Mechanism of Cooperativity and Nonlinear Release Kinetics in

Oct 20, 2015 - Despite extensive studies on drug delivery using multivalent complexation systems, the biophysical basis for release kinetics remains p...
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Mechanism of Cooperativity and Nonlinear Release Kinetics in Multivalent Dendrimer−Atropine Complexes Jhindan Mukherjee, Pamela T. Wong, Shengzhuang Tang, Kristina Gam, Alexa Coulter, James R. Baker, Jr., and Seok Ki Choi* Michigan Nanotechnology Institute for Medicine and Biological Sciences, and Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: Despite extensive studies on drug delivery using multivalent complexation systems, the biophysical basis for release kinetics remains poorly defined. The present study addresses this aspect involved in the complexation of a fifth generation poly(amidoamine) (PAMAM) dendrimer with atropine, an essential antidote used for treating organophosphate poisoning. First, we designed 1H NMR titration studies for determining the molecular basis of the drug complexation with a glutarate-modified anionic dendrimer. These provide evidence pointing to a combination of electrostatic and hydrophobic interactions as the driving forces for dendrimer complexation with the alkaloid drug molecule. Second, using LC−MS/MS spectrometry, we determined the dissociation constants (KD) at steady state and also measured the drug release kinetics of atropine complexes with four negatively charged dendrimer types. Each of these dendrimers has a high payload capacity for up to ∼100 atropine molecules. However, the affinity of the atropine to the carrier was highly dependent on the drug to dendrimer ratio. Thus, a complex made at a lower loading ratio (≤0.1) displayed greater atropine affinity (KD ≈ μM) than other complexes prepared at higher ratios (>10), which showed only mM affinity. This negative cooperative variation in affinity is tightly associated with the nonlinear release kinetics observed for each complex in which drug release occurs more slowly at the later time phase at a lower loading ratio. In summary, the present study provides novel insights on the cooperativity as the mechanistic basis for nonlinear release kinetics observed in multivalent carrier systems. KEYWORDS: PAMAM dendrimer, atropine, host−guest complexes, cooperativity, nonlinear release kinetics

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INTRODUCTION

kinetics associated with this multivalent complexation system (Figure 1). A large number of delivery systems that employ complexation as the mode for carrying a payload have been reported with diverse types of molecules ranging from nucleic acid oligomers11,15,25 and proteins7,26 to small therapeutic molecules including anticancer therapeutics such as paclitaxel,1,13 doxorubicin,12,14 methotrexate,3 5-fluorouracil (5-FU),27 and other types of small drug molecules.20,21 This complexation strategy, in particular, offers unique benefits for improving the physicochemical properties and pharmacokinetic parameters of certain small guest molecules that suffer from poor aqueous solubility,1,13,18 enzymatic degradation,7,26 or rapid renal clearance,16,17 which contributes to their short plasma halflives. The effectiveness of this delivery strategy relies on the structure, design, and loading capacity of the carrier. Most of the existing delivery systems have utilized nanomaterial carriers

Complexation with nanometer-sized carriers constitutes one of the principal mechanisms in the controlled delivery of therapeutic genes, proteins, pharmaceutical agents, and imaging molecules.1−9 The biophysical basis for such guest complexation or release in these systems is unique given the multivalent nature of the carrier in which each carrier presents multiple binding sites, either peripheral or internal, that engage in simultaneous binding with multiple guest molecules.10−12 Thus, the kinetic features of payload release from these systems share the similarity of following nonlinear or exponential release kinetics in which release occurs more rapidly in the initial phase as compared to the late phase.1,3,13,14 Despite its significant impact on the design of controlled delivery systems, the biophysical mechanism of such nonlinear release kinetics remains largely undetermined relative to the large number of studies focused on the biochemical or molecular mechanisms of host−guest interactions1,3,11−22 involved in the complexation systems. In this article, we investigate the mechanistic basis of atropine23 complexation with poly(amidoamine) (PAMAM)24 dendrimer nanoparticles and present evidence for the biophysical determinants, which explain the nonlinear release © XXXX American Chemical Society

