In vivo biodistribution and small animal PET of 64Cu-labeled antimicrobial peptoids

Article abstract of DOI:10.1021/bc300091d

Peptoids are a rapidly developing class of biomimetic polymers based on oligo-N-substituted glycine backbones, designed to mimic peptides and proteins. Inspired by natural antimicrobial peptides, a group of cationic amphipathic peptoids has been successfully discovered with potent, broad-spectrum activity against pathogenic bacteria; however, there are limited studies to address the in vivo pharmacokinetics of the peptoids. Herein, ⁶⁴Cu-labeled DOTA conjugates of three different peptoids and two control peptides were synthesized and assayed in vivo by both biodistribution studies and small animal positron emission tomography (PET). The study was designed in a way to assess how structural differences of the peptidomimetics affect in vivo pharmacokinetics. As amphipathic molecules, major uptake of the peptoids occurred in the liver. Increased kidney uptake was observed by deleting one hydrophobic residue in the peptoid, and ⁶⁴Cu-3 achieved the highest kidney uptake of all the conjugates tested in this study. In comparison to peptides, our data indicated that peptoids had general in vivo properties of higher tissue accumulation, slower elimination, and higher in vivo stability. Different administration routes (intravenous, intraperitoneal, and oral) were investigated with peptoids. When administered orally, the peptoids showed poor bioavailability, reminiscent of that of peptide. However, remarkably longer passage through the gastrointestinal (GI) tract without rapid digestion was observed for peptoids. These unique in vivo properties of peptoids were rationalized by efficient cellular membrane permeability and protease resistance of peptoids. The results observed in the biodistribution studies could be confirmed by PET imaging, which provides a reliable way to evaluate in vivo pharmacokinetic properties of peptoids noninvasively and in real time. The pharmacokinetic data presented here can provide insight for further development of the antimicrobial peptoids as pharmaceuticals.

Full text of DOI:10.1021/bc300091d

Article
pubs.acs.org/bc
64
In Vivo Biodistribution and Small Animal PET of Cu-Labeled
Antimicrobial Peptoids
Jiwon Seo,#,§ Gang Ren,#,† Hongguang Liu,† Zheng Miao,† Minyoung Park,⊥ Yihong Wang,†
Tyler M. Miller,‡ Annelise E. Barron,*,‡ and Zhen Cheng*,†
†Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Bio-X Program, Canary Center at Stanford
for Cancer Early Detection, Stanford University, California, 94305-5344, United States
⊥
‡Department of Bioengineering, Department of Chemical and Systems Biology, Stanford University, California,
94305-5440, United States
§School of General Studies, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea
S
* Supporting Information
ABSTRACT: Peptoids are a rapidly developing class of bio-
mimetic polymers based on oligo-N-substituted glycine back-
bones, designed to mimic peptides and proteins. Inspired by
natural antimicrobial peptides, a group of cationic amphipathic
peptoids has been successfully discovered with potent, broad-
spectrum activity against pathogenic bacteria; however, there are
limited studies to address the in vivo pharmacokinetics of the
peptoids. Herein, 64Cu-labeled DOTA conjugates of three
different peptoids and two control peptides were synthesized
and assayed in vivo by both biodistribution studies and small
animal positron emission tomography (PET). The study was
designed in a way to assess how structural differences of the
peptidomimetics affect in vivo pharmacokinetics. As amphipathic molecules, major uptake of the peptoids occurred in the liver.
Increased kidney uptake was observed by deleting one hydrophobic residue in the peptoid, and 64Cu-3 achieved the highest
kidney uptake of all the conjugates tested in this study. In comparison to peptides, our data indicated that peptoids had general
in vivo properties of higher tissue accumulation, slower elimination, and higher in vivo stability. Different administration routes
(intravenous, intraperitoneal, and oral) were investigated with peptoids. When administered orally, the peptoids showed poor
bioavailability, reminiscent of that of peptide. However, remarkably longer passage through the gastrointestinal (GI) tract without
rapid digestion was observed for peptoids. These unique in vivo properties of peptoids were rationalized by efficient cellular
membrane permeability and protease resistance of peptoids. The results observed in the biodistribution studies could be
confirmed by PET imaging, which provides a reliable way to evaluate in vivo pharmacokinetic properties of peptoids
noninvasively and in real time. The pharmacokinetic data presented here can provide insight for further development of the
antimicrobial peptoids as pharmaceuticals.
