Investigation of Lysine-Functionalized Dendrimers as Dichlorvos

Article pubs.acs.org/Biomac

Investigation of Lysine-Functionalized Dendrimers as Dichlorvos Detoxification Agents Esteban F. Durán-Lara,†,§ Jennifer L. Marple,† Joseph A. Giesen,† Yunlan Fang,‡ Jacobs H. Jordan,† W Terrence Godbey,‡ Adolfo Marican,§ Leonardo S. Santos,§ and Scott M. Grayson*,† †

Department of Chemistry, ‡Department of Chemical and Biomolecular Engineering, Tulane University, New Orleans, Louisiana 70118 United States § Laboratory of Asymmetric Synthesis, Chemistry Institute of Natural Resources; Nanobiotechnology Division at University of Talca, Fraunhofer Chile Research Foundation - Center for Systems Biotechnology, FCR-CSB, Talca University, P.O. Box 747, Talca, Chile S Supporting Information *

ABSTRACT: Lysine-containing polymers have seen broad application due to their amines’ inherent ability to bind to a range of biologically relevant molecules. The synthesis of multiple generations of polyester dendrimers bearing lysine groups on their periphery is described in this report. Their hydrolytic stabilities with respect to pH and time, their toxicity to a range of cell lines, and their possible application as nanodetoxification agents of organophosphate compounds are all investigated. These zeroth-, first-, and second-generation water-soluble dendrimers have been designed to bear exactly 4, 8, and 16 lysine groups, respectively, on their dendritic periphery. Such monodisperse bioactive polymers show potential for a range of applications including drug delivery, gene delivery, heavy metal binding, and the sequestration of organic toxins. These monodisperse bioactive dendrimers were synthesized using an aliphatic ester dendritic core (prepared from pentaerythritol) and protected amino acid moieties. This library of lysine-conjugated dendrimers showed the ability to efficiently capture the pesticide dichlorvos, confirming the potential of dendrimer-based antidotes to maintain acetylcholinesterase activity in response to poisoning events.

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INTRODUCTION

2,2-bis(hydroxymethyl)propanoic acid (bis-MPA) polyester dendrimers28,29 are currently being evaluated for many in vivo biological applications such as drug and gene delivery.16,30−32 In contrast with conventional polymers and some early developed dendrimers, these bis-MPA dendrimers can be prepared to exhibit an extremely well-defined “perfectly” branched architecture that is truly monodisperse, bearing an exact multiplicity of reactive chain ends. Herein, we report the divergent synthesis of a new amino acid-functionalized, watersoluble library of polyester dendrimers based on a bis-MPA repeating unit. Pentaerythritol was used as a core with 4, 8, and 16 lysine moieties attached to the end groups of zeroth-, first-, and second-generation dendrimers, respectively. In addition, the in vitro biocompatibility of the library of bis-MPA dendrimers was assessed using human cell lines, and the stability of the dendrimers was determined as a function of pH and time. Finally, the capacity of these dendrimers to capture highly toxic organophosphate (OP) compounds, specifically dichlorvos (DCV), has been evaluated. DCV is an OP compound commonly used as a pesticide. Similar OP compounds have been utilized worldwide for many

The use of polymeric carriers for the delivery of therapeutic agents originated from the hypothesis that polymers may be tailored to improve the solubility, increase the blood circulation time, and reduce the toxicity of small molecule drugs.1 Dendritic polymers, however, are of particular interest because they can exhibit a “perfect” symmetric, highly branched architecture, and an exact molecular weight. As a consequence, each molecule exhibits a uniform structure, globular shape, an exact multivalency, and an ease in surface modification due to increased end group accessibility.2 Dendrimers’ branched structure and exceptional synthetic modularity3−5 result in a class of compounds that can be easily tailored to exhibit high solubility,6−8 low viscosity,9,10 biocompatibility,11−14 nonimmunogenicity,15−17 and biodegradability.13,18 In recent decades, amino acid-functionalized polymers, dendrimers,19−21 and hyperbranched polymers22−24 have attracted much attention because of their potential biomedical applications. Morever, the attachment of positively charged amino acids such as lysine or arginine onto hyperbranched cores has been demonstrated to improve cell penetration efficiencies.25 Furthermore, the multivalency of dendrimers affords enhanced binding to various target molecules such as tartaric acid, heavy metals, etc., through synergistic non-covalent interactions. © XXXX American Chemical Society

Received: May 15, 2015 Revised: October 7, 2015

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DOI: 10.1021/acs.biomac.5b00657 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

