Research Article Received: 10 September 2014
Revised: 19 December 2014
Accepted: 30 December 2014
Published online in Wiley Online Library: 19 February 2015
(wileyonlinelibrary.com) DOI 10.1002/psc.2752
The interaction mechanism between lipopeptide (daptomycin) and polyamidoamine (PAMAM) dendrimers Boontarika Chanvorachote,a Jiang Qiu,b Walaisiri Muangsiri,a Ubonthip Nimmannita,c and Lee E. Kirschb* The interaction mechanism of lipopeptide antibiotic daptomycin and polyamidoamine (PAMAM) dendrimers was studied using fluorescence spectroscopy. The fluorescence changes observed are associated with daptomycin–dendrimer interactions. The binding isotherms were constructed by plotting the fluorescence difference at 460 nm from kynurenine (Kyn-13) of daptomycin in the presence and absence of dendrimer. A one-site and two-site binding model were quantitatively generated to estimate binding capacity and affinity constants from the isotherms. The shape of the binding isotherm and the dependence of the estimated capacity constants on dendrimer sizes and solvent pH values provide meaningful insight into the mechanism of interactions. A one-site binding model adequately describes the binding isotherm obtained under a variety of experimental conditions with dendrimers of various sizes in the optimal binding pH region 3.5 to 4.5. Comparing the pH-dependent binding capacity with the ionization profiles of daptomycin and dendrimer, the ionized aspartic acid residue (Asp-9) of daptomycin primarily interact with PAMAM cationic surface amine. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Keywords: fluorescence spectroscopy; daptomycin; lipopeptide; polyamidoamine (PAMAM) dendrimers; peptide delivery; polymeric drug delivery
Introduction
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Daptomycin is a cyclic lipopeptide antibiotic that consists of 13 amino acids and a decanoyl side chain in Figure 1. In vitro, daptomycin possesses bactericidal activity with a post-antibiotic effect (PAE) [1,2]. Daptomycin exhibits bactericidal activity against vancomycin-resistant Enterococci and methicillin-resistant Staphylococci [3–5]. Initial clinical studies were terminated because of treatment failures that were attributed, in part, to protein binding, rapid renal clearance, and inadequate biophase concentrations [6–9]; subsequent clinical trials demonstrated the compound’s utility against methicillin-resistant Staphylococci aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) bacteremia.[10] Our overall goal is to evaluate the utility of daptomycin– biopolymer conjugates and complexes in modifying the pharmacokinetic properties of the antibiotic to improve its biophase concentration–time profile. We expect that the successful development of daptomycin–biopolymer drug delivery systems will overcome a number of shortcomings of current therapy by providing reduced renal clearance of large molecular weight drug–polymer complex and controlled and sustained release of drug from the macromolecular carrier. Previously, we have reported on daptomycin–dextran conjugates. Herein, we present our studies on the pH-dependent complexation of daptomycin and polyamidoamine (PAMAM) dendrimers. Daptomycin has six side chain ionizable groups, four carboxylic acids (three aspartic acids, Asp-3, Asp-7, and Asp-9, and one methyl-glutamic acid, mGlu-12), and two amines (aliphatic amine in Orn-6 and aromatic amine in Kyn-13). In the pH range of 3.0 to 9.0, the dominant species are anionic thus providing an opportunity for electrostatic attachment to cationic carriers. It is possible
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that this type of drug carrier system can provide a pharmacokinetic enhancement of biophase concentrations [11]. Polyamidoamine dendrimers are monodisperse-branched globular macromolecules that carry a multiplicity of functional groups at their periphery [12,13]. PAMAM dendrimers have primary amine end groups on the surface and tertiary amine groups situated at the branching points within the core. The number of surface functional groups, molecular weight, and size of dendrimers are defined by the generation number that describes the number of layers of subunits that have been attached during synthesis [14–16]. Both amines are protonated in the pH range of 3.0 to 10.0. Negatively charged drug molecules can be entrapped in the interior core as well as electrostatically attached to the amine groups on the surface [17]. PAMAM dendrimers have been proposed as noncovalent drug carrier systems because of well-defined structures, molecular monodispersity, and highly charged surface and interior residues [12]. Moreover, dendritic formulations exhibit prolonged blood/plasma retention owing to their hydrophilicity, surface
* Correspondence to: Lee E. Kirsch, Division of Pharmaceutics, College of Pharmacy, The University of Iowa, Iowa City, IA 52242, USA. E-mail: [email protected] a Pharmaceutical Technology (International) Program and Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand b Division of Pharmaceutics, College of Pharmacy, The University of Iowa, Iowa City, IA, 52242, USA c National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), 111 Thailand Science Park, Phahonyothin Road, Klong 1, Klong Luang, Pathumthani, 12120, Thailand
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
THE INTERACTION MECHANISM BETWEEN DAPTOMYCIN AND PAMAM
Figure 1. Chemical structure of daptomycin.
characteristics, and molecular size [18]. These characteristics are potentially beneficial in the design of noncovalent drug delivery systems that may overcome problems associated with the free drug molecule and covalent carrier system. Daptomycin contains two fluorophores, tryptophan (Trp-1), and kynurenine (Kyn-13) side chains. The fluorophores on daptomycin molecules have been used to study various molecular interactions and were demonstrated to be useful for evaluating daptomycin binding and estimating binding constants [19]. The excitation and emission wavelengths of free tryptophan in solution were 285 and 355 nm, respectively. For free kynurenine in solution, the excitation and emission wavelengths were 364 and 461 nm, respectively [11]. Excited at 285 nm, the emission wavelengths of daptomycin are at 355 and 460 nm from Trp-1 and Kyn-13, respectively [20]. Fluorescence spectroscopy has been used as a technique to determine the interaction between a small-molecule ligand and biomacromolecule [19,21,22]. Changes in fluorescence emission characteristics of fluorescence probes, e.g. 1-anilinonapthalene-8sulfonic acid, have been used to estimate binding constants [23]. Several factors have been shown to influence the formation of dendrimeric complexes such as pH, molecular size, and surface structure [24–26]. Objectives of this study were to determine the molecular mechanisms leading to the formation of dendrimeric daptomycin complexes by studying the effects of pH and dendrimer size on binding capacity and affinity and exploit the potential application of this system to improve the poor pharmacokinetics of daptomycin. In addition, these investigations provided insight into the nature of daptomycin interactions with charged macromolecules to control the assembly of daptomycin and PAMAM.