Particularly, short (<40 amino acids), cationic, linear, and α-helical
AMPs are amenable to mimicry, and peptoids are among the
front-runners in mimicking this class of AMPs.
INTRODUCTION
■
Over recent decades, a number of antimicrobial peptides
(AMPs) have been identified as a front-line defense in various
host organisms.1,2 AMPs have drawn great scientific interest
because they not only belong to the intrinsic host-defense
system,3−6 but also are promising candidates for the development
of novel anti-infective agents.7,8 Natural AMP-based therapeutics
demonstrate activities against broad-spectrum pathogens in vitro;
however, they exhibit major drawbacks in vivo such as their low
resistance against proteolysis and rapid clearance by the
kidneys.9,10 Recently, the challenges associated with peptides are
being addressed owing to the use of non-natural amino acids and
modified peptide structures;11,12 hence, various peptidomimetics
that are designed to reproduce critical AMP structural character-
istics, such as spatially segregated cationicity and hydrophobicity
in an amphipathic secondary structure, have been developed.13−18
Peptoids (oligo-N-substituted glycines) are a novel class of
peptidomimetics that differ from peptides only in that side
chains are attached to the backbone amide nitrogen instead of
the α-carbon (Figure 1).19,20 This unique structural feature of
peptoids leads to quite different pharmacological properties
from natural peptides: (1) they are highly stable against
proteases;21,22 (2) they lack hydrogen-bonding potential, which
further prevents backbone-driven aggregation and thus increases
bioavailability;23,24 (3) they show increased cell permeability over
Received: February 23, 2012
Revised: April 3, 2012
Published: April 9, 2012
© 2012 American Chemical Society
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Figure 1. (A) Comparison of peptide and peptoid. (B) Chemical structures of peptoid submonomers and DOTA. (C) Sequences of the synthesized
DOTA conjugates and corresponding compound names.
peptides;23,25 and (4) they may evade immune recognition.26
Peptoids can be readily synthesized using a conventional solid-
phase peptide synthesis (SPPS) method, and diverse sequences of
peptoids can be prepared at relatively low cost. They are also
precisely tunable in conjugation with established synthetic
methods.27 Since their development in the early 1990s, peptoids
have become attractive scaffolds for many different biological
applications.28−30 For example, the use of peptoids as
antimicrobial agents,31−36 molecular transporters for intracellu-
lar drug delivery,37 or ligands for tumor receptor binding has
been reported.25
not affected by multidrug resistance (MDR) developed in
cancer cells.44 Therefore, cationic amphipathic peptoids
represent an excellent molecular platform for the development
of antimicrobials as well as cytotoxic chemotherapeutics and
warrant systematic investigations on their in vivo behaviors.
The in vivo pharmacokinetic studies on antimicrobial
peptides have been reported using radiolabeled peptides;45,46
however, we cannot simply extrapolate peptoid biodistribution
from previous peptide biodistribution data. First, due to the
lack of hydrogen-bond donors in the peptoid backbone, the
hydrodynamic radii of peptides and peptoids are expected to be
significantly different.24 Second, peptoids are protease-resistant
and metabolically more stable than peptides.21,22 For these
reasons, the pharmacokinetic profiles of peptoids are likely to be
different from what was observed with peptides, and a new set of
pharmacokinetic data are required for better understanding the
in vivo behavior of this unique family of peptidomimetics.
Recently, we found that antimicrobial peptoids showed
different in vitro antimicrobial activity depending on their
degree of helicity, chain length, and hydrophobicity (Table 1;
note that the biological data are for oligomers without DOTA
conjugation). Then, we reasoned that the different structural
and physicochemical properties could also cause distinct in vivo
pharmacokinetic profiles of the peptoids. Herein, three cationic
amphipathic peptoids and two control peptides were selected,
and their in vivo profiles were evaluated using both
biodistribution and small animal positron emission tomography
(PET). Peptoids 1, 2, and 3 represent N-terminal 1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) con-
jugates of cationic amphipathic peptoids (Table 1 and Figure 1).