Received: September 5, 2015 Revised: October 10, 2015 Accepted: October 20, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00684 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

spherical macromolecule (diameter = 5.4 nm24) and has a welldefined core−shell architecture, which provides an array of hydrophobic cavities and repetitive amine-terminated branches (theoretically 12824). This multivalent architecture confers the dendrimer nanoparticle with the ability to interact simultaneously with multiple guest molecules. Application of the PAMAM dendrimers in controlled delivery via complexation has been extensively demonstrated with a diverse class of guest molecules including small drug molecules,3,14,16,17,19,21,28 DNA for gene delivery,11,15,31,32 and proteins33 in vitro and in vivo. Despite the validation of such a wide breadth of applications, most of the studies on dendrimer-based delivery systems have focused on cell-based assays and thus lack fundamental insight on the biophysical basis of host−guest interactions. Only a few studies have investigated the molecular basis of dendrimer− drug interactions, which are attributed to electrostatic association,21 hydrogen bonding,20,34 and hydrophobic interactions.14,18,35 Recently, we investigated the complexation of G5 PAMAM dendrimers with pralidoxime (2-PAM) and obidoxime, each an oxime-based antidote used for the treatment of organophosphate (OP) intoxication.16,17 We provided evidence supporting the significant role of electrostatic interactions and multivalent cooperativity in complex formation with these oxime drug molecules. In this study, we focus on dendrimer carrier design and perform mechanistic investigations on the release kinetics of atropine, an acetylcholine receptor antagonist that serves as an essential antidote in the treatment of OP poisoning.23 The therapeutic efficacy of atropine is attributed to its potent activity in blocking the overstimulation of muscarinic acetylcholine receptors, which otherwise causes adverse symptoms associated with OP poisoning.36 However, atropine suffers from a short half-life because of its rapid renal excretion and susceptibility to enzymatic metabolism (t1/2 ≈ 1−2 h in human

Figure 1. (A) Structure of atropine, an alkaloid molecule (pKa = 10.0) that exists exclusively in the protonated form under physiological conditions. (B) Compound 1 G5(GA), a fifth generation (G5) PAMAM dendrimer fully derivatized with glutarate (GA), and a proposed model for its complexation with atropine driven by electrostatic and van der Waals interactions.

such as polymers,13 polymer micelles,1 dendrimers,3,4,15−17,28,29 and porous nanoparticles.12 Of those nanocarriers, our laboratory along with numerous others have explored the use of a fifth generation (G5) PAMAM24 dendrimer for applications in controlled drug delivery.2,3,5,6,28,30 This G5 dendrimer is a nanometer-sized

Figure 2. Structural models for a G5 PAMAM dendrimer G5(NH2)n (n = 128 (theoretical); 114 (experimental)) and its surface modified dendrimers 1 G5(GA), 2 G5(GHA), 3 G5(GAcp), and 4 G5(PEG). Abbreviation: GA = glutaric acid, GHA = glutaryl hydroxamic acid, GAcp = cyclopentane (cp)-fused glutaric acid, PEG = polyethylene glycol terminated with carboxylic acid. Given the symmetry of dendritic branches, only a pair of two branches in close proximity are illustrated for clarity. In 4, the remaining terminal branches are composed of 33 GA residues on a mean basis (not shown). B

DOI: 10.1021/acs.molpharmaceut.5b00684 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics plasma).37 Thus, the complexation of atropine with the G5 dendrimer can serve as a potential strategy for addressing such undesired pharmacokinetics as the resulting dendrimer complex is anticipated to follow polymer pharmacokinetics, which is characterized by a long circulation half-life of the dendrimer (≥24 h).38−40 This delivery approach can prevent premature drug degradation and extend the duration of drug action through the slow controlled release of loaded atropine molecules. The present study reports a first delivery system for atropine designed by the modification of G5 PAMAM dendrimer and discusses the binding parameters and release kinetics. These studies provide evidence for multivalent cooperativity in the complexation of drug and demonstrate its role in the nonlinear release kinetics observed in this system.