value of 6.50. The detector mass range was set to 200-8000 Da in order to exclude high intensity peaks (matrix noise) from the lower mass range. The laser intensity was set to the lowest value possible to acquire high resolution spectra. The instrument was calibrated using SpheriCal calibrants obtained from Polymer Factory Sweden AB (Stockholm). A THF solution of trans-2-[3-(4-tert-butylphenyl)-2methyl-2-propenylidene]malononitrile (DCTB) (10 mg/mL) was used as matrix and Na/TFA (2mg/mL) was used as the cation source for the analysis of the protected (with tert-butyloxycarbonyl (Boc) and carboxybenzyl (Z) groups) dendrimers in a THF solution (2 mg/mL). Samples were prepared in a 2:2:1 matrix:salt:sample volume ratio, and 1.5 μL was spotted on the sample plate. A THF solution of α-cyano-4-hydroxycinnamic acid (CHCA) (10 mg/mL) was used as matrix for the deprotected (amino acid-terminated) dendrimers in a THF solution (2 mg/mL) and spotted using the sample ratio and spotting volume as the protected dendrimers. The obtained mass spectra were analyzed with FlexAnalysis (Bruker Daltonics; Bremen, Germany, version 2.2). Proton nuclear magnetic resonance (1H NMR) experiments were performed on a Bruker AV 300 MHz NMR instrument, a Varian 400 MHz NMR instrument, and a Bruker 500 MHz NMR instrument. 1H NMR spectra were acquired with a spectral window of 20 ppm, an acquisition time of 5.3 s, a relaxation delay of 5 s, and 64 scans. Carbon-13 nuclear magnetic resonance (13C NMR) spectra were acquired with a spectral window of 240 ppm, an acquisition time of 1.8 s, a relaxation delay of 2 s, and as many scans as necessary to obtain complete spectra. Spectra were recorded in CDCl3 for the protected (with tert-butyloxycarbonyl (Boc) and carboxybenzyl (Z) groups) dendrimers and (CD3)2SO solutions for the deprotected (amino acid-terminated) dendrimers at 25 °C temperature, and the chemical shifts were calibrated against the residual solvent peak. Gel permeation chromatography (GPC) was performed on a Waters model 1515 series pump (Milford, MA) with three column series from Polymer Laboratories, consisting of PLgel 5 μm Mixed D (300 mm × 7.5 mm, molecular weight range 200− 400 000), PLgel 5 μm 500 Å (300 mm × 7.5 mm, molecular weight range 500−30 000), and PLgel 5 μm 50 Å (300 mm × 7.5 mm, molecular weight range up to 2000) columns. The system was fitted with a Model 2487 differential refractometer detector, and HPLC grade THF was used as the mobile phase (1 mL min−1 flow rate). The calculated molecular weight was based on a calibration using linear polystyrene standards. Data were collected and processed using Precision Acquire software. Procedure for the Synthesis of First-Generation Protected Dendrimer, 2, and General Procedure for Adding a Dendritic Layer. The compound 2, [G-1]-Ph4, was synthesized according to Ihre et al.29 Pentaerythritol, 1 (100 mg, 0.734 mmol, 1.0 equiv), and 4(dimethylamino)pyridine (DMAP) (45 mg, 0.36 mmol, 0.5 equiv) were dissolved in 7 mL of CH2Cl2, and 4.5 mL of pyridine was added. The benzylidene protected bis-MPA anhydride synthesized according to Ihre29 (1.5 g, 3.5 mmol, 4.8 equiv) was added, and the reaction mixture was stirred at room temperature overnight. The excess anhydride was quenched by stirring the reaction mixture with 8 mL of a 1:1 pyridine:water solution overnight. The organic phase was diluted with 60 mL of CH2Cl2 and extracted with 1 M NaHSO4 (3 × 40 mL), 10% NaHCO3 (3 × 40 mL), and saturated brine (40 mL). The organic phase was dried with MgSO4, filtered, and the filtrate evaporated to yield 702 mg (95%) of 2 as a glassy solid. Spectroscopic data agreed with those previously reported.29 Procedure for the Synthesis of the First-Generation Deprotected Dendrimer, 3a, and General Procedure for Removal of the Benzyldene Protecting Groups. The compound 3a, [G-1]-Ph4, was synthesized according to Ihre et al.29 Compound 2 (300 mg, 0.32 mmol) was dissolved in MeOH (15 mL); CH2Cl2 (20 mL) and 75 mg of 10% Pd/C were added. The apparatus for catalytic hydrogenation was evacuated and filled with H2 three times. After vigorous stirring overnight, the completion of the deprotection reaction was confirmed by MALDI-ToF mass spectra acquired from crude aliquots. The catalyst was removed via filtration through a plug of Celite in a glass fritted filter followed by multiple washings of the Celite with ethyl acetate. The filtrate was evaporated to yield 271 mg (98%) of

years to protect plants and prevent crop damage. Because of their inherent toxicity, many locations, such as the European Union, have set maximum residue limits (MRLs) for pesticides in plants and food.33 Ideally, a pesticide should achieve its intended effect without harming human health or the environment. In practice, however, pesticides are not deposited exclusively on the target and often contaminate soil, groundwater, rivers, lakes, etc.,34 resulting in toxicity to nearby human and animal populations. OP pesticides/herbicides are organic molecules containing phosphate groups that have the capacity to irreversibly inactivate the enzyme acetylcholinesterase (AChE).35 Human exposure to OP compounds can decrease the activity of vital neurotransmitters resulting in incapacitating symptoms that vary from rhinorrhea, excessive salivation, perspiration and lacrimation, headaches, nausea and vomiting, abdominal pain, chest tightness and dyspnea, involuntary urination and defecation, muscle fasciculation, seizures, coma, and potentially even death.36,37 Although specific antidotes have been developed for OP poisoning, they are not without shortcomings, including limited biodisponibility, stock shortages in situ (i.e., owed to limited shelf life or cost), and toxic side effects (preventing their use as pretreatments in high risk environments). As a consequence, pretreatments are uncommon, and early treatments are usually limited to less effective measures such as emptying the stomach of the patient or administering activated charcoal. The standard drug treatments are based, generally, on anticholinergic therapies using atropines.38,39 Atropines antagonize the central and muscarinic effects of neurotransmitters by blocking these receptors. However, atropines do not bind to nicotinic receptors; hence, muscular weaknesses, including respiratory muscle weakness, are not affected.40 For this reason, nanocarriers have emerged as a viable alternative over traditional treatments for detoxification in cases where no antidote is available.41 Nanocarriers, to be effective, must remain in the blood long enough to sequester the toxic material and/or their metabolites, and the toxin-bound complex must remain stable until it is removed from the bloodstream. Dendrimers have demonstrated the ability to function as effective carriers with a very low toxicity and fast renal clearance in in vivo studies.42,43 This work aims to synthesize, characterize, and evaluate the stability and biocompatibility of well-defined lysine-functionalized polyester dendrimers. The long-term goal of this research is to evaluate these dendrimers’ potential as OP nanodetoxification agents to be applied in agriculture, the food industry, and biomedicine.

