic and lactic acid in the copolymers ALa and ALt (A/LaL) as compared to the feed ratios. In 1H NMR spectra of PSL the ratio of CH2 and CH3 integrals is equal to A/LaL. However, the signals of DMSO protons overlap the CH2 region, so the A/LaL ratio was approximately (± 10%) estimated from 13C NMR spectra by integrating the succinimide and lactic acid CH signals (Table 2). The percentage of end groups in the copolymers, NC, (Table 2) was estimated from 1H NMR spectra with ± 10% precision, due to overlapping signals. The proton sig- a) b) Figure 1. 13C-NMR spectra of PLS ALa and ALt in DMSO-d6, CO (a) and CH region (b). nals, representing end units and all comonomer units, were integrated and NC was calculated using the equation NC = (a + b + c + d/2) / (f + g + h + d/2) (Scheme 1). NC, thc absolute number average molar mass determined by SEC-MALS, Mn, and the average molar mass of comonomer units, Mn-unit, were used to calculate the number of end co-monomer units per copolymer chain via Nchain = Mn ■ NC / Mn-unit (Table 2). With Mn > 100,000 g/mol, NC from 8 to 9% and Mn-unit from 75 to 90 g/mol, the number of end units per chain was > 100. More than one hundred end co-monomer units per chain indicates a branched copolymer structure. 3. 2. Interactions Between Polymer Ligands and Zn2+ Fluorescence spectroscopy is a relative method, based upon a calibration curve (F* = f([Zn2+]f)), where F* denotes the relative fluorescence intensity and is calculated via Equation (1). Polymer ligands for binding of Zn2+ ions were PA with 100% aspartic acid units (A), PAL ALa (90% of A) and PAL ALt (10% of A), Table 2. The fluorescence method was critically evaluated with regard to photobleaching of the indicator FluoZin-1, precision, accuracy, linearity, Fmin and Fmax. The results indicated no photobleaching of FluoZin-1 over a time period of 300 s, which is long enough to accurately prepare the sample solutions. The precision of the measurements was tested with preparations in 6 and 4 replicates and measurements of solutions containing 50 and 36 pmol/L free Zn2+ of [Zn(ADA)2] solutions, respectively; N-(carbamoyl-methyl)iminodiacetic acid buffer (ADA) was used to mimic the conditions in polymer solutions. The error dF*/F* (Equation (3)) was ± 2.5% and the relative standard deviation less than ± 1%. Deviations from the average of duplicate measurements of F of ZnL solutions showed an accuracy of ± 2.5% (Table A3 in the Supplementary material, column [Zn2+]f**/<[Zn2+]j>), which is not higher than the dF*/F* error range mentioned above. Therefore the fluorescence measurements were performed at four concentrations of total zinc ions and more than ten concentrations of total polymer ligands in duplicate for each L and the error was attributed to be ± 2.5%. The accuracy of the proposed method was evaluated by comparing the slopes of the calibration curves from solutions of [Zn(ADA)2] with those of ZnCl2. With ordinate cross-sections at 0, the slopes of the calibration curves of [Zn(ADA)2] and ZnCl2 were equivalent to 0.041 and 0.036, respectively (Figure 2). With regard to simplified calculations of [Zn2+]f in solutions of [Zn(ADA)2] (Equation (A1) in the Supplementary material), we consider the slopes to be in good agreement and can be used to calculate total Zn2+ in aqueous copolymer solutions. The solutions of ZnL were therefore prepared without addition of ADA. with ADA „ » X*' without ADA 0 10 2D 30 40 50 60 concentration of free / ■*':. ii .1J Figure 