scattering (DLS) and zeta potential (ZP) measurements at room temperature on a Zetasizer Nano ZS (Malvern).43,44 1 H NMR Spectroscopy. All 1H NMR studies that include standard structural characterization, two-dimensional (2D) correlation spectroscopy (COSY), and atropine titration experiments were performed at 297.3 K (±0.2) at 500 MHz (Varian NMR spectrometer) as described previously.16,17 Chemical shift (δ) values are reported in ppm units relative to an internal standard 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (DSS; δ = 0.00). The 1H NMR titration experiment was performed by adding the solution of atropine acetate in incremental amounts (1−120 mol equiv) to a fixed amount of 1 G5(GA) (0.25 mM, 0.55 mL) in deuterated PBS pH 7.4 (Figure S3; Supporting Information). LC−MS/MS Spectrometry. Mass spectrometric analysis was performed on a Waters H Class Acquity UPLC system coupled to a TQ detector mass spectrometer.19,41 UPLC separation was performed using a Waters XBridge column (XBridge BEH C18 2.5 μm; 2.1 × 50 mm): flow rate = 0.41 mL/min; column temperature = 40 °C; sample injection volume = 10 μL. A linear gradient method was applied starting with a mobile phase at 99:1 (v/v) water (A)/acetonitrile (B), each with formic acid (0.1% v/v). The initial gradient at 1% B (0−1.28 min) was followed by a linear increase to 2% B (1.28− 1.30 min), and then to 95% B (1.30−2.49 min), and finally a linear decrease to 1% B (2.49−7 min). For atropine analysis, a stock solution (25 μM) was prepared in 60% aqueous methanol and diluted to a working concentration of 100 nM in the same solvent. Atropine was analyzed using the method of positive electrospray ionization in a multiple reaction mode: source temperature at 150 °C, desolvation temperature 400 °C, cone voltage = 8 V. Atropine was detected at tR = 2.45 min and its area under curve (AUC) was quantified by focusing the molecular species at this tR. The total ion chromatogram (TIC) of atropine was analyzed by MS/MS that showed two fragmentations (124.07, 93.03). A calibration curve for atropine was generated by analysis of serially diluted standard solutions (2−70 nM), each in triplicate (Figure S4; Supporting Information). The limit of detection (LOD) determined for atropine was ≥2 nM. Mass Spectrometric Analysis of Atropine Complexes. Each dendrimer/atropine complex was prepared by adding a solution of atropine in ethanol (fixed volume = 0.01 mL) to a solution of 1 G5(GA) dendrimer in water (1 mL, 136 μM) (Figure S5; Supporting Information). The concentration of atropine added ([Ap] = 136 μM to 163 mM) was varied to make complexes at various ratios of [Ap]/[D] (= 0.1, 1, 10, 20, 40, 60, 80, 120) where Ap refers to atropine and D refers to dendrimer. After incubation for 30 min at room temp, each mixture was transferred to a centrifugal filter unit (Amicon; MWCO 3000−5000) and spun down very shortly (≤30 s). Each filtrate (≤0.05 mL) was analyzed by LC−MS/MS spectrometry, and the amount of free atropine in the filtrate was determined. Mass Spectrometric Analysis of Release Kinetics. A representative procedure for release experiments is illustrated with a complex of atropine with 3 G5(GAcp) (Figure S6; Supporting Information): A solution of 3 in water (1 mL; 136 μM) was mixed with an atropine solution in ethanol (0.01 mL; 163 mM), and the mixture was left for 10 min at room temperature. For atropine alone, the same amount of atropine (0.01 mL; 163 mM) was mixed with 1.0 mL of blank water without the dendrimer. This solution served as a control for the

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MATERIALS AND METHODS Materials. Details for materials and solvents are described in the Supporting Information. G5 PAMAM dendrimer was purchased as a methanol solution (17.5% w/w; Dendritech, Inc.) and purified prior to use by membrane dialysis (molecular weight cutoff (MWCO) = 10,000; Spectrum Laboratories) against deionized water as described elsewhere.16 The average number (n) of primary amines per dendrimer molecule for G5(NH2)n is 128 (theoretical24) and 114 (experimental by potentiometric titration16). Modification of Dendrimer G5(NH2). Each of four PAMAM dendrimers 1−4 (Figure 2) was prepared by the surface modification of the parent G5(NH2) following standard methods.17,41,42 First, a fully glutaric acid (GA)-terminated anionic dendrimer 1 G5(GA)n=108 was made by amide formation of G5(NH2) with an excess amount of glutaric anhydride (260 molar equiv) and triethylamine in methanol.42 Second, 2 G5(GHA), a dendrimer terminated with glutaryl hydroxamate (GHA) (66 GHA residues per dendrimer), was prepared in two steps: (i) N-(3-(dimethylamino)propyl)-N′ethylcarbodiimide hydrochloride (EDC)-based activation of 1 G5(GA) and (ii) a subsequent reaction with O-tertbutyldimethylsilyl (TBDMS) protected hydroxylamine.41 Third, 3 G5(GAcp), a dendrimer terminated with cyclopentane (CP)-fused glutarate GAcp, was prepared by reaction of G5(NH2) with 3,3-tetramethyleneglutaric anhydride (228 molar equiv) and trimethylamine in methanol (details in Supporting Information). Finally, a PEG (polyethylene glycol)modified dendrimer 4 G5(PEG), which contains 53 PEG residues per dendrimer on a mean basis, was prepared following a similar conjugation method by reaction with an activated ester of poly(ethylene glycol) bis(carboxymethyl) ether (average Mn = 600) prepared by treatment with benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 1-hydroxybenzotriazole (HOBt) in DMF (see Supporting Information). After this PEGylation, unreacted amines of the dendrimer were further capped with glutaric anhydride, providing 4 G5(PEG). Each modified dendrimer was purified by membrane dialysis (MWCO 10,000) against phosphate buffered saline (PBS, pH 7.4; 2 × 12 h) and deionized water (2 × 12 h). Its material characterization was performed by 1H NMR spectroscopy, ultra performance liquid chromatography (UPLC), gel permeation chromatography (GPC), and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (Figures S1 and S2; Supporting Information). The size and charge distribution of dendrimers were determined for each conjugate at 5 μM (1 mM HEPES pH 7.0) by dynamic light C