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MATERIALS AND METHODS

Materials. Unless otherwise noted, all reagents were purchased from Sigma-Aldrich and used without further purification. Solvents were removed under reduced pressure using a rotary evaporator or by vacuum pump evacuation. Compounds 2, 3a, 4, and 5a were synthesized according to published procedures.29 Dichlorvos PESTANAL analytical standard was purchased from Sigma-Aldrich. Characterization. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) data were acquired on a Bruker AutoFlex III MALDI-ToF MS equipped with a nitrogen (N2) laser (337 nm) and a gridless ion source. All spectra below m/z = 4000 were acquired using a reflector-positive acquisition method with a constant ion source 1 value of 19.00, an ion source 2 value that fluctuated between 16.55 and 17.65 depending on the dendrimer mass, a lens value of 8.50, and a reflector voltage of 11.36 kV. All spectra above m/z 4000 were acquired using a linear-positive acquisition method with an acceleration voltage of 1.27 kV and a lens B


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Biomacromolecules compound 3a as white crystals. Spectroscopic data agreed with those previously reported.29 Procedure for the Synthesis of the Second-Generation Protected Dendrimer, 4. The compound 4 was synthesized following the general procedure for adding a dendritic layer, but instead using as the starting material compound 3a (150 mg, 0.255 mmol, 1.0 equiv) with DMAP (61.35 mg, 0.5 mmol, 0.5 equiv) in 7 mL of CH2Cl2 and 4.5 mL of pyridine, followed by the addition of the benzylidene protected bisMPA anhydride (1.043 g, 2.44 mmol, 9.6 equiv). The product was isolated following the general procedure to yield 340 mg (95%) of 4 as a glassy solid. Spectroscopic data agreed with those previously reported.29 Procedure for the Synthesis of the Second-Generation Deprotected Dendrimer, 5a. Following the general deprotection procedure, compound 4 (659 mg 0.39 mmol) was dissolved in MeOH (15 mL); CH2Cl2 (20 mL) and 120 mg of 10% Pd/C were added. The filtrate was evaporated to yield 585 mg (98%) of 5a as white crystals. Spectroscopic data agreed with those previously reported.29 General Procedure for the Conjugation of Dendrimers with Protected Lysine Groups (1b, 3b, and 5b). One equivalent of the hydroxylated dendrimer core, 1.2 equiv of Boc-Lys(Z)-OH, and 10 wt % of 4-(dimethylamino)pyridine (DMAP) (with respect to the core) were dissolved in DMF to give a 0.3 M solution with respect to the core. Once dissolved, 1.2 equiv of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) was added per hydroxyl. The coupling was monitored by MALDI-TOF MS, and once complete, the reaction mixture was diluted 150-fold with diethyl ether. The organic layer was rapidly washed with saturated aqueous NaHSO4 (2 × 150 mL), saturated aqueous NaHCO3 (2 × 150 mL), and saturated brine (1 × 150 mL). The organic layer was then dried over sodium sulfate, filtered, and the solvent removed by rotary evaporation. Flash column chromatography was used to purify the protected lysine-functionalized dendrimers using a gradient of 1−6% MeOH/CHCl3. Procedure for the Synthesis of the Protected Lysine-Functionalized Zeroth-Generation Dendrimer, [G-0]-(Boc-Lys(Z))8 (1b). Pentaerythritol, 1a (509.6 mg, 3.74 mmol), 4.8 equiv of Boc-Lys(Z)OH (6.13 g, 16.1 mmol), and 4-(dimethylamino)pyridine (DMAP) (49.6 mg, 0.406 mmol) were dissolved in DMF (11 mL), and then 1ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) (2.276 g, 0.012 mol) was added. After 1.5 h, the reaction was complete as determined by MALDI and worked up according to the general procedure to yield a crude white solid (2.019 g, 36%). MALDI calculated [1b + Na]+: m/z = 1607.80. Found: 1607.80. GPC (1b): Mw = 1980, Mn = 1970; Đ = 1.01. Synthesis of the Protected, Lysine-Functionalized First-Generation Bis-MPA Dendrimer, [G-1]-(Boc-Lys(Z))8 (3b). Dendrimer 3a (500 mg, 0.033 mmol), Boc-Lys(Z)-OH (3.063 g, 8.05 mmol), and 4(dimethylamino)pyridine (DMAP) (54.3 mg, 0.437 mmol) were dissolved in 8 mL of DMF. EDC (1.56 g, 8.16 mmol) was added to the solution. After 4 h another quarter equivalent per hydroxyl was added of both Boc-Lys(Z)-OH (0.654 g, 1.72 mmol) and EDC (0.3191 g, 1.66 mmol) to the solution. The reaction was determined complete 4 h later by MALDI and worked up according to the general procedure to yield a crude white solid (2.06 g, 70%). MALDI calculated [3b + Na]+: m/z = 3520.73. Found: 3520.79. GPC (3b): Mw = 3620, Mn = 3600; Đ = 1.01. Synthesis of the Protected, Lysine-Functionalized SecondGeneration Bis-MPA Dendrimer, [G-2]-(Boc-Lys(Z))16 (5b). Dendrimer 5a (407 mg, 0.266 mmol), Boc-Lys(Z)-OH (2.6370 g, 6.93 mmol), and 4-(dimethylamino)pyridine (DMAP) (38.3 mg, 0.313 mmol) were dissolved in 3 mL of DMF. After 4 h another quarter equivalent per hydroxyl was added of both both Boc-Lys(Z)-OH (0.457 g, 1.20 mmol) and EDC (0.198 g, 1.03 mmol) to the solution. The reaction was determined complete 12 h later by MALDI and worked up according to the general procedure to yield a crude white solid (0.925 g, 48%). MALDI calculated [5b + Na]+: average m/z = 7351.4. Found: 7351.3. GPC (5b): Mw = 6370, Mn = 6140; Đ = 1.03. General Procedure for Removal of the CBz and Boc Deprotection of Lysine Groups. The protected lysine functionalized dendrimer was