Materials and Methods Reagents Daptomycin obtained from Eli Lilly Research Laboratories (Indianapolis, IN) was used as received. Standard sodium hydroxide and standard hydrochloric acid solutions were purchased from Fisher Scientific (Fair Lawn, NJ). 1,4-Diaminobutane core PAMAM generation 3 (PAMAM 3) in 20% (w/v), generation 5 (PAMAM 5) and generation 6 (PAMAM 6) in 10% (w/v) methanol solutions were purchased from Sigma-Aldrich (St. Louis, MO).
scanned from 320 to 540 nm in order to observe the emission responses from both Trp-1 and Kyn-13. PAMAM–Daptomycin Complex Fluorescence Properties In this experiment (as in subsequently described titration studies), the commercially obtained methanolic solutions of PAMAM dendrimers were used to prepare aqueous dendrimer stock solutions. The methanol was evaporated by purging nitrogen gas, and the dendrimers were redissolved in double-distilled water. Daptomycin and PAMAM solutions were separately prepared at the same target pH values adjusted using 0.1 M sodium hydroxide or hydrochloric acid solutions. PAMAM 5 (0.18 μM) and daptomycin (3 μM) aqueous solutions at pH 4.0 and an equal volume mixture of PAMAM 5 and daptomycin solutions were prepared to evaluate the fluorescence properties of PAMAM–daptomycin complex. Binding Isotherm Construction Daptomycin–PAMAM complex binding isotherms were generated by the addition of aqueous daptomycin to PAMAM solutions using various size dendrimers (PAMAM 3, 5, and 6). As described in the preceding texts, the ligand (daptomycin) and substrate (PAMAM) solutions were prepared separately by adjusting each solution to a target pH value in the range of 3 to 9 using 0.1 M sodium hydroxide and/or hydrochloric acid solutions. A 3 ml aliquot of the PAMAM solutions (Table 1) was placed in a fluorescence quartz cell (10 mm path length) equipped with a magnetic stirrer. Typically, 15 aliquots (5 μl) of aqueous daptomycin solutions were added incrementally using a Hamilton microliter syringe to a final daptomycin maximum concentration as described in Table 1. After the addition of each aliquot, the mixtures were stirred for 3 min, and then, the fluorescence spectra were collected. As in the preceding texts, the control titrations were performed by adding 5 μl aliquots of daptomycin to the same pH value aqueous solutions in the absence of dendrimers and stirred for 3 min prior to spectroscopic analysis. All solutions were pH adjusted to the desired value prior to the addition, and the pH values of the titration mixtures were constant (≤0.1 pH) throughout each titration experiment. The binding isotherms were constructed by plotting the fluorescence intensity differences (ΔF) at 460 nm in the presence and absence of dendrimers against the total daptomycin concentrations to yield binding isotherms.
Fluorescence Spectroscopy
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Estimation of the Binding Parameters The mathematical models used to estimate binding constants from the isotherms have been previously described [19]. Briefly a
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Fluorescence spectra were recorded using a LS-55 Luminescence Spectrometer (Perkin Elmer Instruments, Norwalk, CT). The excitation wavelength was set at 285 nm, and the emission spectra were
CHANVORACHOTE ET AL. 1
Table 1. Dissociation constant (Kd; μM ) and capacity constant (n; molecules of daptomycin per one molecule of PAMAM dendrimer) at various pH ranges of the binding interaction between daptomycin and various PAMAM dendrimer generation estimated by simultaneous fitting using WinNonlin software with fixing the molar signal coefficient (ΔE1 and ΔE2) as a constant at 240.13 and 46.65, respectively Estimated binding parameters Substrate (PAMAM) Generation size
Ligand (daptomycin)
Second binding site 1
Maximum conc. (μM)
pH
Kd (μM )
n
Kd (μM )
n
0.14 0.14 0.27 0.05 0.05 0.05 0.27 0.27 0.27 0.27 0.27 0.01 0.01 0.01 0.01 0.01 0.01
74.07 74.07 11.61 5.80 5.80 5.80 8.21 8.21 7.06 11.61 11.61 74.07 74.07 74.07 74.07 74.07 74.07
4.0 7.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 3.5 4.0 4.5 5.0 6.0 7.0
0.76 0.01
13 55
0.26
13
0.33 0.06 0.05 0.13 0.13 0.11
49 59 58 12 10 8.0
0.06 0.22 0.12 0.07 0.07 0.01 0.01
6.8 44 66 57 57 35 19
1.73 2.00
82 91
5
6
quadratic equation was derived for one-site binding model that relates in the total ligand (daptomycin) concentration to fluorescence differences as described in the Eqn (1).
ΔF ¼
The utility of the model equation to distinguish between the binding mechanism and estimate the binding parameters including the difference in molar extinction coefficient (ΔE) between the
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 ΔE ½daptomycinT þ Kd þ R ± ½daptomycinT þ Kd þ R 4R½daptomycinT 2
where [daptomycin]T, ΔE, Kd, and R represent the total daptomycin concentration, the difference in molar extinction coefficient between the free and bound fluorophores, the equilibrium dissociation constant, and the total number of independent binding sites, respectively. For a two-site binding model, an Eqn (2) was derived to describe fluorescence difference titration curves in terms of the molar extinction parameters, total daptomycin concentration, and binding parameters. ΔF ¼ ΔE1
1
Conc. (μM)
3
First binding site
R1 ½daptomycinF Kd1 þ ½daptomycinF
þ ΔE2
R2 ½daptomycinF Kd2 þ ½daptomycinF (2)
where [daptomycin]F, a, b, c, and θ are given by the following relationships and are defined in terms of binding parameters and the total daptomycin concentration. a 2pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi θ ½daptomycinF ¼ þ ða2 3bÞcos 3 3 3 b ¼ Kd1 Kd2 þ Kd2 R1 þ Kd1 þ R2 -ðKd1 þ Kd2 Þ½daptomycinT c ¼ Kd1 Kd2 ½daptomycinT
314
2a3 þ 9ab 27c θ ¼ arccos qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi3 2 ða2 3bÞ
ð0 < θ < π Þ
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(1)
free and bound fluorophores, dissociation constant (Kd), and the total number of independent binding sites (R, or R1, and R2) has been previously described [19]. The nonlinear regression analysis was used to estimate binding parameters (WinNonlin Softer Version 5.0.1, Pharsight Corporation).
Results Fluorescence Changes Associated with PAMAM–Daptomycin Binding Shown in Figure 2 are the fluorescence spectra of daptomycin, PAMAM, and PAMAM–daptomycin complex at pH 4.0 aqueous solutions. Daptomycin has intrinsic fluorescence emissions at 355 nm from Trp-1 and 460 nm from Kyn-13, but PAMAM dendrimer shows no fluorescence emission maxima over the wavelength range between 320 and 540 nm. The PAMAM–daptomycin complex had a 14-fold increase in emission intensity at 460 nm from Kyn-13 and a 50% decrease at 355 nm from Trp-1. The significant fluorescence emission enhancement at 460 nm could be the self-association aggregation of daptomycin or the interaction of daptomycin with dendrimers. However, previous studies using a variety of orthogonal analytical approaches including NMR, fluorescence, and dynamic light scattering have demonstrated that the minimum critical aggregation concentration for daptomycin in the pH range 3 to 7 was always more than 100 μM [27]. The existing daptomycin
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
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THE INTERACTION MECHANISM BETWEEN DAPTOMYCIN AND PAMAM Effect of pH and Dendrimer Size on the Binding Characteristics of Daptomycin–Dendrimer Complex
Figure 2. The fluorescence spectra of daptomycin (3.0 μM), PAMAM 5 (0.18 μM), and an equal volume mixture of daptomycin (3.0 μM) and PAMAM 5 (0.18 μM) at pH 4.0 and 25 °C (cuvette path length 10 mm; excitation wavelength at 285 nm).
concentration here was only 3.0 μM. Therefore, the fluorescence enhancements observed herein could be unequivocally attributed to daptomycin–dendrimer interactions because the maximum daptomycin concentrations in all studies were less than 100 μM. The similar fluorescence enhancements at 460 nm from Kyn-13 have also been reported in the interaction of daptomycin and phospholipids [28]. The fluorescence changes observed were associated with daptomycin and dendrimer interactions. Typical fluorescence intensity changes in the absence and presence of dendrimers (PAMAM 5) as a function of daptomycin concentrations in pH 5.0 aqueous solutions are depicted in Figure 3. In the absence of dendrimer (PAMAM 5), the fluorescence intensities at 460 nm increased linearly over the range of daptomycin concentrations. Moreover, these increases were colinear in all pH value solutions. In the presence of PAMAM 5, the fluorescence emission intensities at 460 nm displayed a steep increase at low daptomycin concentrations and a gradual linear increase at higher concentrations.