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Molecular Pharmaceutics Table 1. Molecular Parameters of Four Dendrimers 1−4 and Atropine dendrimer; molecule G5(NH2) 1 G5(GA) 2 G5(GHA) 3 G5(GAcp) 4 G5(PEG) atropine

molecular weight (g·mol−1)a b

27600 (26270 ; PDI = 1.010) 40200 (40850b; PDI = 1.046) 41200 45400 62500 (73,530b; PDI = 1.138) 289.40

terminal or key functional group (pKa) NH2 (9.0−10.77; ref 47,48) CO2H (4.59c) CO2H (4.59) + C(O)NHOH (9.0−9.5; ref 46) CO2H (4.59) CO2H (4.59) + NH2 (9.0−10.77) tropane amine (10.03; ref 45)

size d (nm)d 5.4 4.0 6.5 4.2 6.1

(ref 24) (±0.1) (±0.6) (±0.2) (±0.3)

ZP (mV)e 43.3 (ref 49) −35.0 (±6.31) −17.7 (±10.0) −51.3 (±9.88) 26.4 (±5.42)

a Mr measured by MALDI-TOF mass spectrometry. bNumber-averaged molecular weight (Mn) determined by GPC; polydispersity index (PDI) = Mw/Mn. cCalculated by Advanced Chemistry Development (ACD/Laboratories) Software V8.14. dHydrodynamic diameter (number-averaged) by dynamic light scattering. eZeta potential.

phobic residues such as the bicyclic and phenyl ring. The latter can make additional contributions through van der Waals interactions in the hydrophobic cavities of the dendrimer. Surface Modified PAMAM Dendrimers. Based on these binding motifs, we were interested in using a G5 PAMAM dendrimer modified with negatively charged branches because of its complementarity to the positive charge of atropine. We focused on a series of G5 dendrimers terminated with glutaric acid (GA; pKa = 4.59) at its peripheral termini (Figure 2). This dendrimer series includes a fully GA-terminated dendrimer 1 G5(GA)17,42 and 2 G5(GHA)a G5(GA) dendrimer further modified with glutaryl hydroxamic acid (GHA), which is sufficiently acidic (pKa = 9.0−9.5)46 for electrostatic association with the basic tropane amine (pKa = 10.0). In addition to its utility in atropine delivery, use of G5(GHA) could be of potential benefit for the treatment of OP intoxication given the additional catalytic activity of GHA for destroying reactive OP molecules such as paraoxon.41 Two other dendrimers 3 G5(GAcp) and 4 G5(PEG) were designed for further modulating the physicochemical properties at the GA branch. Thus, in each of these dendrimers, the GA residue is replaced with a hydrophobic cyclopentane-fused GAcp moiety or an amphiphilic PEG chain (Mn = 600) terminated with carboxylic acid, respectively. In summary, four types of negatively charged G5 dendrimers were designed through branch modification at the peripheral amine site for investigating the effects on atropine complexation. Each of the dendrimers 1−4 was prepared following surface modification methods17,41 applied for G5(NH2) by treatment with anhydride or an activated ester as described in the experimental section and Supporting Information. The modified dendrimer was characterized for its polymer purity (>95%) by UPLC and for structural identity and molecular properties by 1H NMR, MALDI-TOF, GPC, and DLS (Figures S1, S2, and S3; Supporting Information). Table 1 provides a summary of their molecular weights (MALDI, Mr; GPC, Mn), polydispersity indices (PDI = Mw/Mn), hydrodynamic diameters (d, nm), and charge properties (ZP, mV). In 2 and 4, the dendritic branches are composed of two or three different types, GA/GHA or GA/PEG/NH2, and the branch fraction was determined by 1H NMR and MALDI-TOF methods.41 Specifically, the number (valency, n) of GHA per dendrimer in 2 G5(GHA)n (n = 66) was calculated on an average basis by comparison of an NMR integration value for its GHA protons (middle CH2, δ 1.87 ppm) to that of the remaining GA protons (δ 1.82 ppm): n = ∑CH2GHA/(∑CH2GA + ∑CH2GHA) × 108 = 66.41 In 4 G5(PEG)n, the PEG valency (n = 53) was calculated by comparison of its methylene protons (OCH2C(O), δ 3.96 ppm) to that of the dendrimer methylene protons (CH2C(O), δ 2.6 ppm): n = ∑CH2PEG/(∑CH2G5)