dissolved in 33% HBr/HOAc, and the solution was stirred at room temperature for approximately 1 h according to Okarvi et al.44 Evaporation of the solvent afforded the product as an oil, which was then used in the following reaction. Then, a solution of 4M HCl/ dioxane in a 25 mL round-bottom flask equipped with a magnetic stirrer was cooled by an ice−water bath under nitrogen, and the product from the previous step was added and stirred. The ice bath was removed, and the mixture was stirred for approximately 1 h. TLC was used to confirm that the reaction was completed; the reaction mixture was condensed by rotary evaporation under high vacuum at room temperature. The residue was then washed with dry ethyl ether and collected by filtration to afford the deprotected product (for oil products, a simple decantation was used instead).11,45 Synthesis of the Deprotected, Lysine-Functionalized FirstGeneration Bis-MPA Dendrimer, [G-0]-(Lys)8 (1c). Following the general procedure for the lysine deprotection, compound 1b (48 mg, 0.030 mmol) was deprotected by successive reaction with 33% HBr/ HOAc (4 mL) and then HCl/dioxane (8 mL) to afford 17 mg of 1c (92% yield). MALDI calculated [1c + Na]+: exact mass m/z = 671.44. Found: 671.55 (1H and 13C NMR data available in the Supporting Information). Synthesis of the Deprotected, Lysine-Functionalized FirstGeneration Bis-MPA Dendrimer, [G-1]-(Lys)8 (3c). Following the general procedure for the lysine deprotection, compound 3b (180 mg, 0.051 mmol) was deprotected by successive reaction with 33% HBr/ HOAc (14 mL) and then HCl/dioxane (28 mL) to afford 75 mg of 3c (89% yield). MALDI calculated [3c + Na]+: exact mass m/z = 1648.01. Found: 1648.74 (1H and 13C NMR data available in the Supporting Information). Synthesis of the Deprotected, Lysine-Functionalized SecondGeneration Bis-MPA Dendrimer, [G-2]-(Lys)16 (5c). Following the general procedure for the lysine deprotection of compound 1b, compound 5b (82 mg, 0.011 mmol) was deprotected by successive reaction with 33% HBr/ HOAc (8 mL) and then HCl/dioxane (16 mL) to afford 40 mg of 5c (99% yield). MALDI calculated [5c + Na]+: average mass m/z = 3603.3. Found: 3602.1 (1H and 13C NMR data available in the Supporting Information). Synthesis of the Deprotected, Lysine-Functionalized PEG 600 Diol, (Lys-PEG600-Lys) (6c). Following the general procedure for the lysine deprotection of compound 1b, compound 6b (123 mg, 0.090 mmol) was deprotected by successive reaction with 33% HBr/HOAc and then HCl/dioxane to afford 47.63 mg of (6c) (90% yield). MALDI calculated for 14-mer [6cn=14 + H]+: m/z = 891.57. Found: 891.87. GPC: Mn = 870, Mw = 890, Đ = 1.01 Assessment of Dendrimer Degradation. The dendrimers 1c, 3c, and 5c (1.00 mmol) were each dissolved into 10 mL of three separate solutions of buffer. The buffers used were acetate buffer (0.10 M), phosphate buffer (0.10 M), and Tris-HCl buffer (0.10 M) at pH 5.5, 7.0, and 8.5, respectively, and each was held at constant ionic strength (I = 0.15 M) by addition of KCl. The solutions were kept at 20 °C, and aliquots were taken at regular time intervals to monitor degradation by MALDI-ToF MS analysis. Biocompatibility. The murine urothelial carcinoma cell line MB49 (Anthony Atala, Wake Forest University Baptist Medical Center, Winston Salem, NC), the murine colon carcinoma cell line CT26. WT [American Type Culture Collection (ATCC), Manassas, VA], and the human normal foreskin cell line HFF-1 (ATCC) were used for cell viability assays. MB49 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA), 100 U mL−1 penicillin, and 100 U mL−1 streptomycin (Invitrogen). CT26.WT (ATCC CRL-2638) cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin, and 100 U mL−1 streptomycin. HFF-1 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum, 100 U mL−1 penicillin, and 100 U mL−1 streptomycin. All cells were grown in 75 cm2 culture flasks that were kept in a humidified 37 °C incubator with 5% CO2. C


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Biomacromolecules

Scheme 1. General Synthesis and Deprotection of the Zeroth-, First-, and Second-Generation Lysine-Functionalized Polyester Dendrimers (1c, 3c, and 5c)

Affinity Assays. For each dendrimer, experiments were carried out to determine the extent of binding and fractional binding of DCV in methanol solutions. Briefly, functionalized dendrimers (1c, 3c, 5c) adjusted at 0.045, 0.0045, 0.0009, and 0.000 45 mM were mixed in a 1:1 v/v ratio with a 0.045 mM (10 ppm) of DCV to give a final dendrimer:DCV molar ratio of 1:1, 0.1:1, 0.02:1, and 0.01:1, respectively. The assays were performed at pH 4.5−5.5. The samples were mixed for 45 min with dendrimer derivatives at constant room temperature (25 °C) and then centrifuged at 10 000 rpm for 10 min. The concentrations of DCV in supernatants separated from precipitated were analyzed by HPLC. The adsorption efficiency of DCV by the lysine-functionalized dendrimers were evaluated by determining the percentage decrease in the absorbance at each specific maximum absorbance wavelength using the equation