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Figure 4. The binding isotherms of the interaction between PAMAM 5 and daptomycin in the range of pH 3.0 to 9.0. The normalized ΔF values were the ratio of ΔF value to PAMAM 5 concentration.
Figure 5. The binding isotherms of the interaction between PAMAM 6 and daptomycin in the range of pH 3.5 to 7.0. The normalized ΔF values were the ratio of ΔF value to PAMAM 6 concentration.
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Figure 3. The fluorescence intensities of daptomycin at 460 nm in the absence (solid square) and in the presence (solid circle) of PAMAM 5 at pH 5.0 (cuvette path length 10 mm; excitation wavelength at 285 nm).
Ionic interaction between daptomycin and dendrimer was anticipated to be a significant factor in daptomycin–dendrimer complex. Thus, factors affecting the ionic states of both compounds were investigated using various size dendrimers PAMAM 3, 5, and 6 in the pH range from 3 to 9. The generation number of dendrimers determines the size and amount of surface functional groups. The binding isotherm was constructed by plotting the difference between the fluorescence intensity in the presence and absence of dendrimer (ΔF) at each daptomycin concentration versus the total daptomycin concentration. The magnitude of the maximum fluorescence enhancement varied with pH and necessitated using different concentrations of ligand and substrate at different experimental conditions (Table 1). Therefore, the binding isotherms in the different pH solutions were generated in Figures 4, 5, and 6 by plotting the normalized fluorescence difference (ΔF/[PAMAM]) in the presence and absence of dendrimer at each daptomycin concentration versus the total daptomycin concentration
CHANVORACHOTE ET AL.
Figure 6. The binding isotherm of the interaction between PAMAM 3 and daptomycin at pH 4 and 7. The normalized ΔF values were the ratio of ΔF value to PAMAM 6 concentration.
In the presence of PAMAM 5 (Figure 4), the typical binding isotherms of the normalized fluorescence differences from pH 3.5 to 8 showed a steep increase at low concentration and a plateau at higher concentrations of daptomycin, which were consistent with one-site binding model [19]. In addition, the magnitude of the maximum values ( F/[PAMAM]) in the pH range 3.5 to 4.5 was 4-fold to 5-fold greater than in the pH range 5 to 8. The titrations of daptomycin to PAMAM 6 were conducted in the pH range of 3.5 to 7. In a pH range of pH 3.5 to 5 (Figure 5), the normalized fluorescence differences steeply increased and attained plateaus at higher daptomycin concentrations as described in PAMAM 5, which conformed to the one-site binding model. In the pH range of 6 to 7, the isotherm shapes were different in that the normalized fluorescence difference values increased at low daptomycin concentration attained a maximum value and then gradually decreased with increasing in daptomycin concentrations. This isotherm shape suggested change in binding at higher concentrations either because of a loss in binding capacity or a second binding site that was associated with a loss in fluorescence intensity. The resultant binding isotherms of PAMAM 3 at pH 4 and 7 are depicted in Figure 6. At pH 4, the binding isotherm shape appeared to be consistent with one-site binding model, whereas the binding isotherm at pH 7 was consistent with a two-site model. However, a little initial lag phase was observed from the one-site binding isotherm at pH 4. This may be a consequence of asymmetry structure of PAMAM 3 [29]. Binding Parameters
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The shapes of the isotherms were used to deduce the nature of the binding process and estimate the binding parameters. The binding parameters, molar extinction coefficient (ΔE), dissociation constant (Kd), and the total number of independent binding sites (R), were estimated by simultaneously fitting the model equation to the binding isotherm by nonlinear regression analysis. The molar extinction coefficient (ΔE) obtained from the fitting of the binding isotherms of PAMAM 5 titration in the pH ranges of 3.5 to 8 were not significantly different (p-value = 0.053). The constant value of ΔE indicated that the fluorescence characteristics of the interaction between daptomycin and PAMAM 5 were identical throughout the pH range of 3.5 to 8. Consequently, the ΔE value was fixed for all binding isotherms at a constant mean value of
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240.1. Then, the Eqn (1) was refitted to all binding isotherms with just two adjustable parameters in an attempt to attribute pH differences to the binding parameters, Kd (the equilibrium dissociation constant) and R (the total number of independent binding sites). The capacity constant (n, the number of bound daptomycin molecules on per PAMAM dendrimer molecule) of each individual pH range was then calculated based on the ratio of the estimate R value to the total concentration of PAMAM dendrimer. The binding parameters were reported in Table 1. The capacity constants (n) appeared to change dramatically as a function of pH, whereas the dissociation constants were not notably pH dependent. The one-site binding isotherms obtained from the titration of PAMAM 6 with daptomycin at low pH range were described. Model predicted curves were shown to describe the experiment data of the titration at pH range of 3.5 to 5. This suggested that the binding interaction at the pH ranges of 3.5 to 5 was consistent with one-site binding model. Two-site model equations [Eqn (2)] were used to describe the data obtained at pH 6 and 7. The binding parameters were estimated by simultaneously fitting the model equation to the binding isotherm data using nonlinear regression analysis in WinNonlin. The model-predicted isotherms agreed well with experiment data. Furthermore, the residual plots did not show region of systematic bias. The molar signal coefficient (ΔE), the dissociation constant (Kd), and the capacity constant (n) that belong to two different types of binding interaction were successfully estimated at each pH value. The values for ΔE1 and ΔE2 were essentially constant over the pH range studied (p-value of ΔE1 = 0.159 and p-value of ΔE2 = 0.768). The ΔE1 appear to be in the same range as the ΔE estimated using the data obtained from PAMAM 5. Therefore, these values were fixed in Eqn (1) (ΔE = 240.1) and Eqn (2) (ΔE1 at 240.1 and ΔE2 at 46.65), and the values for affinity and capacity constants were re-estimated at each condition by nonlinear regression thereby assigning any remaining variability to the binding properties. All of the predicted curves based on model equations and estimated parameters agreed with the experiment data. The estimated binding parameters were reported in Table 1. Comparison of the Kd1 values by one-way ANOVA indicated that these constants were discernibly pH dependent and that they could be separated into three groups, estimates associated with pH conditions 3.5, 4–5, and 6–7. The dissociation constant at the second type of binding interaction (Kd2) were not discernibly pH dependent. For the interaction of PAMAM 3 and daptomycin, a one-site binding model [Eqn (1)] was used to estimate binding parameters using data generated at pH 4, whereas, the binding isotherm at pH 7 corresponded to a two-site binding model [Eqn (2)]. The model equations used at each pH condition fit the experimental data without systematic residual bias and reasonably high correlation coefficients. The estimated binding parameters are displayed in Table 1.