diffusion kinetics of atropine. The atropine solution alone or atropine/G5 complex solution was loaded into a Float-A-Lyser membrane tube (Amicon; MWCO 3000−5000), and the tube was immediately immersed in a beaker containing deionized water (300 mL). The water in each beaker was magnetically stirred and a series of aliquots, each 0.2 mL, were taken out at specific time points after tube immersion (0, 2, 5 10, 15, 30 min, and then 1, 2, 3, 6, and 24 h). After each aliquot was removed, the same volume of deionized water (0.2 mL) was replenished into the beaker to maintain the initial volume (300 mL) of water. Each of these aliquots was analyzed by LC−MS/ MS spectrometry, and the amount of atropine released into the outside compartment was determined using an established calibration curve of atropine. Cell Culture. The KB carcinoma cell line (ATCC) was used for all in vitro cell based studies. Cells were grown and maintained in RPMI 1640 medium with no folic acid (FA) (Life Technologies) with 10% heat-inactivated fetal bovine serum (FBS), 100 IU penicillin, and 100 mg/mL streptomycin. All studies were performed in media lacking FA. In Vitro Cytotoxicity Assay. The biocompatibility of the dendrimers was assessed by a cell based assay in vitro using the KB cell line.11 Three thousand KB cells/well were seeded in a 96-well cell-culture plate in FA-free RPMI 1640 media and grown for 2 days at 37 °C. Growth media was removed and replaced with serial dilutions of dendrimers 1−4 in media (3− 100 μM) and incubated at 37 °C for 24 h. The media was removed and replaced with fresh media, and the cells were allowed to grow for another 4 days. The cellular viability was then measured using a XTT (sodium 3-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate) assay according to the manufacturer’s protocol (Roche Life Science).

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RESULTS AND DISCUSSION Binding Motifs. Design of PAMAM dendrimers that are able to engage in noncovalent complexation with atropine requires understanding the structural motifs of the atropine molecule. Atropine belongs to a tropane (azabicyclic) class of alkaloid natural products with a basic tertiary amine moiety (pKa value = 10.0; Figure 1).45 It exists exclusively as a protonated cationic species under the physiological pH of 7.4 ([protonated]/[neutral] = 10(pKa−pH) = 398). This positive charge thus can provide a strong driving force toward complexation with the negatively charged residues of the dendrimer through electrostatic attraction. Given the bulky size of atropine, most of such electrostatic interactions are expected to occur on the external surface rather than in the inner core of the dendrimer. In addition, atropine carries other binding motifs including H-bond donor/acceptor moieties and hydroD

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Figure 3. (A) 1H NMR spectral assignment for 1 G5(GA) dendrimer and atropine acetate. (B) 1H−1H COSY spectrum of atropine acetate. Cross peaks marked in dashed rectangles refer to the protons in scalar coupling. Each spectrum was acquired in deuterated PBS, pD 7.4: [G5(GA)] = 0.25 mM; [Atropine] = 34.6 mM. DSS (4,4-dimethyl-4-silapentane-1-sulfonate sodium) was used as an internal standard (δ = 0.0 ppm).

= 53; ∑CH2PEG/(∑CH2GA) = 1.584 (Figure S2, Supporting Information). These values from the NMR analysis are in good agreement with those calculated from the mass increase in its MALDI-TOF (Mr = 62,500 g mol−1) relative to G5(NH2) (Mr = 27,600 g mol−1). Each of these dendrimers was stable in aqueous media such as PBS pH 7.4, as no decomposition products were detected by UPLC analysis over several days. NMR Analysis of Atropine Complexes with G5(GA). The 1H NMR titration method has been frequently employed for analyzing the complex formation between dendrimers and small guest molecules.14,16−18,20,21 In our earlier studies, we performed 1H NMR titration experiments to determine the structural motifs involved in the complexation of PAMAM dendrimers with the oxime class of antidotes including pralidoxime and obidoxime.16,17 In the present study, we used this method for investigating the mechanism of complexation between atropine and 1 G5(GA) as a representative dendrimer. Prior to the titration experiments, we analyzed two 1H NMR spectra for 1 G5(GA) and atropine alone as shown in Figure 3a

since complete assignment of proton signals in each spectrum is of critical importance for the accurate interpretation of the titration experiments. Proton signals for 1 G5(GA) were assigned as described previously.17,42 Atropine proton signals, however, appear complicated in this 1D spectrum due to its complex patterns of spin−spin couplings and signal overlaps, in particular, in those protons associated with the tropane ring structure. In order for more accurate assignment, we performed a 2D 1H−1H COSY experiment for atropine (Figure 3B). This COSY spectral data allowed us to build the bond connectivity among multiple sets of spin−spin coupled protons. As illustrated, those cross peaks marked in red squares suggest that these coupled protons belong to either the tropane ring or 3-hydroxy-2-phenylpropanoate. The NMR titration experiment was performed by adding a solution of atropine acetate to a fixed amount of 1 G5(GA) at a variable molar equivalent (Figure 4A). Upon atropine addition, several subsets of the dendrimer protons shifted upfield as a function of the [Ap]/[D] ratio. Such shifts occurred with the protons (marked as d−h) in close proximity to the tertiary E

