1. 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 nano-
detoxification 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.
■ INTRODUCTION
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
non-
immunogenicity,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.
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, water-
soluble 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
Received: May 15, 2015
Revised: October 7, 2015
Published: October 12, 2015
Article
pubs.acs.org/Biomac
© 2015 American Chemical Society 3434 DOI: 10.1021/acs.biomac.5b00657
Biomacromolecules 2015, 16, 3434−3444

2. 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, ground-
water, 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, nano-
carriers 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-function-
alized polyester dendrimers. The long-term goal of this research
is to evaluate these dendrimers’ potential as OP nano-
detoxification agents to be applied in agriculture, the food
industry, and biomedicine.
■ 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 PESTA-
NAL 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
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)-2-
methyl-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 (1
H 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. 1
H 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 (13
C 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
Biomacromolecules Article
DOI: 10.1021/acs.biomac.5b00657
Biomacromolecules 2015, 16, 3434−3444
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3. 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 bis-
MPA 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 Depro-
tected 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-Function-
alized 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 1-
ethyl-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-Gener-
ation 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 Second-
Generation Bis-MPA Dendrimer, [G-2]-(Boc-Lys(Z))16 (5b). Den-
drimer 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 First-
Generation 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 (1
H and 13
C NMR data available in the Supporting
Information).
Synthesis of the Deprotected, Lysine-Functionalized First-
Generation 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 (1
H and 13
C NMR data available in the
Supporting Information).
Synthesis of the Deprotected, Lysine-Functionalized Second-
Generation 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 (1
H and 13
C 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.
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4. 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
=
−
×
A A
A
adsorption (%) 1000
0 (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 hexadecafunc-
tional 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
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.
■ 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 zeroth-
generation dendrimer 1b (Scheme 1) after acidic and basic
aqueous extractions. The GPC and MALDI characterization
data for the protected lysine dendrimer confirmed the well-
Scheme 1. General Synthesis and Deprotection of the Zeroth-, First-, and Second-Generation Lysine-Functionalized Polyester
Dendrimers (1c, 3c, and 5c)
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5. defined nature of this product (Figure 1, Figures S1−S3, and
Table 1). Compound 1b was then deprotected using the
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, 1
H NMR, 13
C 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
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 (1
H NMR, 13
C
NMR, and MALDI mass spectra detailed in Figure 2 and
Figures S10−S12).
Synthesis of 5c. The second-generation hydroxyl-function-
alized 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
protecting groups on compound 5b could be removed using
acid-catalyzed deprotection conditions to afford the lysine-
functionalized 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 (1
H NMR, 13
C 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, pH-
triggered 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 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 1b 8 1584.81 1584.81 1980 1970 1.01
1 3b 16 3497.74 3497.80 3620 3600 1.01
2 5b 32 7323.59 7323.5 6370 6140 1.03
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.
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.
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6. 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
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 degrada-
tion 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
Figure 4. MALDI-ToF mass spectra of the 14-mer region of 6c and that of the entire mass spectrum of 6c (inset).
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7. 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 lysine-
functionalized 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 (HFF-
1). 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 Den-
drimers in Model Solutions. The adsorption behaviors of
DCV by the lysine-functionalized dendrimers was studied by
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.
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8. 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
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 lysine-
functionalized dendrimers were compared to their unfunction-
alized 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-function-
alized 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 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.
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).
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9. 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.
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 lysine-
functionalized 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
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 nanomateri-
als 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
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.
Table 2. Percentage of Trapping from Each Generation
Regarding Their Molar Ratio (Dendrimers:DCV)
% of trapping
compd
no. of
lysine
molar ratio
1:1
molar ratio
0.1:1
molar ratio
0.02:1
molar ratio
0.01:1
1c 4 100 52 25 5
3c 8 100 64 34 10
5c 16 100 83 56 25
Table 3. Bis-Lysine PEG Controls for Each of the
Dendrimers at a 0.1 to 1.0 Molar Ratio
% of DCV trapped
compd
lysine conc
(M)
dendrimers to DCV
molar ratio 0.1:1.0
PEG 6a lysine equivalent
ratio 0.1:1.0
1c 0.009 52 14
3c 0.018 64 24
5c 0.036 83 31
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10. provide both the desired size in solution while maintaining the
multiplicty of functional groups and the exact number of
functional groups associated with dendrimers.
■ CONCLUSION
Three biodegradable lysine−polyester dendrimers of zeroth,
first, and second generation have been synthesized in a scalable
process yielding well-defined dendrimers, though chromato-
graphic 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 lysine-
functionalized polyester dendrimers exhibit enhanced DCV-
capturing 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.
■ ASSOCIATED CONTENT
*S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.5b00657.
1
H NMR, 13
C NMR, and representative HPLC
chromatogram of the dichlorvos standard (PDF)
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: sgrayson@tulane.edu (S.M.G.).
Author Contributions
E.F.D.-L. and J.L.M. contributed equally.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by a grant from E. Durán and L. S.
Santos FONDECYT (Postdoctoral Grant N°3120178) and
Innova Chile CORFO Code FCR-CSB 09CEII-6991. Addi-
tional 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.
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Biomacromolecules Article
DOI: 10.1021/acs.biomac.5b00657
Biomacromolecules 2015, 16, 3434−3444
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