Discussion Assessment of Relationship Between the Ionic Properties of Daptomycin, PAMAM Dendrimer, and Binding Capacity Daptomycin contains six ionizable groups, four side chain carboxylic groups (three aspartic acids, Asp-3, Asp-7, and Asp-9, and one methyl-glutamic acid, mGlu-12), and two primary amines (an aromatic amine in Kyn-13 and an aliphatic amine in Orn-6). Based on the 2D 1H TOCSY NMR pH titration methods [20], the sequence specific pKa values for Asp-7, Kyn-13, Asp-9, Asp-3, and mGlu-12 were estimated to be 1.01, 1.30, 3.85, 4.15, and 4.55, respectively. The aliphatic amine Orn-6 in daptomycin has a pKa value of 10.7 and
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THE INTERACTION MECHANISM BETWEEN DAPTOMYCIN AND PAMAM therefore is always protonated at pH values below 10 [11]. In the pH range of 0.8 to 3.5, the major ionic species is a zwitterion (H4A±) form as a result of the deprotonation of Asp-7 and the cationic Orn-6 (Figure 7). Monoanion (H3A) and dianion (H2A2) are the primary ionic species in the pH range of 3.5 to 4.7 as a result of the overlapping deprotonation of side chain carboxylic acids in Asp-9 and Asp-3 (Figure 7). Above pH 4.7, the trianion (HA3) is the predominant ionic form as mGlu-12 residue is deprotonated. PAMAM dendrimers possess two types of cationic amino groups: an outer shell of primary amines with a pKa value in the range of 7–9 and internal, tertiary amines with pKa values between 3 and 6 [16,30–32]. PAMAM 5 has 128 primary amines and 126 tertiary amines wherease PAMAM 6 has 256 primary and 254 tertiary amines [16,33]. The protonation of PAMAM dendrimers involves protonation of primary amine groups at the outer rim of the dendrimer over the entire range of interest. The tertiary amine groups in the dendrimer core are protonated at lower pH but deprotonate in the acidic pH range. Comparisons of daptomycin ionization profile and the pHdependent binding capacity parameters obtained using PAMAM 5 and 6 (Figure 7) suggest that the primary determinants in the magnitude of the binding capacity are associated with the predominating presence of the monoanion (H3A) and dianion (H2A2) in the pH range 4 to 5. Only small binding capacity changes can be attributed to changes in the ionic state of the tertiary amine residues in the dendrimer because the binding capacities remain high in the pH region wherein the tertiary amine are progressively less charged. Thus, the major sites for electrostatic interaction are between Asp-9 and cationic moieties on the dendrimer surface. Furthermore, these results suggest that additional anionic charges dianion and special trianion as a result of the protonation of Asp3 and mGlu-12 on daptomycin may weaken, rather than strengthen, daptomycin–dendrimer interactions. Interestingly, the binding capacity pH profile for PAMAM 5 is similar to the pH distribution of monoanionic daptomycin, which may explain why PAMAM 5 is consistent with the one-site binding model throughout the pH range of 3.5 to 8. The capacity constants decreased at pH values > 4.7 for PAMAM 5 and at pH values > 5.2 for PAMAM 6. The difference in the pH
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Assessment of Molecular Model for the Interaction Between Daptomycin and PAMAM Dendrimer An attempt to further develop the spatial and orientation relationships involved in daptomycin–PAMAM dendrimer interactions was undertaken by examining the conformations of these two molecules and predicting the maximum capacity constants based on molecular orientation and size. The conformation of free daptomycin was downloaded from RCSB protein data bank. This conformation was proposed using nuclear overhauser enhancement spectroscopy techniques [38]. A space-filling or CPK model of daptomycin was regenerated using Chem3D Ultra 10.0. The model showed atoms as three-dimensional spheres whose radii are scaled to the atomic van der Waals radii. The two limiting case daptomycin orientations considered were longitudinal and latitudinal orientations. The molecular radii were estimated to be 24.5 and 14.9 Å, respectively. The maximum theoretical binding capacity for ligand bound to dendrimer surface is given by the ratio of the dendrimer surface areas to the cross-sectional area of the ligand. Assuming a spherical PAMAM dendrimer and published PAMAM dendrimer diameters [39], the estimated dendrimer surface areas of PAMAM 3, 5, and 6 are 3.02 × 103, 8.82 × 103, and 14.1 × 104 Å2, respectively. Based on the two limiting case orientations described in the preceding texts, circular cross-sectional areas were estimated to be 471 and 174 Å2 for longitudinal and latitudinal orientation, respectively. The maximum theoretical binding capacities of various dendrimers for daptomycin molecules oriented latitudinally or longitudinally (Figure 8) were predicted and are compared with the experimentally measured maximum capacity constants (Table 2). For all of the dendrimers, the predicted theoretical capacities using a latitudinal orientation are in reasonable agreement with the
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Figure 7. Comparison of the distribution of ionic forms of daptomycin with the estimated capacity constants for its interactions with PAMAM 5 and 6. ± Fraction of daptomycin ionization varied from zwitterion (H4A ), anion (H3A ), 2 3 dianion (H2A ), and trianion (HA ), respectively. Each point represented the capacity constant (molecule of daptomycin/molecule of PAMAM) obtained from binding interaction between daptomycin and PAMAM 5 and 6 at various pH solutions.
dependence of the binding capacities for these two different-sized dendrimers could be explained by the effect of pH on the size of dendrimer. As the dendrimer becomes less ionic, it collapses and the geometry of the polymer changes [34]. Increasing the solution pH value from 5 to 8 results in a 2.7-fold decrease of dendrimer (PAMAM 6) valence [35]. Interior electrostatic repulsions are present at acidic pH values causing full extension of dendrimer branches and a large surface area. However, at alkaline pH, the lack of interior electrostatic repulsion decreases the dendrimer volume and surface area. Thus, the decrease in the estimated capacity constants observed for PAMAM 5 (pH > 4.7) and 6 (pH > 5.2) may be because of the combination of reduced dendrimer charge and surface area [34,36]. Both the monoanion and dianions contribute to daptomycin binding to PAMAM 6 in the pH range of 3.5 to 5. Because large dendrimers have a greater number of amines that can bind daptomycin, the overall binding constant on a per dendrimer basis increases with dendrimer generations from PAMAM 5 to 6 [37]. For PAMAM 6, the decrease in the estimated capacity constant observed in the pH range 5 to 7 appears to coincide with the deprotonation of the dendrimer tertiary amines. In the pH range of 6 to 7, the interaction between daptomycin with PAMAM 6 conforms to the two-site model. For PAMAM 3, 5, and 6, the optimal binding occurs in the pH region 3.5 to 4.5 wherein both the external and internal dendrimer amines are protonated and daptomycin is in its monoanionic and dianionic forms. The intermolecular interaction is based on electrostatic attraction, wherein the deprotonated Asp-9 of daptomycin primarily interacts with surface amine on the dendrimers.
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2
3
4
5
6 7
Figure 8. Space-filling model showing the measurement of daptomycin diameter in longitudinal and latitudinal dimension using Chem3D.
8 9
Table 2. The calculated theoretical capacity and observed capacity obtained from fluorescence titration Substrate Generation size 3 5 6
Theoretical capacity
10
Latitudinal
Longitudinal
Observed capacity
17 51 81
6 19 30
13 59 66 11
observed values suggesting that daptomycin is capable of nearly completely covering the dendrimer surface under pH conditions wherein binding is optimal (pH 3.5 to 4.5).
12
Conclusions
14
The fluorescence changes of the enhanced fluorescence resonance energy transfer between Trp-1 and Kyn-13 were observed upon PAMAM solutions titrated with daptomycin, which are because of the alteration of daptomycin conformation. The shapes of the binding isotherms and the dependence of the estimated capacity constants on dendrimer size and solvent pH values provide meaningful insight into the interaction mechanism of daptomycin and dendrimers. By comparing the ionization profile of daptomycin based on the estimated sequence-specific pKa values with the pH-dependent binding capacity, the ionized Asp-9 residue of daptomycin primarily interact with PAMAM cationic surface amines.