adsorption (%) =

A0 − A × 100 A0

Application Version 2.13 (NICOMP Particle Sizing Systems, Santa Barbara, CA), which employed proprietary NICOMP distribution analysis as the inversion of the Laplace transform (ILT) and nonlinear least-squares (NLLS) analysis. A distribution of hydrodynamic diameters was obtained for the diffusion coefficient. All distributions were weighted by volume and number. The scattering angle was set to 90° while the temperature was held at 20 °C. Prior to analysis, all solutions were filtered through Whatman 0.20 μm PTFE membrane filter (GE Life Siences, Pittsburgh, PA) and centrifuged for 10 min at 10 000 rpm on an Eppendorf centrifuge 5424 (Eppendorf North America, Hauppauge, NY) (Serial #5424AN741084). For preparation of 5.0 mM G0, 1800 μL of chloroform was added to 14.3 mg of G0 to give 5.02 mM solution. For preparation of 5.0 mM G1, 1030 μL of chloroform was added to 18.0 mg of G1 to give 5.00 mM solution. For preparation of 5.0 mM G2, 1120 μL of chloroform was added to 41.0 mg of G2 to give 5.00 mM solution. 400 μL was added to 6 mM disposable glass culture tubes, and data were collected over 30 min of autocorrelation acquisition. The volume-weighted distributions obtained were G0 = 1.6 ± 0.2, G1 = 2.1 ± 0.5, and G2 = 2.9 ± 0.7. The number-weighted distributions obtained were G0 = 1.5 ± 0.2, G1 = 2.0 ± 0.3, and G2 = 2.7 ± 0.4.

(1)

where A0 is the initial absorbance at specific wavelength and A is the observed absorbance at the same wavelength of each OP compound. All three lysine-functionalized dendrimers at 1:0.1 were compared to their unfunctionalized analogues to confirm that the peripheral lysine units, not the dendritic core, were predominantly responsible for the observed binding to DCV. To further elucidate the effect of the dendritic architecture on binding, an equivalent lysine functional poly(ethylene glycol), 6c, was used as a control with functionalized lysine dendrimers. Briefly, the bis-lysine PEG, 6c, was adjusted to concentrations so as to exhibit an equivalent number of lysines as each of the functionalized dendrimers (at 0.0045 mM), e.g., 0.009 mM (to compare to tetrafunctional G0), 0.018 mM (to compare to the octafunctional G1), and 0.036 mM (to compare to the hexadecafunctional G2). Dynamic Light Scattering. Dynamic light scattering (DLS) analyses were performed on a NICOMP Z3000 particle sizer (Serial #1409301) with calculations of size distributions and distribution averages performed using NICOMP software package ZPW388

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RESULTS AND DISCUSSION Polyester dendrimers based on bis-MPA monomer units were selected as a scaffold because they are nonimmunogenic, biodegradable, and nontoxic.13,14,46 Three generations of dendrimers were prepared to investigate their utility toward DCV binding. Synthesis of 1c. The tetrafunctional pentaerythritol core 1a was modified with a Boc-Lys(Z)-OH to afford the zerothgeneration dendrimer 1b (Scheme 1) after acidic and basic aqueous extractions. The GPC and MALDI characterization data for the protected lysine dendrimer confirmed the wellD


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Biomacromolecules

Table 1). Finally, compound 3b was deprotected using the standard acid-catalyzed protocol for removal of the Cbz and Boc groups to afford polyester dendrimer 3c as its HCl salt bearing exactly eight peripheral lysine groups (1H NMR, 13C NMR, and MALDI mass spectra detailed in Figure 2 and Figures S10−S12). Synthesis of 5c. The second-generation hydroxyl-functionalized dendrimer 5a was prepared as described previously29 and functionalized with 16 Boc-Lys(Z)-OH groups (Scheme 1) to afford the functionalized dendrimer 5b. This compound could be obtained after simple aqueous extractions and purified by flash chromatography, as judged by GPC and MALDI-ToF MS data (Figure 3, Figures S13−S15, and Table 1). The amino acid

defined nature of this product (Figure 1, Figures S1−S3, and Table 1). Compound 1b was then deprotected using the

Figure 1. MALDI-ToF mass spectra of (a) 1b before and (b) 1c after Boc/Cbz deprotection. (c) GPC chromatogram of 1b.

Table 1. Polyester Dendrimers Bearing Protected Amino Acids and Their Characterization, MALDI-TOF MS Data Subtracting Ionizing Cations group

name

no. of NH2

theor MW

MW (MALDI)

Mw (GPC)

Mn (GPC)

Đ (GPC)

0 1 2

1b 3b 5b

8 16 32

1584.81 3497.74 7323.59

1584.81 3497.80 7323.5

1980 3620 6370

1970 3600 6140

1.01 1.01 1.03

Figure 3. MALDI-ToF mass spectra of (a) 5b before and (b) 5c after Boc/Cbz deprotection. (c) GPC chromatogram of the protected precursor 5b.

optimized acid-catalyzed conditions for removal of the Cbz and Boc groups. The resultant product 1c was isolated as its HCl salt and displayed exactly four lysine substituents on the periphery of the pentaerythritol core (see Figure 1, Figures S4− S6, 1H NMR, 13C NMR, and MALDI mass spectra in the Supporting Information). Synthesis of 3c. The first-generation dendrimer, 3a, was prepared from pentaerythritol (1a) by dendronization to first yield compound 2 and subsequent deprotection, according to previously described procedures29 (Scheme 1). Compound 3a was then functionalized with Boc-Lys(Z)-OH via an EDC coupling to afford the first-generation dendrimer 3b (Figure 2, Figures S7−S9). This compound could be isolated via simple aqueous extractions and purified by flash chromatography, based on both GPC and MALDI-ToF MS data (Figure 2 and