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Figure 4. (A) 1H NMR titration experiments for the complex formation between 1 G5(GA) dendrimer (D) and atropine (Ap) acetate in deuterated PBS, pD 7.4. The drug was added to a fixed amount of the dendrimer ([D] = 2.5 × 10−4 M) at a variable molar ratio ([Ap]/[D]). The signals highlighted in colored circles refer to the protons that showed changes in chemical shift values. (B) A proposed model for the atropine complex as suggested by the 1H NMR titration experiments above.

amine flanked with two GA-terminated branches (Figure 4B). Of those, the protons attached to the carbon directly adjacent to the tertiary nitrogen showed the greatest shifts in Δ (= δdrug added − δfree): e ≈ f > h > d. These shifts suggest that the tertiary amine domain may constitute a receptor site for interacting with the atropine molecule. On the guest side, a certain fraction of atropine signals showed line broadening or upfield shifts in response to variation in the [Ap]/[D] ratio. These signal changes occurred notably in the tropane ring protons including N-CH3 (δ 2.72 ppm), 6β and 7β (δ 2.14 ppm) as highlighted in light blue or green, respectively. Proposed Model for the Atropine Complex. Based on the 1H NMR titration experiment above, we propose a model for the dendrimer−atropine complex in which an atropine molecule binds in a peripheral hydrophobic cavity composed of a tertiary amine flanked by terminal GA branches (Figure 4B). This model is consistent with the binding motifs discussed above in which the electrostatic interactions between the protonated N-CH3 (pKa = 10.0) and the terminal carboxylate

ions (pKa = 4.59) constitute one of the primary driving forces of complexation. This proposed interaction is supported by the upfield shift of the tropane N-CH3 signals, which may be attributable to its partial charge transfer to the carboxylate ion in the dendrimer. In addition, van der Waals interaction also could play a role in the complexation as suggested by line broadening in those protons (6β, 7β) that belong to the hydrophobic tropane ring (cLogP = 1.70). We believe that this tropane binding could occur in the cavity around a tertiary amine, which is believed to remain neutral due to its weak basicity (pKa = 6.3−6.85 for its conjugate acid47). LC−MS/MS Analysis of Atropine Complexes. In the NMR titration study, we presented a molecular picture for atropine binding in 1 G5(GA) as a model dendrimer. We next performed quantitative analysis of the biophysical parameters that dictate individual binding events, which constitute global complexation. We prepared various atropine complexes with each of the four dendrimers, 1−4, and employed LC−MS/MS spectrometry to determine the fraction of free (unbound) F

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Figure 5. LC−MS/MS spectrometric analysis of atropine complexes with dendrimer 1 G5(GA), 2 G5(GHA), 3 G5(GAcp), and 4 G5(PEG). Each complex was prepared at a fixed dendrimer concentration ([D]fixed = 136 μM) with variation in the [Ap]/[D] ratio. (A) Bound atropine (%) = 100 × [Ap]bound/[Ap]total added. (B) Fraction (θ) of receptor occupancy. A dotted line refers to a theoretical line for maximal occupancy in which all added atropine molecules are assumed to be bound. (C) A representative Scatchard plot analysis for the atropine complex with 1 G5(GA). (D) Plots of dissociation constants (KD) for various complexes as a function of [Ap]added/[D] ratio. Each data point in A−C refers to a mean value ± standard deviation (SD; n ≥ 3).

atropine molecules isolated by using flash membrane filtration (Figure 5). Use of mass spectrometry enabled detection of atropine with high sensitivity (LOD ≥ 2 nM) and precision (SD = 2−12%) (Figure S4). Such detection capabilities are not readily achievable by standard HPLC analysis due to the lack of strong UV−vis absorption by atropine. In addition, this LC− MS/MS method is also compatible with the analysis of release kinetics experiments, which will be described later. For each dendrimer, a series of complexes were prepared by varying the ratio of [Ap]/[D] (molar equiv = 0.1, 1, 10, 20, 40, 60, 80, 120). The amount of free drug ([Ap]free) measured by LC−MS/MS was used to calculate the amount of bound drug ([Ap]bound) in each complex series as summarized in Figure 5A. The bound fractions were also used to derive the fraction of occupancy (θ, Figure 5B), which is defined as the number of occupied sites (= drug molecules bound) relative to the number of total receptor (cavity) sites per dendrimer (= 108). Given the variation in the branch structure between the four dendrimers, this calculation is based on an assumption that one terminal branch presents a receptor site as proposed in Figures 1 and 2.