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Revised: 19 December 2014
Accepted: 30 December 2014
Published online in Wiley Online Library: 19 February 2015
(wileyonlinelibrary.com) DOI 10.1002/psc.2752
The interaction mechanism between lipopeptide (daptomycin) and polyamidoamine (PAMAM) dendrimers Boontarika Chanvorachote,a Jiang Qiu,b Walaisiri Muangsiri,a Ubonthip Nimmannita,c and Lee E. Kirschb* The interaction mechanism of lipopeptide antibiotic daptomycin and polyamidoamine (PAMAM) dendrimers was studied using fluorescence spectroscopy. The fluorescence changes observed are associated with daptomycin–dendrimer interactions. The binding isotherms were constructed by plotting the fluorescence difference at 460 nm from kynurenine (Kyn-13) of daptomycin in the presence and absence of dendrimer. A one-site and two-site binding model were quantitatively generated to estimate binding capacity and affinity constants from the isotherms. The shape of the binding isotherm and the dependence of the estimated capacity constants on dendrimer sizes and solvent pH values provide meaningful insight into the mechanism of interactions. A one-site binding model adequately describes the binding isotherm obtained under a variety of experimental conditions with dendrimers of various sizes in the optimal binding pH region 3.5 to 4.5. Comparing the pH-dependent binding capacity with the ionization profiles of daptomycin and dendrimer, the ionized aspartic acid residue (Asp-9) of daptomycin primarily interact with PAMAM cationic surface amine. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Keywords: fluorescence spectroscopy; daptomycin; lipopeptide; polyamidoamine (PAMAM) dendrimers; peptide delivery; polymeric drug delivery
Introduction
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Daptomycin is a cyclic lipopeptide antibiotic that consists of 13 amino acids and a decanoyl side chain in Figure 1. In vitro, daptomycin possesses bactericidal activity with a post-antibiotic effect (PAE) [1,2]. Daptomycin exhibits bactericidal activity against vancomycin-resistant Enterococci and methicillin-resistant Staphylococci [3–5]. Initial clinical studies were terminated because of treatment failures that were attributed, in part, to protein binding, rapid renal clearance, and inadequate biophase concentrations [6–9]; subsequent clinical trials demonstrated the compound’s utility against methicillin-resistant Staphylococci aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) bacteremia.[10] Our overall goal is to evaluate the utility of daptomycin– biopolymer conjugates and complexes in modifying the pharmacokinetic properties of the antibiotic to improve its biophase concentration–time profile. We expect that the successful development of daptomycin–biopolymer drug delivery systems will overcome a number of shortcomings of current therapy by providing reduced renal clearance of large molecular weight drug–polymer complex and controlled and sustained release of drug from the macromolecular carrier. Previously, we have reported on daptomycin–dextran conjugates. Herein, we present our studies on the pH-dependent complexation of daptomycin and polyamidoamine (PAMAM) dendrimers. Daptomycin has six side chain ionizable groups, four carboxylic acids (three aspartic acids, Asp-3, Asp-7, and Asp-9, and one methyl-glutamic acid, mGlu-12), and two amines (aliphatic amine in Orn-6 and aromatic amine in Kyn-13). In the pH range of 3.0 to 9.0, the dominant species are anionic thus providing an opportunity for electrostatic attachment to cationic carriers. It is possible
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that this type of drug carrier system can provide a pharmacokinetic enhancement of biophase concentrations [11]. Polyamidoamine dendrimers are monodisperse-branched globular macromolecules that carry a multiplicity of functional groups at their periphery [12,13]. PAMAM dendrimers have primary amine end groups on the surface and tertiary amine groups situated at the branching points within the core. The number of surface functional groups, molecular weight, and size of dendrimers are defined by the generation number that describes the number of layers of subunits that have been attached during synthesis [14–16]. Both amines are protonated in the pH range of 3.0 to 10.0. Negatively charged drug molecules can be entrapped in the interior core as well as electrostatically attached to the amine groups on the surface [17]. PAMAM dendrimers have been proposed as noncovalent drug carrier systems because of well-defined structures, molecular monodispersity, and highly charged surface and interior residues [12]. Moreover, dendritic formulations exhibit prolonged blood/plasma retention owing to their hydrophilicity, surface
* Correspondence to: Lee E. Kirsch, Division of Pharmaceutics, College of Pharmacy, The University of Iowa, Iowa City, IA 52242, USA. E-mail: [email protected] a Pharmaceutical Technology (International) Program and Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand b Division of Pharmaceutics, College of Pharmacy, The University of Iowa, Iowa City, IA, 52242, USA c National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), 111 Thailand Science Park, Phahonyothin Road, Klong 1, Klong Luang, Pathumthani, 12120, Thailand
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THE INTERACTION MECHANISM BETWEEN DAPTOMYCIN AND PAMAM
Figure 1. Chemical structure of daptomycin.
characteristics, and molecular size [18]. These characteristics are potentially beneficial in the design of noncovalent drug delivery systems that may overcome problems associated with the free drug molecule and covalent carrier system. Daptomycin contains two fluorophores, tryptophan (Trp-1), and kynurenine (Kyn-13) side chains. The fluorophores on daptomycin molecules have been used to study various molecular interactions and were demonstrated to be useful for evaluating daptomycin binding and estimating binding constants [19]. The excitation and emission wavelengths of free tryptophan in solution were 285 and 355 nm, respectively. For free kynurenine in solution, the excitation and emission wavelengths were 364 and 461 nm, respectively [11]. Excited at 285 nm, the emission wavelengths of daptomycin are at 355 and 460 nm from Trp-1 and Kyn-13, respectively [20]. Fluorescence spectroscopy has been used as a technique to determine the interaction between a small-molecule ligand and biomacromolecule [19,21,22]. Changes in fluorescence emission characteristics of fluorescence probes, e.g. 1-anilinonapthalene-8sulfonic acid, have been used to estimate binding constants [23]. Several factors have been shown to influence the formation of dendrimeric complexes such as pH, molecular size, and surface structure [24–26]. Objectives of this study were to determine the molecular mechanisms leading to the formation of dendrimeric daptomycin complexes by studying the effects of pH and dendrimer size on binding capacity and affinity and exploit the potential application of this system to improve the poor pharmacokinetics of daptomycin. In addition, these investigations provided insight into the nature of daptomycin interactions with charged macromolecules to control the assembly of daptomycin and PAMAM.