2. Calibration curves of [ZnCl2] and [Zn(ADA)2], pH 6, [Zn2+]f 0-55 |mol/L. The deviation from linearity of the calibration curve of ZnCl2 solutions in the concentration range from 0 to 100 |imol/L Zn2+, R2, was 0.998. F. and Fmax of • ' ' min max [Zn(ADA)2], ZnCl2 and ZnL solutions were the averages of duplicate measurements of the solutions listed in Table A1 in the Supplementary material. At concentrations higher than 1000 |imol/l Zn2+, small differences in Fmax (± 1%) produced large errors in calculating F* (± 100%). To avoid such errors, we prepared samples of ZnL in the concentration range of [Zn2+]t = 20-80 |imol/L. Fmin and Fmax (Table A1 in the Supplementary material), the time dependence of F (Table A2 in the Supplementary material) and the accuracy of preparing the ZnL solutions (Table A3 in the Supplementary material for PA) were defined. F values of solutions with constant [Zn2+]t (at 20, 40, 60 and 80 | mol/L) were measured and the values of [L]t, F*, [Zn2+]f, [Zn2+]b ([Zn2+]t = [Zn2+]b + [Zn2+]f), [Zn2+]b/[Zn2+]f, and ln[Zn2+]f were calculated (Table A4 in the Supplementary material for PA). From plots of Type 1 and 2 (Figures 3 and 4) equations for calculating Zn2+ bound to L for any [L]t and [Zn2+]t (Table 3), and stability constants for binding Zn2+ to L (Table 4) and the concentrations of bound polymer ligands (Table 5) were determined. 24 and 96 hours after the preparation of aqueous solutions of ZnL fluorescence intensities were again measured (Table A2 in the Supplementary material). Due to different voltages in the photomultiplier, F values of the solutions were not comparable, therefore relative values F* were calculated and compared. The results showed substantially more Zn2+ bound to PA 96 hours after preparation of the solutions (smaller values of F* after 96 h). The time between preparation of the ZnL solutions and measurement of fluorescence intensity was therefore kept constant, at 96 hours. In Table 3 the linear equations of Type 1 plots at [Zn2+]t = 40 |imol/L for all ZnL, together with deviations from linearity, R2, are listed. Figure 3 shows the Type 1 plot at [Zn2+]t 40 |mol/L for ZnL with PA, PAL ALa and PAL ALt. With [L]t 5000, the concentration of Zn2+ bound on PA, PAL ALa and PAL ALt is equal to 39, 29 and 11 |mol/L, respectively. Table 3. Dependence of [L]t and [Zn2+]f at [Zn2+]t = 40 |imol/L calculated from Type 1 plots, [L]t = f(ln[Zn2+]f) and deviations from linearity, R2 L PA PAL ALa PAL ALt equation [PA ]t = -1438 ■ ln[Zn2+]f [PAL ALa] = -11940 ■ ln[Zn2+]f [PAL ALt] =-23650 ■ ln[Zn2+]f + 5347 +15275 + 84280 R2 0.9969 0. 9831 0. 9894 N 11 11 9 N = number of different concentrations of [L]t Figure 3. Type 1 plot showing dependence of [L]t on ln[Zn2+]f, pH 6, [Zn2+]t 40 |imol/L. Figure 4. Type 2 plot showing dependence of [Zn ]b/[Zn ]f on [L]t, pH 6, [Zn2+]t 40 |imol/L. Figure 4 shows the Type 2 plot for ZnL at [Zn2+]t = 40 |imol/L. The straight line can be expressed by the equations [Zn2+]b/[Zn2+]f = KL[L]t - KL[L]b. The values in Type 2 plots at low concentrations of [L]t are negative due to the complexity of the calculations, through which experimental errors are summed. Because our main interest was in the slopes of these plots which represent the stability constants for binding of Zn2+ to polymer ligands, these Type 2 plots are presented without corrections. Table 4. Stability constants of polymer ligands PA, PAL ALa and PAL ALt at pH 6 and [Zn2+]t = 20, 40, 60 and 80 |imol/L with descriptive