Peptoids 1 and 3, where the latter is almost identical to 1 except
that it has one less C-terminal Nspe monomer, are linear peptoid
Among many peptidomimetic systems under study, peptoids
are particularly well suited for structural mimicry of AMPs31,32
because they can readily form helical secondary structures via a
periodic incorporation of bulky, α-chiral side chains.38−40 The
structures of the peptoid helices have been characterized to be
stable polyproline type-I-like helices which exhibit a pitch of
́
6.0−6.7 Å and a periodicity of approximately three residues per
turn.41−43 The threefold periodicity of the peptoid helix makes
it easier to mimic the facially amphipathic structures similar
to those found in many AMPs; for example, the trimer repeat
(X-Y-Z)n forms a peptoid helix with three faces, composed of
X, Y, and Z residues. Previously, a group of cationic amphipathic
peptoids, with broad-spectrum antimicrobial activity against
both Gram-positive and Gram-negative bacteria including
clinically relevant biosafety level 2 (BSL-2) pathogens, has
been discovered.31−34 Interestingly, these antimicrobial pep-
toids also show potential in treating multidrug resistant strains
such as Pseudomonas aeruginosa biofilms35 and Micobacterium
tuberculosis.36 Furthermore, we recently found that the cationic
amphipathic peptoids were potent against several cancer cell
lines at low micromolar concentrations, and their actions were
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Table 1. Properties of 1−5
e
antimicrobial activity
a
b
d
f
compounds
class
chain length
secondary structure
helical
hydrophobicity (tR in minutes)
E. coli MIC, μM
selectivity ratio
1
2
3
4
5
peptoid
peptoid
peptoid
peptide
peptide
12
12
11
12
25.0
24.1
23.9
24.0
3.5−6.3
6.3
6.0
almost unstructured
helical
205
132
ND
24
6.3
helix-like
ND
c
22
random coil
21.3
3.1−6.3
a
b
c
Number of peptoid or peptide residues. Determined by circular dichroism spectroscopy. Fold into helical conformation upon binding to lipid
membranes.47,48 Detailed HPLC conditions are provided in the Experimental Procedure section. HPLC chromatograms are shown in the
d
e
Supporting Information (tR: retention time in reverse phase HPLC column). Biological data are for peptoids or peptides without DOTA
conjugation. MIC (minimum inhibitory concentration) values and selectivity ratios for 1 and 5 are from ref 32, and for 2 and 3 are unpublished
f
results. Selectivity ratio = (HD10)/(E. coli MIC). HD10: 10% hemolytic dose.
helices. Peptoid 3 (without DOTA) has lower hydrophobicity
than peptoid 1 (without DOTA) and was recently found to have
substantially higher selectivity in killing bacterial cells without
significant loss in potency (Table 1). Peptoid 2 (without DOTA)
is a nonstructured peptoid, which nonetheless can adopt a helical
conformation in the presence of the anionic bacterial membrane
that serves as an organizing surface. For comparison, we tested
two cationic amphipathic peptide controls: (1) peptide 4 has a
direct sequence homology to peptoid 1, and the effect of the
peptoid backbone versus the peptide backbone was examined;
and (2) peptide 5 is based on a well-known antimicrobial peptide,
namely, pexiganan,47,48 which advanced to phase 3 clinical trials.49
Also known as MSI-78, pexiganan exhibits a random coil
conformation, which folds into a helical conformation upon
binding to a bacterial membrane (Table 1). To obtain kinetic data
for the biodistribution and clearance for 24 h, a radionuclide with
an appropriate half-life was needed. Therefore, DOTA-chelated
64Cu was used due to its relatively long half-life (762 min) and
numerous precedent biodistribution data of the DOTA-64Cu
labeled peptides.50−52 With the radiolabeled peptoids and
peptides, three different administration routes (intravenous,
intraperitoneal, and oral) were examined. The results reported
here can provide insight for future development of peptoids as
antimicrobial chemotherapeutics.