Materials and Methods Reagents Daptomycin obtained from Eli Lilly Research Laboratories (Indianapolis, IN) was used as received. Standard sodium hydroxide and standard hydrochloric acid solutions were purchased from Fisher Scientific (Fair Lawn, NJ). 1,4-Diaminobutane core PAMAM generation 3 (PAMAM 3) in 20% (w/v), generation 5 (PAMAM 5) and generation 6 (PAMAM 6) in 10% (w/v) methanol solutions were purchased from Sigma-Aldrich (St. Louis, MO).
scanned from 320 to 540 nm in order to observe the emission responses from both Trp-1 and Kyn-13. PAMAM–Daptomycin Complex Fluorescence Properties In this experiment (as in subsequently described titration studies), the commercially obtained methanolic solutions of PAMAM dendrimers were used to prepare aqueous dendrimer stock solutions. The methanol was evaporated by purging nitrogen gas, and the dendrimers were redissolved in double-distilled water. Daptomycin and PAMAM solutions were separately prepared at the same target pH values adjusted using 0.1 M sodium hydroxide or hydrochloric acid solutions. PAMAM 5 (0.18 μM) and daptomycin (3 μM) aqueous solutions at pH 4.0 and an equal volume mixture of PAMAM 5 and daptomycin solutions were prepared to evaluate the fluorescence properties of PAMAM–daptomycin complex. Binding Isotherm Construction Daptomycin–PAMAM complex binding isotherms were generated by the addition of aqueous daptomycin to PAMAM solutions using various size dendrimers (PAMAM 3, 5, and 6). As described in the preceding texts, the ligand (daptomycin) and substrate (PAMAM) solutions were prepared separately by adjusting each solution to a target pH value in the range of 3 to 9 using 0.1 M sodium hydroxide and/or hydrochloric acid solutions. A 3 ml aliquot of the PAMAM solutions (Table 1) was placed in a fluorescence quartz cell (10 mm path length) equipped with a magnetic stirrer. Typically, 15 aliquots (5 μl) of aqueous daptomycin solutions were added incrementally using a Hamilton microliter syringe to a final daptomycin maximum concentration as described in Table 1. After the addition of each aliquot, the mixtures were stirred for 3 min, and then, the fluorescence spectra were collected. As in the preceding texts, the control titrations were performed by adding 5 μl aliquots of daptomycin to the same pH value aqueous solutions in the absence of dendrimers and stirred for 3 min prior to spectroscopic analysis. All solutions were pH adjusted to the desired value prior to the addition, and the pH values of the titration mixtures were constant (≤0.1 pH) throughout each titration experiment. The binding isotherms were constructed by plotting the fluorescence intensity differences (ΔF) at 460 nm in the presence and absence of dendrimers against the total daptomycin concentrations to yield binding isotherms.
Fluorescence Spectroscopy
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Estimation of the Binding Parameters The mathematical models used to estimate binding constants from the isotherms have been previously described [19]. Briefly a
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Fluorescence spectra were recorded using a LS-55 Luminescence Spectrometer (Perkin Elmer Instruments, Norwalk, CT). The excitation wavelength was set at 285 nm, and the emission spectra were
CHANVORACHOTE ET AL. 1
Table 1. Dissociation constant (Kd; μM ) and capacity constant (n; molecules of daptomycin per one molecule of PAMAM dendrimer) at various pH ranges of the binding interaction between daptomycin and various PAMAM dendrimer generation estimated by simultaneous fitting using WinNonlin software with fixing the molar signal coefficient (ΔE1 and ΔE2) as a constant at 240.13 and 46.65, respectively Estimated binding parameters Substrate (PAMAM) Generation size
Ligand (daptomycin)
Second binding site 1
Maximum conc. (μM)
pH
Kd (μM )
n
Kd (μM )
n
0.14 0.14 0.27 0.05 0.05 0.05 0.27 0.27 0.27 0.27 0.27 0.01 0.01 0.01 0.01 0.01 0.01
74.07 74.07 11.61 5.80 5.80 5.80 8.21 8.21 7.06 11.61 11.61 74.07 74.07 74.07 74.07 74.07 74.07
4.0 7.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 3.5 4.0 4.5 5.0 6.0 7.0
0.76 0.01
13 55
0.26
13
0.33 0.06 0.05 0.13 0.13 0.11
49 59 58 12 10 8.0
0.06 0.22 0.12 0.07 0.07 0.01 0.01
6.8 44 66 57 57 35 19
1.73 2.00
82 91
5
6
quadratic equation was derived for one-site binding model that relates in the total ligand (daptomycin) concentration to fluorescence differences as described in the Eqn (1).
ΔF ¼
The utility of the model equation to distinguish between the binding mechanism and estimate the binding parameters including the difference in molar extinction coefficient (ΔE) between the
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 ΔE ½daptomycinT þ Kd þ R ± ½daptomycinT þ Kd þ R 4R½daptomycinT 2
where [daptomycin]T, ΔE, Kd, and R represent the total daptomycin concentration, the difference in molar extinction coefficient between the free and bound fluorophores, the equilibrium dissociation constant, and the total number of independent binding sites, respectively. For a two-site binding model, an Eqn (2) was derived to describe fluorescence difference titration curves in terms of the molar extinction parameters, total daptomycin concentration, and binding parameters. ΔF ¼ ΔE1
1
Conc. (μM)
3
First binding site
R1 ½daptomycinF Kd1 þ ½daptomycinF
þ ΔE2
R2 ½daptomycinF Kd2 þ ½daptomycinF (2)
where [daptomycin]F, a, b, c, and θ are given by the following relationships and are defined in terms of binding parameters and the total daptomycin concentration. a 2pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi θ ½daptomycinF ¼ þ ða2 3bÞcos 3 3 3 b ¼ Kd1 Kd2 þ Kd2 R1 þ Kd1 þ R2 -ðKd1 þ Kd2 Þ½daptomycinT c ¼ Kd1 Kd2 ½daptomycinT
314
2a3 þ 9ab 27c θ ¼ arccos qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi3 2 ða2 3bÞ
ð0 < θ < π Þ
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(1)
free and bound fluorophores, dissociation constant (Kd), and the total number of independent binding sites (R, or R1, and R2) has been previously described [19]. The nonlinear regression analysis was used to estimate binding parameters (WinNonlin Softer Version 5.0.1, Pharsight Corporation).
Results Fluorescence Changes Associated with PAMAM–Daptomycin Binding Shown in Figure 2 are the fluorescence spectra of daptomycin, PAMAM, and PAMAM–daptomycin complex at pH 4.0 aqueous solutions. Daptomycin has intrinsic fluorescence emissions at 355 nm from Trp-1 and 460 nm from Kyn-13, but PAMAM dendrimer shows no fluorescence emission maxima over the wavelength range between 320 and 540 nm. The PAMAM–daptomycin complex had a 14-fold increase in emission intensity at 460 nm from Kyn-13 and a 50% decrease at 355 nm from Trp-1. The significant fluorescence emission enhancement at 460 nm could be the self-association aggregation of daptomycin or the interaction of daptomycin with dendrimers. However, previous studies using a variety of orthogonal analytical approaches including NMR, fluorescence, and dynamic light scattering have demonstrated that the minimum critical aggregation concentration for daptomycin in the pH range 3 to 7 was always more than 100 μM [27]. The existing daptomycin
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THE INTERACTION MECHANISM BETWEEN DAPTOMYCIN AND PAMAM Effect of pH and Dendrimer Size on the Binding Characteristics of Daptomycin–Dendrimer Complex
Figure 2. The fluorescence spectra of daptomycin (3.0 μM), PAMAM 5 (0.18 μM), and an equal volume mixture of daptomycin (3.0 μM) and PAMAM 5 (0.18 μM) at pH 4.0 and 25 °C (cuvette path length 10 mm; excitation wavelength at 285 nm).
concentration here was only 3.0 μM. Therefore, the fluorescence enhancements observed herein could be unequivocally attributed to daptomycin–dendrimer interactions because the maximum daptomycin concentrations in all studies were less than 100 μM. The similar fluorescence enhancements at 460 nm from Kyn-13 have also been reported in the interaction of daptomycin and phospholipids [28]. The fluorescence changes observed were associated with daptomycin and dendrimer interactions. Typical fluorescence intensity changes in the absence and presence of dendrimers (PAMAM 5) as a function of daptomycin concentrations in pH 5.0 aqueous solutions are depicted in Figure 3. In the absence of dendrimer (PAMAM 5), the fluorescence intensities at 460 nm increased linearly over the range of daptomycin concentrations. Moreover, these increases were colinear in all pH value solutions. In the presence of PAMAM 5, the fluorescence emission intensities at 460 nm displayed a steep increase at low daptomycin concentrations and a gradual linear increase at higher concentrations.