statistics - averages (AVG) and relative standard errors (RSE) (determined in fourfold repetition) descriptive statistics PA PAL ALa PAL Alt 100% A 90% A 10% A AVG Kl (L/mol) 1445 671 86 RSE (%) 2 3 8 Table 5. The concentration of bound polymer ligands, [L]b, PA, PAL ALa and PAL ALt, at different [Zn2+]t and pH 6 (determined for approx. ten concentrations of total L in duplicate repetition) [Zn2+]t (^mol/L) [L]b (mmol/L) PA PbAL ALa PAL ALt 100% A 90% A 10% A 20 25 100 510 40 145 255 670 60 285 600 630 80 360 600 670 From Type 2 plots KL and [L]b were calculated (Tables 4 and 5). The ability of a polymer ligand to bind a metal ion is dependent on its capacity to donate an electron pair, though this is reduced by steric hindrance14 such as methyl groups of lactic acid in PAL ALa and PAL ALt, branching of chains and lowering of the molar mass. PA has a Mw around 3000 g/mol and is approximately linear, whereas PAL ALa and PAL ALt have Mw around 166,000 and 365,000 g/mol with 140 and 390 end units per chain, respectively; see Table 2. From time dependencies of F values we deduced that PA, PAL ALa and PAL ALt gradually or step-by-step bind Zn2+ (Table A2 in the Supplementary material) and therefore the duration between preparation of the samples and measurement was kept constant. The interactions between Zn2+ and PA, PAL ALa and PAL ALt were compared by Type 1 plots (Figure 3 and Table 3), the concentrations of bound L, [L]b, and stability constants, KL (Tables 4 and 5). With the help of the equations for Type 1 plots (Table 3) we can relate [Zn2+]t and [Zn2+]b at constant [L]t, and for any [Zn2+]t and [L]t calculate [Zn2+]b. From the linear dependencies in Type 2 plots (Figure 4) we conclude that zinc ions coordinate on PA, PAL ALa and PAL ALt by the same mechanism, independent of the higher degree of branching in ALa and Alt, and the lower Mw of PA. According to Flory's principle,19 metal ions bind to polymer ligands as if the polymer chains were infinitely long; the reactivity of ligand groups is namely independent of their position on the polymer chain. The binding of metal ions in this manner is similar to the gradual binding of low molecular weight ligands. Flory's equation for unidentate polymer ligands, our Type 2 plot, is [Zn2+]b/[Zn2+]f = Kl ■ [L]t - KL ■ [L]b. The slope in Type 2 plots is equal to the product of the stability constant and [L]t, and intersect on the ordinate is the product of KL and [L]b. A lower concentration of bound L indicates a more stable coordination compound and a higher KL a higher amount of bound Zn2+ per polymer unit.14 Zn2+ at pH 6 mainly binds to side groups in polymer ligands.19,24,25 This means that Zn2+ at pH 6 predominantly binds to carbon of the -COO- and COOH side groups of aspartic acid units of the copolymers of aspartic and lactic acids. The results show that the stability constants for binding of Zn2+ are practically independent of the concentration of total zinc ions (RSE in Table 4), whereas in the curve in Figure 5 they depend on the number of aspartic acid units in the polymer ligand. Namely, PA has 100%, ALa 90% and ALt 10% aspartic acid units and the stability constant for binding of Zn2+ on PA, KPA, is twice as high as Kpal ALa and seventeen-times higher than KPAL ALt. The results in Figures 3, 4 and 5 and Table 4 indicate that at pH 6, in comparison to PAL ALa and Alt, PA has the highest capacity for binding of Zn2+, as expected. Figure 5. Stability constant at pH 6 for binding of