Fractional Occupancy. Figure 5A summarizes the fractions of bound atropine molecules for a series of complexes made with 1−4, each plotted as a function of [Ap]/[D] ratio. In general, higher percentages of bound atropine (∼90−95%) were observed at lower ratios (≤1) than higher ratios (≥10) as anticipated from previous studies with oximes.16,17 Thus, only small variations were observed in the lower loading ratios regardless of the dendrimer types, suggesting that the fractional loading efficiency is greater when fewer drug molecules are present. However, larger variations were observed at the higher ratios. The loading capacity, the maximal number of drug molecules bound per dendrimer, varied by dendrimer type with the following rank order in capacity: 3 G5(GAcp) > 2 G5(GHA) > 4 G5(PEG) ≈ 1 G5(GA). We also calculated the fractional occupancy (θ) as shown in Figure 5B. The θ values calculated at the highest ratio of [Ap]/[D] = 120 follow the same order as the loading capacity: 3 (θ ≈ 0.85) > 2 (θ ≈ 0.80) > 4 (θ ≈ 0.75) > 1 (θ ≈ 0.70). Thus, in complex 3, a maximum of 92 sites (= 0.85 × 108) could be occupied from all the available sites (108 per dendrimer). These fractional analyses suggest approximately 1:1 binding stoichiometry G

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Molecular Pharmaceutics

Figure 6. LC−MS/MS analysis for the atropine release kinetics of dendrimer complexes with 1−4. A Float-A-Lyser membrane tube (MWCO 3000) containing an aqueous solution (IN) of atropine alone or atropine/G5 complex was immersed in a beaker containing water (OUT). A series of aliquots were taken out from the water (OUT) at specific time points for mass spectrometric analysis. (A) Representative release kinetics for atropine alone (initial [Ap]IN = 1.63 × 10−3 M) or in complex with 3 G5(GAcp) (initial [D]IN = 1.36 × 10−5 M; [Ap]/[D] = 120). (B) Normalized atropine release kinetics for various dendrimer complexes, each prepared at a fixed [Ap]/[D] ratio (= 120). Normalized (fractional) Ap release = [Ap]OUT (complex)/[Ap]OUT (atropine alone). Each data point in A−B refers to a mean value ± standard deviation (SD; n = 3).

Affinity and Multivalent Cooperativity. We determined the affinity constants (KD) for bound drug molecules in the complexes made at various ratios. In our earlier studies with oxime molecules,16,17 we demonstrated that this variation in affinity is closely related with the degree of cooperativity between individual binding events that occur in the multivalent dendrimer system. Figure 5C shows a representative Scatchard analysis performed for the complexes with 1 G5(GA). In this plot, the slope of θ/[Ap]free vs θ corresponds to an affinity constant (KA= 1/KD = −slope). This Scatchard plot shows a nonlinear decay curve that varies as a function of fractional occupancy (θ). Thus, drug binding to an identical dendrimer occurs with various affinity constants (KD1 − KDn) instead of a single value as illustrated in Figure 5C. Affinity constants determined for each of the four complexes are plotted in Figures 5D and S7 (Supporting Information). It is notable that the variation of affinities is largely influenced by the [Ap]/[D] ratio. In general, higher affinities are observed at lower loading ratios (KD ≥ 10−4−10−6 M at [Ap]/[D] ≤ 1) than higher ratios (KD = 10−2−10−4 M at [Ap]/[D] = 10− 100). Thus, the affinities are inversely correlated with the fractional occupancy, indicating negative cooperativity between successive binding events. A Hill plot analysis performed for 1 G5(GA) complexes provides a cooperativity coefficient (n) of 0.92, which points to negative cooperativity (n < 1). This result is consistent with our earlier observations in the multivalent complexation of G5 PAMAM dendrimers with small oxime drug molecules.16,17 We believe that this negative cooperativity might be attributable to the effect of repulsive steric interactions between multiple drug molecules bound in close proximity. This cooperativity also accounts for the variation in affinities of the same dendrimer for atropine at different [Ap]/[D] ratios despite the homogeneous nature of the cavities. Release Kinetics. We hypothesized that the dependence of the binding affinity on the complex ratio as discussed above