protecting groups on compound 5b could be removed using acid-catalyzed deprotection conditions to afford the lysinefunctionalized dendrimer 5c as its HCl salt. The presence of only the Na+ adduct signal in the MALDI-ToF mass spectrum confirmed the complete deprotection and high purity of the product (1H NMR, 13C NMR, and MALDI mass spectra detailed in Figure 3 and Figures S16−S18). Synthesis of 6c. In addition, in order to provide a nondendritic lysine-functionalized polymer as a control for binding studies, a bis-functional poly(ethylene glycol) was also functionalized with lysine groups attached at both ends. Although not monodisperse, the MALDI-ToF mass spectrum enabled identification of three series of signals, corresponding to the H+ (major), Na+, and K+ adducts (Figure 4). Assessment of Degradation of 1c, 3c, and 5c Dendrimers as a Function of pH and Time. The first detailed investigations of degradable dendrimers47−49 emerged less than two decades ago. Since then, a number of examples of dendrimers that cleave in response to stimuli such as light, transition metals, catalytic antibodies, reducing agents, and pH change have been developed.50,51 Among these stimuli, pHtriggered degradation is perhaps most useful for site-specific biological applications, as the pH varies predictably inside the various regions of the body.52 Although sequestration agents should be of sufficient size to exhibit sufficient blood circulation times, a reasonably fast renal elimination rate is critical to prevent bioacculmulation; therefore, the timely degradation of polymer therapeutics into nontoxic oligomeric byproducts is important. In a very recent study, Reul et al. investigated the degradation profiles of three Boltorn hyperbranched polyesters (HBPs),53 namely unmodified hydroxyl-terminated HBPs, fatty

Figure 2. MALDI-ToF mass spectra of (a) 3b before and (b) 3c after Boc/Cbz deprotection. (c) GPC chromatogram of the protected precursor 3b. E


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Biomacromolecules

Figure 4. MALDI-ToF mass spectra of the 14-mer region of 6c and that of the entire mass spectrum of 6c (inset).

perhaps the more sterically crowded environment in the larger dendrimers inhibits the hydrolysis reaction, though the backfolding of the chain ends toward the core at higher generations may also play a role. It is important to note as well that observed degradation reactions correspond to the loss of the peripheral lysine units, but there is little evidence for degradation of the ester linkages within the dendrimer core during the time frame investigated. This suggests that the lysine units are preferentially hydrolyzed, perhaps via a backbiting of the lysine amino side chain to generate an amino-lactam byproduct.54 In addition, the increased localized steric hindrance of the pivalate esters within the dendritic core is believed to play a significant role in inhibition of their hydrolysis. Additional degradation assays of the three generations of lysine-functionalized dendrimers were carried out in buffers of pH 5.5, 7.0, and 8.5 at 22 °C (Figure 6, Figures S22 and S23). For each study, the removal of one of the lysine end groups appears to facilitate access to other terminal ester bonds, accelerating the hydrolysis of subsequent lysine groups. The initiation of the ester hydrolysis was evident only after 6 h at pH 5.5 for each of the three generations. The majority of dendrimer starting materials appear to have initiated degradation within the first 24 h, exhibiting the loss of at least one lysine unit. As observed for the degradation in neutral water, the formation of a series of new signals corresponding to the loss of additional lysine units (146 g mol−1 per lysine unit) reveals that the initial hydrolysis occurs predominantly among the peripheral lysine units. Although degradation of the ester

acid-modified HBPs, and amphiphilic HBPs. They discovered that the unmodified polymer displayed a faster degradation rate, presumably due to the presence of free hydroxyl groups accelerating the ester hydrolysis. Here, the degradation rate of amino acid-modified dendrimer analogues 1c, 3c, and 5c dendrimers were investigated by MALDI-ToF MS as a function of both time and pH. Such MS techniques cannot provide quantitative degradation rates without rigorous control studies because the species observed may have different ionization efficiencies. However, a direct comparison of the mass spectrum of an equimolar mixture of 3a and 3c (Figure S19) confirms that their ionization efficiencies are sufficiently similar to enable a qualitative MS degradation study (suggesting that the presence of the lysine groups does not substantially bias the observed MS signal). Stock solutions of each of the three generations were prepared at a concentration of 10 mol L−1 and an ionic strength of 0.15 mol L−1. First, the stability of each dendrimer was evaluated with respect to time in deionized water at 22 °C. The degradation reaction was slow and was easily monitored by MALDI-TOF MS. These results confirm that the first signs of degradation in water occur within 24 h and correspond to the loss of one of the lysine units via hydrolysis of its ester linkage to the dendrimer (Figure 5, Figures S20 and S21). While 1c shows signs of complete hydrolysis (all four lysine units) after 1 week, 3c shows the loss of about half (∼4) of the lysine end groups after 1 week and 5c only one-quarter (∼4) of the lysine units. As the linker chemistry for each of the lysines is identical in each case, the retarded degradation for the higher generation suggests that F


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Biomacromolecules

Figure 5. MALDI-ToF mass spectra monitoring the hydrolytic degradation of the zeroth generation dendrimer, 1c, in deionized water over a series of time points.

units (116 g mol−1 per repeat unit) is also possible, especially at increased or reduced pH, the lysine units appear to be cleaved selectively, confirming the relative stability of the polyester dendrimer scaffold under these conditions. Similar results are observed for the degradation behavior in buffered solutions at pH of 7.5 and 8.5 (Figure 6, Figures S22 and S23). Again, the hydrolysis is observed predominantly among the peripheral lysine units during the first 3 days, with the degradation at a pH of 8.5 being slightly more rapid than at the other pHs, similar to what had been previously reported for the hydrolytic degradation of dendrimer cores by themselves.13 It is important to reiterate that the pivalate ester bonds of each of the dendrimers seem to withstand hydrolytic degradation during the first 3 days of each of the degradation studies. Again, this is attributed to a combination of the increased accessibility of the periphery, the proposed lactam-forming lysine hydrolysis via backbiting, and the increased steric hindrance of the core pivalate esters compared to the amino acid ester linkages. However, degradation studies by Feliu et al. have confirmed that the dendritic cores eventually exhibit biodegradation at

each of these pHs and the bis-MPA dendrimers by themselves are generally biocompatible.13 Biocompatibility. The biocompatibility of the dendrimers was also investigated with multiple cell viability assays. These viability studies confirmed the negligible toxicity of the lysinefunctionalized dendrimers. Figure 7 shows cell viability after 24 h in three tested cell lines: murine colon carcinoma (CT26), murine bladder carcinoma (MB49), and human foreskin (HFF1). Cell viability is represented in this figure as cell numbers in treated groups normalized to that in a saline only (sham) group. For all generations of the lysine-functionalized dendrimers the observed cell viability is comparable to sham group for each of three cell lines. Additionally, there does not appear to be any generation dependence on the observed cell viability, which were compared as a constant weight percent per sample (1.71 μg/mL); this confirms that the lysine dendrimers themselves do not exhibit significant toxicity at this concentration. Adsorption of DCV by Lysine-Functionalized Dendrimers in Model Solutions. The adsorption behaviors of DCV by the lysine-functionalized dendrimers was studied by G