Zn2+ on polymer ligands of PA, PAL ALa and PAL ALt (with standard error intervals of fourfold repetition) as a function of aspartic acid moieties in the polymer ligands; PA contains 100%, PAL ALa 90% and PAL ALt 10% of aspartic acid units; the dotted line on the graph is theoretical value. lative fluorescence intensity as a function of the concentration of Zn2+, F* = f([Zn2+]t), - verifying the potential time dependence of the relative fluorescence intensity F* of the polymer coordination compounds (Table A2), - searching for linear dependences in plots such as for example [L]t= f(ln[Zn2+]t) and [Zn2+]b/[Zn2+]f = f([L]t) = Kl ■ [L]t - Kl ■ [L]b, in order to calculate stability constants of the polymer coordination compounds and the concentration of bound zinc at constant concentrations of total zinc and polymer ligand. 4. Conclusions The products of aspartic and lactic acid or lactide synthesis and basic hydrolysis were poly(succinimide-co-lactic acid) and water-soluble poly(sodium a,P-aspartate-co-lactic acid), respectively. The average molecular weights (Mw) of the copolymers, determined by SEC-MALS, were > 100,000 g/mol. More than 100 end groups per chain were found by 1H NMR analysis, indicating the branched structure of the copolymers. Since the equilibrium of free and bound Zn2+ in aqueous solutions of polymer ligands prevents complete removal of free Zn2+, interactions between polymer ligands and zinc ions in aqueous solution were followed by a method with fluorescent spectroscopy using the fluorescent indicator dye FluoZin-1 with minimal equilibrium intervention. The protocol is listed in Results and Discussion. By this method we could calculate the stability constants for binding of zinc to the copolymer ligands and furthermore, at constant total ligand and zinc concentrations we could denote the concentration of zinc bound to polymer. We found that zinc ions gradually or step-by-step bind to the copolymer ligands by the same mechanism, independent of the higher degree of branching in ALa and Alt, and the lower Mw of PA. With increase of aspartic acid units in the polymer ligands the values of the stability constants increase. Hence, we propose that by modifying the ratio of aspartic acid moieties we can forecast the quantity of coordinated Zn2+. [L]b increases with increasing [Zn2+]t from 20 to 80 pmol/L (Table 5). The lowest values of Zn2+ bound on polymer ligands are for PA, followed by PAL ALa and PAL Alt, in an increasing order. This could indicate that coordination compounds with PA are the most stable in comparison to PAL ALa and PAL ALt. In short, the protocol for the method development was as follows: - determining the fluorescence intensity of the blank (F .) and saturation of the indicator (Fmax), v mm max' ' Table A1, the error of measurements (Equation (3)) and defining the calibration curve - i.e. the re- 5. Acknowledgements The authors gratefully acknowledge Vojmir France-tic for discussions on the behavior of zinc ions in aqueous solutions and the Ministry of Education, Science and Sport of the Republic Slovenia for the financial support. 6. References 1. G. Kwon, K. Kataoka, Adv. Drug Delivery Rev. 1995, 16, 295-309. 2. K. E. Uhrich, S. M. Cannizzaro, R. S. Langer, K. M. Shakes-heff, Chem. Rev. 1999, 99, 3181-3198. 3. K. Tabata, H. Abe, Y. Doi, Biomacromolecules 2000, 1, 157-161. 4. H. Shinoda, Y. Asou, A. Suetsugu, K. Tanaka, Macromol. Biosci. 2003, 3, 34-43. 5. J. P. Pennings, H. Dijkstra, A. J. Pennings, Polymer 1993, 34, 942-951. 