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Figure 4. The binding isotherms of the interaction between PAMAM 5 and daptomycin in the range of pH 3.0 to 9.0. The normalized ΔF values were the ratio of ΔF value to PAMAM 5 concentration.
Figure 5. The binding isotherms of the interaction between PAMAM 6 and daptomycin in the range of pH 3.5 to 7.0. The normalized ΔF values were the ratio of ΔF value to PAMAM 6 concentration.
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Figure 3. The fluorescence intensities of daptomycin at 460 nm in the absence (solid square) and in the presence (solid circle) of PAMAM 5 at pH 5.0 (cuvette path length 10 mm; excitation wavelength at 285 nm).
Ionic interaction between daptomycin and dendrimer was anticipated to be a significant factor in daptomycin–dendrimer complex. Thus, factors affecting the ionic states of both compounds were investigated using various size dendrimers PAMAM 3, 5, and 6 in the pH range from 3 to 9. The generation number of dendrimers determines the size and amount of surface functional groups. The binding isotherm was constructed by plotting the difference between the fluorescence intensity in the presence and absence of dendrimer (ΔF) at each daptomycin concentration versus the total daptomycin concentration. The magnitude of the maximum fluorescence enhancement varied with pH and necessitated using different concentrations of ligand and substrate at different experimental conditions (Table 1). Therefore, the binding isotherms in the different pH solutions were generated in Figures 4, 5, and 6 by plotting the normalized fluorescence difference (ΔF/[PAMAM]) in the presence and absence of dendrimer at each daptomycin concentration versus the total daptomycin concentration
CHANVORACHOTE ET AL.
Figure 6. The binding isotherm of the interaction between PAMAM 3 and daptomycin at pH 4 and 7. The normalized ΔF values were the ratio of ΔF value to PAMAM 6 concentration.
In the presence of PAMAM 5 (Figure 4), the typical binding isotherms of the normalized fluorescence differences from pH 3.5 to 8 showed a steep increase at low concentration and a plateau at higher concentrations of daptomycin, which were consistent with one-site binding model [19]. In addition, the magnitude of the maximum values ( F/[PAMAM]) in the pH range 3.5 to 4.5 was 4-fold to 5-fold greater than in the pH range 5 to 8. The titrations of daptomycin to PAMAM 6 were conducted in the pH range of 3.5 to 7. In a pH range of pH 3.5 to 5 (Figure 5), the normalized fluorescence differences steeply increased and attained plateaus at higher daptomycin concentrations as described in PAMAM 5, which conformed to the one-site binding model. In the pH range of 6 to 7, the isotherm shapes were different in that the normalized fluorescence difference values increased at low daptomycin concentration attained a maximum value and then gradually decreased with increasing in daptomycin concentrations. This isotherm shape suggested change in binding at higher concentrations either because of a loss in binding capacity or a second binding site that was associated with a loss in fluorescence intensity. The resultant binding isotherms of PAMAM 3 at pH 4 and 7 are depicted in Figure 6. At pH 4, the binding isotherm shape appeared to be consistent with one-site binding model, whereas the binding isotherm at pH 7 was consistent with a two-site model. However, a little initial lag phase was observed from the one-site binding isotherm at pH 4. This may be a consequence of asymmetry structure of PAMAM 3 [29]. Binding Parameters
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The shapes of the isotherms were used to deduce the nature of the binding process and estimate the binding parameters. The binding parameters, molar extinction coefficient (ΔE), dissociation constant (Kd), and the total number of independent binding sites (R), were estimated by simultaneously fitting the model equation to the binding isotherm by nonlinear regression analysis. The molar extinction coefficient (ΔE) obtained from the fitting of the binding isotherms of PAMAM 5 titration in the pH ranges of 3.5 to 8 were not significantly different (p-value = 0.053). The constant value of ΔE indicated that the fluorescence characteristics of the interaction between daptomycin and PAMAM 5 were identical throughout the pH range of 3.5 to 8. Consequently, the ΔE value was fixed for all binding isotherms at a constant mean value of
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240.1. Then, the Eqn (1) was refitted to all binding isotherms with just two adjustable parameters in an attempt to attribute pH differences to the binding parameters, Kd (the equilibrium dissociation constant) and R (the total number of independent binding sites). The capacity constant (n, the number of bound daptomycin molecules on per PAMAM dendrimer molecule) of each individual pH range was then calculated based on the ratio of the estimate R value to the total concentration of PAMAM dendrimer. The binding parameters were reported in Table 1. The capacity constants (n) appeared to change dramatically as a function of pH, whereas the dissociation constants were not notably pH dependent. The one-site binding isotherms obtained from the titration of PAMAM 6 with daptomycin at low pH range were described. Model predicted curves were shown to describe the experiment data of the titration at pH range of 3.5 to 5. This suggested that the binding interaction at the pH ranges of 3.5 to 5 was consistent with one-site binding model. Two-site model equations [Eqn (2)] were used to describe the data obtained at pH 6 and 7. The binding parameters were estimated by simultaneously fitting the model equation to the binding isotherm data using nonlinear regression analysis in WinNonlin. The model-predicted isotherms agreed well with experiment data. Furthermore, the residual plots did not show region of systematic bias. The molar signal coefficient (ΔE), the dissociation constant (Kd), and the capacity constant (n) that belong to two different types of binding interaction were successfully estimated at each pH value. The values for ΔE1 and ΔE2 were essentially constant over the pH range studied (p-value of ΔE1 = 0.159 and p-value of ΔE2 = 0.768). The ΔE1 appear to be in the same range as the ΔE estimated using the data obtained from PAMAM 5. Therefore, these values were fixed in Eqn (1) (ΔE = 240.1) and Eqn (2) (ΔE1 at 240.1 and ΔE2 at 46.65), and the values for affinity and capacity constants were re-estimated at each condition by nonlinear regression thereby assigning any remaining variability to the binding properties. All of the predicted curves based on model equations and estimated parameters agreed with the experiment data. The estimated binding parameters were reported in Table 1. Comparison of the Kd1 values by one-way ANOVA indicated that these constants were discernibly pH dependent and that they could be separated into three groups, estimates associated with pH conditions 3.5, 4–5, and 6–7. The dissociation constant at the second type of binding interaction (Kd2) were not discernibly pH dependent. For the interaction of PAMAM 3 and daptomycin, a one-site binding model [Eqn (1)] was used to estimate binding parameters using data generated at pH 4, whereas, the binding isotherm at pH 7 corresponded to a two-site binding model [Eqn (2)]. The model equations used at each pH condition fit the experimental data without systematic residual bias and reasonably high correlation coefficients. The estimated binding parameters are displayed in Table 1.