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Figure 6. MALDI-ToF mass spectra of the degradation products of the 5c dendrimers in three different buffers (pH 5.5, 7.0, and 8.5) with respect to time.

to the lysine-functionalized polyester dendrimers 1c, 3c, and 5c yielded an excellent affinity, with 100% trapping observed for all three dendrimers. This means that all of the DCV was bound to dendrimers (by encapsulation into the dendrimers or superficial interaction with them), and no measurable amount of the free DCV was observed in the HPLC chromatograms. However, when the molar ratio of the dendrimer is reduced 10-fold to 0.10 equiv dendrimer to 1 equiv DCV, the trapping percentages of 1c, 3c, and 5c were 52, 64, and 83%, respectively. This confirmed that with a 10-fold excess of DCV (per dendrimer, rather than per lysine), each of the dendrimers was capable of binding to multiple molecules of DCV and, as the number of lysine groups increased, the percentage entrapped (per molecule) also increased. This trend continued at the 0.02 equiv dendrimer to 1 equiv DCV where the observed percentages of trapping of 1c, 3c, and 5c were 25, 34, and 56%, respectively. Finally, when the molar ratio of DCV to dendrimer was changed to 0.01 equiv dendrimer to 1 equiv DCV, the percentages of trapping observe for 1c, 3c, and 5c were 5, 10, and 25%, respectively. To clarify the origin of these results, all three lysinefunctionalized dendrimers were compared to their unfunctionalized analogues (dendrimers without lysine) to confirm that the peripheral lysine units, not the dendritic core, were predominantly responsible for the observed binding to DCV (Figure 8). The unfunctionalized dendrimers exhibited little of the affinity observed with their lysine-functionalized analogues, confirming the critical role of the lysine units in DCV binding. Direct comparison between the different lysine-functionalized dendrimers is complicated by the fact that with each increasing dendritic generation the number of lysines increases 2-fold. Therefore, the enhanced trapping percentages that are observed with increasing generation number are a function of the increased number of lysines but are also affected by the increased size and steric hindrance of the larger dendrimers. A more realistic comparison that removes the difference in effective lysine concentration would be comparing the first-

Figure 7. Cell viability of the G1, G2, and G3 lysine-functionalized dendrimers normalized to sham group in three cell lines. Statistical analysis was performed one-way ANOVA followed by Tukey post hoc test (***p < 0.001).

determining the association of DCV with the dendrimers, isolated as their HCl salts, using a HPLC binding assay, relative to DCV alone (Figure S24). Because the DCV binding is judged by the precipitation and centrifugation of bound DCV− dendrimer complexes, this technique is not expected to be directly applicable to in vivo DCV sequestration. However, this protocol provides a useful tool to quantify the generation dependence of binding which can be used to inform future optimization of detoxification agents. The observed affinity of DCV for lysine-functionalized dendrimers is likely due to the multiple hydrogen bonds that can occur between the phosphoryl group of DCV and primary ammoniums of the lysine-functionalized dendrimers (Figure 8). Additional factors that may influence the binding include van der Waals forces, electrostatic bonds, and hydrophobic interactions as described in previously reported investigations.55,56 In particular, it was observed that the trapping of DCV upon a 1:1 molar exposure H


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Figure 8. Experimental assays indicating the percentage of affinity of the three lysine-functionalized dendrimers to DCV. For assays of 1c, 3c, and 5c, 0.045 mM DCV was used, and the amount of dendrimer used is expressed as molar equivalents to DCV (actual concentrations: 0.045, 0.0045, 0.0009, and 0.000 45 mM). 1a, 3a, and 5a refer to the control assay for the unfunctionalized dendrimers (without lysine) with hydroxy end groups, measured at 0.0045 mM. Finally, 6c was used as a nondendritic lysine-functionalized control. In order to compare with different dendrimer generations, the concentrations were converted to the lysine equivalents of the dendrimers to which they were compared, namely 0.009 mM for comparing to 1c at 0.1:1, 0.018 mM for comparing to 3c at 0.1:1, and 0.036 mM for comparing to 5c at 0.1:1. The statistical analysis was used with the software Prism 5; values are mean ± SD; n = 3, *p < 0.05.

generation, “G1”, dendrimer at 0.01 M (0.08 M lysine equivalents) that exhibited 10% trapping against the G0 at 0.02 M (0.08 M lysine equivalents) that exhibited 25% trapping. A similar comparison can be made between the G2 at 0.01 M (0.16 M lysine equivalents) that exhibited 25% trapping against the G1 at 0.02 M (0.16 M lysine equivalents) that exhibited 34% trapping. In both cases, the smaller dendrimers appear more effective when normalized by lysine equivalents. These two comparisons suggest that any synergistic benefit that might be gained by the multiplicity lysine groups on the dendrimer is outweighted by steric factors or other generation-dependent inhibiting effects.