between the atropine guest molecule and each receptor site, which is consistent with the binding model proposed in Figure 1. We believe that such variations in the loading capacity might be attributable to the difference in the functional groups and structural properties of each receptor unit as well as to the relative conformational flexibility of the unit to allow minimization of the increasing effects of unfavorable steric congestion during drug complexation. First, incorporation of a hydrophobic cyclopentane (cp) moiety on GA or partial replacement of GA with GHA appears to be effective for maintaining the high loading capacity throughout the [Ap]/[D] ratios tested. Thus, cavities with greater hydrophobicity such as those found in 3 G5(GAcp) might be more favorable for binding the hydrophobic tropane in atropine than 1 G5(GAcp). This was supported by the observation of fractional occupancy (θ) that the number of atropine molecules occupying the binding sites in 3 G5(GAcp) (0.85 × 108 = 92) is greater than that in 1 G5(GA) (0.7 × 108 = 76) by up to 16 drug molecules per dendrimer at [Ap]/[D] = 120. Second, the presence of a neutral flexible PEG chains (MW 600 g mol−1) in the cavity of 4 G5(PEG) might offer complementary binding motifs through its enhancement of the hydrophobicity of the cavity or by providing H-bond acceptor atoms (ether oxygens) for interaction with the ester moiety of atropine. Lastly, it is considerable that such variable loading capacity might be influenced by the difference in global dendrimer properties such as the size (d, Table 1) or more specifically the surface area (calcd; 4πr2 = 50.3 (1), 132 (2), 55.4 (3), 117 nm2 (4)). No correlation is observed between the surface area and bound atropine (%) or the fraction of occupancy in each of the complexes (Figure 5A,B). This analysis suggests that the loading capacity observed here is influenced more by the structural and functional features of the individual receptor unit. H

DOI: 10.1021/acs.molpharmaceut.5b00684 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 7. Effect of the [Ap]/[D] ratio on the release kinetics of atropine in complex with 3 G5(GAcp). Atropine release kinetics from the complexes (A) or diffusion of atropine alone (B) as a control was measured and normalized to atropine release at selected time points (C). Each complex was prepared with variation in the [Ap]/[D] ratio ([D]IN = 13.6 μM; [Ap]/[D] = 10, 20, 60, 120). Normalized Ap release is defined as above.

complex and diffusion of atropine alone are plotted as a function of the [Ap]/[D] ratio along with that of atropine alone performed at the identical concentration as summarized in Figure 7A,B. A representative set of the normalized release data are compared at selected time points in Figure 7C. This plot shows that the release occurs faster at the initial phase (t = 0.08 h) than at the later steady phase (t ≥ 1 h), which is notable, in particular, at the higher ratios ([Ap]/[D] = 120, 60). This observed nonlinear kinetics of atropine release has several implications. First, it provides evidence for the dependence of release rate on drug loading ratio. The complex prepared at the higher ratio shows faster release largely at early time points: [Ap]/[D] = 120 ≈ 60 > 20 > 10. Second, these kinetic results are also consistent with negative cooperativity of binding observed at steady state, which suggests an inverse correlation between atropine affinity and occupancy (loading ratio). Third, this result confirms the conventional notion that the kinetics of drug release is controlled by the physicochemical properties of the designed carrier and also demonstrates the notion that the drug release kinetics for a single nanocarrier type can be controlled by variation of the loading ratio alone. In summary, we provide evidence for first time that the affinity distribution explains the nonlinear release kinetics, which is frequently observed in most multivalent complex systems. In Vitro Cytotoxicity. We evaluated whether dendrimers 1−4 have potential toxicity in cells using KB cells, a human cervical cell line frequently employed for studying compound toxicity.11,17 The XTT assay performed in vitro showed that none of the dendrimers inhibited cell viability at doses of up to 0.025 mM (≈ 1−2 mg/mL, Figure S8; Supporting Information). Thus, this assay result is consistent with our previous observations17,42,50 in which the surface modification of G5(NH2) with excess neutral amides or negatively charged groups abolishes undesired cellular toxicity, which is typically otherwise associated with this highly cationic dendrimer.50

(Figure 5C,D) provides a potential means by which the release kinetics of atropine can be modulated by controlling the ratio of drug to carrier. In order to corroborate this hypothesis, we performed the release experiments using a membrane dialysis setup in combination with the LC−MS/MS analysis of released atropine (Figures 6 and S6, Supporting Information). First, the release kinetics were measured at a single complex ratio ([Ap]/ [D] = 120) for each dendrimer type as illustrated with 3 G5(GAcp) relative to atropine alone as a diffusion control (Figure 6A). Atropine alone freely diffuses through the membrane (MWCO 3000) down its concentration gradient. The atropine−dendrimer complex in contrast, shows release kinetics slower than atropine alone as it involves a two-step process: (i) atropine dissociation from the complex; (ii) its diffusion through the membrane. The differences between the release kinetics of free drug vs the drug in complex with the dendrimer demonstrates the utility of this complex for extended drug release. The release experiments were also performed with the other dendrimer complexes (1−4) at the same complex ratio ([Ap]/ [D] = 120). Figure 6B shows a summary of the release kinetics of these dendrimer−atropine complexes, each shown as a curve normalized to the atropine control (normalized (fractional) release = [Ap]OUT (complex)/[Ap]OUT (atropine alone)). In general, the release rate was not uniform over the entire release period. For each complex, drug release was faster at earlier time points (