6. J. W. Leenslag, A. J. Pennings, R. M. Bos, F. R. Rozema, G. Boering, Biomaterials 1987, 8, 70-73. 7. K. Makino, M. Arakawa, T. Kondo, Chem. Pharm. Bull. 1985, 33, 1195-1201. 8. K. Y. Lee, D. J. Mooney, Chem. Rev. 2001, 101, 1869-1879. 9. S. M. Thombre, B. D. Sarwade, J. Macromol. Sci., Pure Appl. Chem. 2005, 42, 1299-1315. 10. M. Hershfinkel, W. F. Silverman, I. Sekler, Mol. Med. 2007, 13, 331-336. 11. M. J. Jackson, In: C. F. Mills (Ed.), Zinc in human biology, Springer-Verlag, London, 1989, pp. 1-14. 12. J. C. King, D. M. Shames, L. R. Woodhouse, J. Nutr. 2000, 130, 1360S-1366S. 13. D. Wöhrle, A. D. Pomogailo, In: H. S. Nalwa (Ed.), Advanced Functional Molecules and Polymers, Overseas Publishers Association, 2001, 1, pp. 87-161. 14. F. Ciardelli, E.Tsuchida, D. Wöhrle, A. D. Pomogailo, Ma-cromolecule-Metal Complexes, Springer, 1996, pp. 1-112. 15. V. Saudek, S. Stokrova, P. Schmidt, Biopolymers 1982, 21, 1011-1020. 16. M. Gričar, I. Poljanšek, B. Brulc, T. Smigovec, M. Žigon, E. Žagar, Acta Chim. Slov. 2GG8, 55, 575-581. 17. M. Gričar, M. Žigon, E. Žagar, Anal. Bioanal. Chem. 2GG9, 393, 1815-1823. 18. B. L. Rivas, E. D. Pereira, I. M. -Villoslada, Prog. Polym. Sci. 2GG3, 28, 173-208. 19. A. D. Pomogailo, I. E. Uflyand, Adv. Polym. Sci. 199G, 97, 61-105. 20. T. Nakato, M. Yoshitake, K. Matsubara, M. Tomida, Macro-molecules 1998, 31, 2107-2113. 21. K. Matsubara, T. Nakato, M. Tomida, Macromolecules 1998, 31, 1466-1472. 22. K. Matsubara, T. Nakato, M. Tomida, Macromolecules 1997, 30, 2305-2312. 23. J. L. Espartero, L. Rashkov, S. M. Li, N. Manolova, M. Vert, Macromolecules 1996, 29, 3535-3539. 24. R. Nakon, C. R. Krishnamoorthy, Science 1983, 221, 749750. 25. E. A. Lance, R. Nakon, Inorg. Chim. Acta 1981, 55, L1-L3. Povzetek Sintetizirali smo biorazgradljive kopolimere asparaginske in mlečne kisline, ki imajo možno uporabo kot nosilci za nadzirano sproščanje zdravilnih učinkovin. Molekule kopolimerov so bile razvejene, z absolutnimi molskimi masami nad 100.000 g/mol in deležem enot asparaginske kisline 0,9 ter 0,1. Za vezavo zdravilne učinkovine na kopolimere bi lahko uporabili komplekse s kovinskimi (predvsem cinkovimi) ioni, zato smo za opredelitev interakcij med cinkovimi ioni in polimernimi ligandi razvili metodo za določanje prostih cinkovih ionov v vodnih raztopinah polimerov s fluorescenčno spektroskopijo. Določili smo stabilnostne konstante vezave cinka in koncentracije vezanega cinka. Ugotovili smo, da se cinkovi ioni pri pH 6 vežejo predvsem na asparaginske enote kopolimerov asparaginske in mlečne kisline. SUPPLEMENTARY MATERIAL Calibration and method development for determination of interactions between polymer ligands and Zn2+ by fluorescence spectroscopy 1. Method Calibration curves of solutions of ZnCl2 + ADA and ZnCl2 were plotted at pH 6 and their slopes compared. At pH values higher than 6.3, free Zn2+ in aqueous solution is unstable due to precipitation [A1]. In ZnCl2 + ADA solutions at pH 6 two complexes, [Zn(ADA)2] and [Zn(ADA)], are formed, but, [Zn(ADA)2] highly predominates [A2-A4]. [Zn2+]f in solutions of ZnCl2 + ADA can therefore be simply calculated by Equation (A1), using the stability constant of [Zn(ADA)] (K[Zn(ADA)] = 127 nmol/L, when Zn2+ : ADA = 1 : 1), considering twice as much free Zn2+ in solutions of [Zn(ADA)2] than [Zn(ADA)] and 50% dilution with Milli-Q water in the cuvette; "t" denotes total: [Zn2+]f = 0.5 [Zn2+]t - 0.5 [ADA] - ^da + (A1) [(0.5 [Zn2+]t - 0.5 [ADA] - ^da)2 + 2 ^da [Zn2+]t]1/2 2. Results Table A1. Fmin and Fmax of [Zn(ADA)2], ZnCl2 and ZnL solutions at pH 6 solution [Zn(ADA)2] ZnCl2 ZnL F . min F max Fmin 2 min Fmax max Fmin min Fmax max [ADA]t (mol/L) 0.05215 0.04736 - - - - [Zn2+]t (mol/L) 0 0.04867 0 0.052 0 0.052 [Zn2+]t (mol/L) 0 0.0026 0 0.052 0 0.052 [NaCl] (mol/L) 0.048 0.09 0.1 0.048 0.1 0.048 [L]t (mol/L) - - - - max 0 Table A2. Fluorescence intensities (F) 24 and 96 hours after the preparation of solutions at pH 6, and relative F (F*) and F* ratios [Zn2+]t (^mol/L) [PA]t (^mol/L) [Zn2+]t /[PA]t (/) F (rlu) 24 h 96 h 24 h F* 96 h F* /F* r (24 h) (96 h) (%) 11250 500 22.5 336 515 24 13 185 10000 500 20 329 500 15 9 167 125 500 0.25 266 / 3 / / 50 500 0.1 231 316 1.6 1.1 145 4 500 0.008 51 70 0.05 0.03 167 2 500 0.004 40 / 0.01 / / 0.8 500 0.002 40 57 0.01 0 / 0 500 0 38 58 Fmln Fmln Fmln 52000 0 / 349 549 p™" pi^in Fmmlanx max max max Table A3. [Zn2+]t 40, 60 and 80 ^mol/L, 96 hours after the preparation of solutions of ZnCl2 + PA, pH 6: average F* (), deviation from average F* (F*-), average [Zn2+]f of duplicates (<[Zn2+]f >), deviation from average ([Zn2+]f ** ) and ratio between [Zn2+]f** and <[Zn2+]f> [Zn2+]t [PA ]t [Zn2+]/[PA ]t F*— <[Zn2+]f> [Zn2+]f** [Zn2+]f**/<[Zn2+]f> (^mol/L) (^mol/L) (/) (^mol/L) (|umol/L) (%) 40 16 2.5 1.65 0.03 46 0.7 1.5 40 64 0.63 1.52 0.03 42.5 0.6 1.5 40 128 0.31 1.44 0.03 40 0.6 1.4 40 160 0.25 1.38 0.02 39 0.5 1.4 40 192 0.21 1.35 0.02 38 0.5 1.4 40 256 0.16 1.28 0.03 36 0.7 1.9 40 320 0.13 1.18 0.03 33 0.7 2.1 40 384 0.10 1.12 0.02 31 0.4 1.3 40 1344 0.03 0.55 0.01 15 0.1 0.8 40 4000 0.01 0.10 0.00 3 0.0 0.1 40 6400 0.006 0.03 0.00 0.8 0.0 1.1 60 384 0.16 1.74 0.02 48 0.3 0.6 60 480 0.13 1.59 0.00 45 0.2 0.5 60 576 0.10 1.49 0.03 42 0.6 1.4 80 512 0.16 2.20 0.05 61.5 1.1 1.8 80 640 0.13 1.97 0.06 55 1.4 2.5 80 768 0.10 1.83 0.05 51 1.1 2.1 0 6400 0 Fmn Fmin / / / 52000 0 / F™" max F™ max / / / F1* = (F1 - 15) / (415 - F1), F2* = (F2 - 15) / (415 - F2), calibration curve: [Zn2+]f = 28 • F Table A4. Solutions of [Zn2+]t 40 ^mol/L and different concentrations of total PA, pH 6, 96 hours after preparation of the solutions: F and F*, concentrations of free Zn2+, natural logarithm of free Zn2+ and ratio between bound and free Zn2+ [Zn2+]t [PA ]t [Zn2+]/[PA ]t F (rlu) F* [Zn2+]f ln[Zn2+]f [Zn2+]b/[Zn2+]f (^mol/L) (|umol/L) (|umol/L) 40 16 2.50 264 1.65a 46 3.84 -0.14 40 65 0.63 256 1.52a 42.5 3.75 -0.06 40 130 0.31 251 1.44a 40 3.70 -0.01 40 160 0.25 247 1.38a 39 3.66 0.03 40 190 0.21 245 1.35a 38 3.64 0.05 40 260 0.16 239 1.28a 36 3.58 0.12 40 320 0.13 231 1.18a 33 3.50 0.21 40 380 0.10 226 1.12a 31 3.45 0.27 40 560 0.07 232 0.92b 27 3.31 0.46 40 800 0.05 210 0.76b 22 3.11 0.78 40 1040 0.04 191 0.64b 19 2.94 1.12 40 1344 0.03 155 0.54a 15 2.73 1.61 40 2000 0.02 129 0.34b 10 2.30 3.00 40 4000 0.01 50 0.10a 3 0.99 13.81 40 6400 0.01 26 0.03a 0.8 -0.22 49.00 52000 0 / 415 Fmaxa / / / 52000 0 / 468 Fmmaaxxb / / / 0 6400 0 15 Fmmianxa 0 / / 0 6400 0 15 min 0 / / a: F* = ( F - 15) / (415 - F), [Zn2+]f = 28 • F*; b: F* = (F - 15) / (468 - F), [Zn2+]f = 30 • F* The measurements F in Table A4 differ in their date of measurement, which is denoted with "a" and "b". 3. References A1. F. Caillaud, A. Smith, J.-F. Baumard, J. Am. Ceram. Soc. 1993, 76, 998-1002. A2. E. A. Lance, C. W. Rhodes, III, R. Nakon, Anal. Biochem. 1983, 133, 492-501. A3. R. Nakon, C. R. Krishnamoorthy, Science 1983, 221, 749-750. A4. E. A. Lance, R. Nakon, Inorg. Chim. Acta 1981, 55, L1-L3.