Table 3. Bis-Lysine PEG Controls for Each of the Dendrimers at a 0.1 to 1.0 Molar Ratio % of DCV trapped

% of trapping compd

molar ratio 1:1

molar ratio 0.1:1

molar ratio 0.02:1

molar ratio 0.01:1

1c 3c 5c

4 8 16

100 100 100

52 64 83

25 34 56

5 10 25

lysine conc (M)

dendrimers to DCV molar ratio 0.1:1.0

PEG 6a lysine equivalent ratio 0.1:1.0

1c 3c 5c

0.009 0.018 0.036

52 64 83

14 24 31

enhanced binding with respect to the PEG control at a concentration with an identical molarity of lysine groups. These data confirm that there is a unique synergistic effect offered by dendrimers for DCV binding, relative to nondendritic polymer analogues. This comparison also confirms a slight inhibition of binding for the higher generation dendrimers, with the G0 dendrimer outperforming the linear PEG lysine dimer 3.7-fold (per lysine equivalent), while the G2 dendrimer outperforms the linear PEG lysine dimer by only 2.7-fold. From these results, it can be concluded that the placement of lysine units in close proximity does provide a synergistic binding to DCV. Interestingly, while the increase in generation enhances the molar binding, it reduces the binding per lysine. This suggests that the steric crowding or the reduced flexibility on the periphery of the higher generation dendrimers may cause inhibition of the binding relative to smaller dendrimers. However, the binding for the second generation dendrimer, 5c, still significantly exceeds that of the PEG control for the same number of lysine groups. This agrees with what has been observed by othersthat enhanced binding in dendritic systems has to be balanced with the steric hindrance and flexibility of the dendritic scaffold in order to optimize their binding to target molecules.57−59 Dendrimer Particle Size. Because the size of nanomaterials is a critical aspect of their behavior in vivo, the size of the protected dendrimers was measured by light scattering. As expected due to their highly compact architecture, the dendrimers were found to be small for their molecular weight, between 1.5 nm, for the zeroth-generation dendrimers, and 2.7 nm, for the second-generation dendrimers (Table 4, Figures S25 and S26). Although polymers in this size range are expected to be cleared rapidly from the bloodstream, the use of dendritic hybrids, such as linear−dendritic systems,29,60 should

Table 2. Percentage of Trapping from Each Generation Regarding Their Molar Ratio (Dendrimers:DCV) no. of lysine

compd

To further elucidate the effect of dendritic architecture relative to the number of lysine units, a linear polymer with two lysine end groups, 6c, was used as a control. Again, this lysinefunctionalized polymer was isolated and evaluated as its ammonium hydrochloride salt. With the two lysine groups at opposite ends of a poly(ethylene glycol) oligomer (average degree of polymerization ∼14 repeat units), this control is expected to exhibit minimal steric inhibition and reduced synergy of binding between the two lysine end groups relative to the dendritic systems where lysine groups are forced in close spatial proximity. In Figure 8, the DCV binding with 6c was found to be less than half of the amount observed for the analogous lysine-functionalized dendrimers (1c, 3c, and 5c). As a representative comparison, compound 1c (the G0 bearing 4 lysines) at a concentration of 0.0045 M was found to bind 52% of a 10-fold excess of DCV (0.1:1.0 dendrimer/DCV molar ratio), while the equivalent amount of lysine groups attached to PEG 6c (0.009 M) only bound to 14% of the same proportion of DCV. Similar results were observed for the higher generation dendrimers (Table 3) where each of these dendrimers exhibits I


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Innova Chile CORFO Code FCR-CSB 09CEII-6991. Additional support by ACS-PRF 53980-ND7 (JLM), NSF-CHE 1412439 (SMG) and a Louisiana Board of Regents Graduate Fellowship (JAG) are acknowledged,. Prof. Bruce Gibb is acknowledged for assistance with the DLS measurements.

Table 4. Size Distributions of the Protected, LysineFunctionalized Dendrimers in Chloroform size distribution compd

generation

no. of lysine

1b 3b 5b

0 1 2

4 8 16

volume-weighted distribution (nm)

number-weighted distribution (nm)

1.6 ± 0.2 2.1 ± 0.5 2.9 ± 0.7

1.5 ± 0.2 2.0 ± 0.3 2.7 ± 0.4

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provide both the desired size in solution while maintaining the multiplicty of functional groups and the exact number of functional groups associated with dendrimers.

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CONCLUSION Three biodegradable lysine−polyester dendrimers of zeroth, first, and second generation have been synthesized in a scalable process yielding well-defined dendrimers, though chromatographic purification was required to generate samples of high purity. The stability of these lysine dendrimers were evaluated with respect to time at different pHs, and it was confirmed that they exhibited modest hydrolytic stability in aqueous solution within the first few days, with the peripheral lysine groups most susceptible to hydrolysis. However, the dendritic core remained robust over a 1 week period, suggesting that alternative linker chemistry might be sufficient to enhance the hydrolytic stability. These compounds also exhibited low toxicity, with no apparent generation dependence on viability for each of the three cell lines assayed. Finally, the dendrimers were evaluated for their ability to trap DCV, a toxic organophosphate compound broadly used in agriculture as an insecticide. While high-generation dendrimers appear to yield some binding inhibition relative to smaller dendrimers (per surface binding group), the binding synergy that results from the use of dendritic scaffolds shows clear advantages over linear polymer scaffolds. This DCV capture assay confirms that lysinefunctionalized polyester dendrimers exhibit enhanced DCVcapturing efficiency in solution, likely due to the spatial synergy of binding groups. While challenges remain in developing effective nanodetoxificatin agents, these results highlight some of the potential advantages of dendrimer-based polymer scaffolds for sequestering OPs.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00657. 1 H NMR, 13 C NMR, and representative HPLC chromatogram of the dichlorvos standard (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (S.M.G.). Author Contributions

E.F.D.-L. and J.L.M. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from E. Durán and L. S. Santos FONDECYT (Postdoctoral Grant N°3120178) and J


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Investigation of Lysine-Functionalized Dendrimers as Dichlorvos

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