Discussion Assessment of Relationship Between the Ionic Properties of Daptomycin, PAMAM Dendrimer, and Binding Capacity Daptomycin contains six ionizable groups, four side chain carboxylic groups (three aspartic acids, Asp-3, Asp-7, and Asp-9, and one methyl-glutamic acid, mGlu-12), and two primary amines (an aromatic amine in Kyn-13 and an aliphatic amine in Orn-6). Based on the 2D 1H TOCSY NMR pH titration methods [20], the sequence specific pKa values for Asp-7, Kyn-13, Asp-9, Asp-3, and mGlu-12 were estimated to be 1.01, 1.30, 3.85, 4.15, and 4.55, respectively. The aliphatic amine Orn-6 in daptomycin has a pKa value of 10.7 and
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THE INTERACTION MECHANISM BETWEEN DAPTOMYCIN AND PAMAM therefore is always protonated at pH values below 10 [11]. In the pH range of 0.8 to 3.5, the major ionic species is a zwitterion (H4A±) form as a result of the deprotonation of Asp-7 and the cationic Orn-6 (Figure 7). Monoanion (H3A) and dianion (H2A2) are the primary ionic species in the pH range of 3.5 to 4.7 as a result of the overlapping deprotonation of side chain carboxylic acids in Asp-9 and Asp-3 (Figure 7). Above pH 4.7, the trianion (HA3) is the predominant ionic form as mGlu-12 residue is deprotonated. PAMAM dendrimers possess two types of cationic amino groups: an outer shell of primary amines with a pKa value in the range of 7–9 and internal, tertiary amines with pKa values between 3 and 6 [16,30–32]. PAMAM 5 has 128 primary amines and 126 tertiary amines wherease PAMAM 6 has 256 primary and 254 tertiary amines [16,33]. The protonation of PAMAM dendrimers involves protonation of primary amine groups at the outer rim of the dendrimer over the entire range of interest. The tertiary amine groups in the dendrimer core are protonated at lower pH but deprotonate in the acidic pH range. Comparisons of daptomycin ionization profile and the pHdependent binding capacity parameters obtained using PAMAM 5 and 6 (Figure 7) suggest that the primary determinants in the magnitude of the binding capacity are associated with the predominating presence of the monoanion (H3A) and dianion (H2A2) in the pH range 4 to 5. Only small binding capacity changes can be attributed to changes in the ionic state of the tertiary amine residues in the dendrimer because the binding capacities remain high in the pH region wherein the tertiary amine are progressively less charged. Thus, the major sites for electrostatic interaction are between Asp-9 and cationic moieties on the dendrimer surface. Furthermore, these results suggest that additional anionic charges dianion and special trianion as a result of the protonation of Asp3 and mGlu-12 on daptomycin may weaken, rather than strengthen, daptomycin–dendrimer interactions. Interestingly, the binding capacity pH profile for PAMAM 5 is similar to the pH distribution of monoanionic daptomycin, which may explain why PAMAM 5 is consistent with the one-site binding model throughout the pH range of 3.5 to 8. The capacity constants decreased at pH values > 4.7 for PAMAM 5 and at pH values > 5.2 for PAMAM 6. The difference in the pH
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Assessment of Molecular Model for the Interaction Between Daptomycin and PAMAM Dendrimer An attempt to further develop the spatial and orientation relationships involved in daptomycin–PAMAM dendrimer interactions was undertaken by examining the conformations of these two molecules and predicting the maximum capacity constants based on molecular orientation and size. The conformation of free daptomycin was downloaded from RCSB protein data bank. This conformation was proposed using nuclear overhauser enhancement spectroscopy techniques [38]. A space-filling or CPK model of daptomycin was regenerated using Chem3D Ultra 10.0. The model showed atoms as three-dimensional spheres whose radii are scaled to the atomic van der Waals radii. The two limiting case daptomycin orientations considered were longitudinal and latitudinal orientations. The molecular radii were estimated to be 24.5 and 14.9 Å, respectively. The maximum theoretical binding capacity for ligand bound to dendrimer surface is given by the ratio of the dendrimer surface areas to the cross-sectional area of the ligand. Assuming a spherical PAMAM dendrimer and published PAMAM dendrimer diameters [39], the estimated dendrimer surface areas of PAMAM 3, 5, and 6 are 3.02 × 103, 8.82 × 103, and 14.1 × 104 Å2, respectively. Based on the two limiting case orientations described in the preceding texts, circular cross-sectional areas were estimated to be 471 and 174 Å2 for longitudinal and latitudinal orientation, respectively. The maximum theoretical binding capacities of various dendrimers for daptomycin molecules oriented latitudinally or longitudinally (Figure 8) were predicted and are compared with the experimentally measured maximum capacity constants (Table 2). For all of the dendrimers, the predicted theoretical capacities using a latitudinal orientation are in reasonable agreement with the
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Figure 7. Comparison of the distribution of ionic forms of daptomycin with the estimated capacity constants for its interactions with PAMAM 5 and 6. ± Fraction of daptomycin ionization varied from zwitterion (H4A ), anion (H3A ), 2 3 dianion (H2A ), and trianion (HA ), respectively. Each point represented the capacity constant (molecule of daptomycin/molecule of PAMAM) obtained from binding interaction between daptomycin and PAMAM 5 and 6 at various pH solutions.
dependence of the binding capacities for these two different-sized dendrimers could be explained by the effect of pH on the size of dendrimer. As the dendrimer becomes less ionic, it collapses and the geometry of the polymer changes [34]. Increasing the solution pH value from 5 to 8 results in a 2.7-fold decrease of dendrimer (PAMAM 6) valence [35]. Interior electrostatic repulsions are present at acidic pH values causing full extension of dendrimer branches and a large surface area. However, at alkaline pH, the lack of interior electrostatic repulsion decreases the dendrimer volume and surface area. Thus, the decrease in the estimated capacity constants observed for PAMAM 5 (pH > 4.7) and 6 (pH > 5.2) may be because of the combination of reduced dendrimer charge and surface area [34,36]. Both the monoanion and dianions contribute to daptomycin binding to PAMAM 6 in the pH range of 3.5 to 5. Because large dendrimers have a greater number of amines that can bind daptomycin, the overall binding constant on a per dendrimer basis increases with dendrimer generations from PAMAM 5 to 6 [37]. For PAMAM 6, the decrease in the estimated capacity constant observed in the pH range 5 to 7 appears to coincide with the deprotonation of the dendrimer tertiary amines. In the pH range of 6 to 7, the interaction between daptomycin with PAMAM 6 conforms to the two-site model. For PAMAM 3, 5, and 6, the optimal binding occurs in the pH region 3.5 to 4.5 wherein both the external and internal dendrimer amines are protonated and daptomycin is in its monoanionic and dianionic forms. The intermolecular interaction is based on electrostatic attraction, wherein the deprotonated Asp-9 of daptomycin primarily interacts with surface amine on the dendrimers.
CHANVORACHOTE ET AL.
2
3
4
5
6 7
Figure 8. Space-filling model showing the measurement of daptomycin diameter in longitudinal and latitudinal dimension using Chem3D.
8 9
Table 2. The calculated theoretical capacity and observed capacity obtained from fluorescence titration Substrate Generation size 3 5 6
Theoretical capacity
10
Latitudinal
Longitudinal
Observed capacity
17 51 81
6 19 30
13 59 66 11
observed values suggesting that daptomycin is capable of nearly completely covering the dendrimer surface under pH conditions wherein binding is optimal (pH 3.5 to 4.5).
12
Conclusions
14
The fluorescence changes of the enhanced fluorescence resonance energy transfer between Trp-1 and Kyn-13 were observed upon PAMAM solutions titrated with daptomycin, which are because of the alteration of daptomycin conformation. The shapes of the binding isotherms and the dependence of the estimated capacity constants on dendrimer size and solvent pH values provide meaningful insight into the interaction mechanism of daptomycin and dendrimers. By comparing the ionization profile of daptomycin based on the estimated sequence-specific pKa values with the pH-dependent binding capacity, the ionized Asp-9 residue of daptomycin primarily interact with PAMAM cationic surface amines.
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