temperature for 20 min were performed. For the displacement
reaction, primary amine (1 mL, 1.5 M in N-methylpyrrolidone
(NMP), 1.5 mmol) was added, and the reactor was agitated
at room temperature for 1 h. The resin was washed with DMF
(6 mL × 5) between each step. Peptide oligomers were
prepared by the automated solid-phase peptide synthesis
according to standard Fmoc/tBu-methodology and HBTU/
HOBt/DIEA coupling.54 Rink amide resin (0.60 mmol/g) was
used to obtain C-terminal amide peptides. After completion of
the solid-phase synthesis, the DOTA chelator was manually
conjugated at the N-terminus of the pepoids or peptides by
activating the carboxylic acid of DOTA-tris(t-Bu ester) with
HATU. Peptoid (or peptide) attached on resin (properly
protected, 0.025 mmol) was swelled in DMF (2 mL). To this
was added DOTA-tris(t-Bu ester) (0.15 mmol), 2-(7-aza-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HATU) (0.30 mmol), and N,N-diisopropylethyl-
amine (DIPEA) (0.75 mmol), and the reaction continued at
room temperature overnight.55 Cleavage from the resin and
deprotection were performed with 95:2.5:2.5 TFA/water/
triisopropylsilane (v/v/v) for 2−3 h at room temperature. It
should be noted that shorter cleavage time resulted in incomplete
tert-butyl ester deprotection of DOTA-tris(t-Bu ester). The
cleavage solution was filtered by solid-phase extraction (SPE)
cartridges (Applied Separations, Allentown, PA, USA) and the
volatiles were removed by a stream of nitrogen. The crude
peptoid was dissolved in acetonitrile/water, lyophilized, and ana-
lyzed by ESI-MS (or MALDI-TOF) and analytical HPLC.
Analytical HPLC was performed on a Waters 2695
separations module system with Waters 2998 photodiode
array detector and Waters Alliance column heater. Phenomenex
Jupiter C18 (250 × 2.0 mm, 5 μ, 300 Å) column was used for
the analytical HPLC and the column was heated at 55 °C.
Sample purity was monitored by absorbance at 220 nm. The
mobile phase was used as follows: [A, water + 0.1% trifluoroacetic
acid (TFA); B, CH3CN + 0.1% TFA] 3 min using 1% B, then a
linear gradient to 99% B over 30 min, a linear gradient back to 1%
B over 10 min, and then 2 min using 1% B. Flow rate of the
analytical HPLC was 0.2 mL/min.
EXPERIMENTAL PROCEDURES
■
Materials. All reagents were purchased from Sigma-Aldrich,
Fisher, and Novabiochem. They were used without further
purification unless stated otherwise. DOTA-tris(t-Bu ester) (or
tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate)
was purchased from Macrocyclics, TX, USA. N,N′-Diisopropyl-
carbodiimide was purchased from Advanced ChemTech, KY,
USA. Radioisotope 64Cu was purchased from the Department of
Medical Physics, University of Wisconsin, Madison, WI, USA. Six-
week-old Balb/c mice were ordered from Harlan, CA, USA.
Synthesis and Purification. The peptoid oligomers were
synthesized by solid-phase submonomer method using an
automated peptide synthesizer (ABI 433A, Applied Biosystems,
CA, USA).20,53 Rink amide resin (0.60 mmol/g, Novabiochem,
San Diego, CA) was used to generate C-terminal amide peptoids.
After Fmoc deprotection, each monomer was added by a series of
bromoacetylation and displacement by an amine. These two steps
were iterated with appropriate amines until the desired peptoid
sequence was obtained. Typically, 0.06 mmol reaction scale was
used (0.1 g of the resin). For bromoacetylation, the addition of
bromoacetic acid (1 mL, 1.2 M in N,N-dimethylformamide
(DMF), 1.2 mmol) followed by N,N′-diisopropylcarbodiimide
(0.15 mL, neat, 1.0 mmol), and then agitation at room
The peptoids or peptides were purified by a preparative HPLC
system (Waters PrepLC system, Waters 2489 UV/Visible
detector, Waters fraction collector III) using a C18 column
(Phenomenex Jupiter C18, 250 × 21.20 mm, 10 μm, 300 Å) at a
flow rate of 25 mL/min. Sample elution was detected by
absorbance at 220 and 260 nm. The purity of the product frac-
tions was confirmed by analytical HPLC, and fractions containing
pure product (>98% purity) were collected, lyophilized, and
stored at −80 °C.
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Figure 2. (A) Peptoid submonomer synthesis protocol. (B) Representative synthetic scheme of DOTA conjugates. (C) Illustrative structure of
64Cu-1 prepared by 64Cu radiolabeling of DOTA conjugate 1.
For electrospray ionization mass spectrometry (ESI-MS)
analysis of compounds, ThermoFinnigan LCQ “Classic” ion
trap LC-MS instrument was used (Stanford University Mass
Spectrometry facility). Voyager-DE RP Biospectrometry instru-
ment was used for matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry (Stanford
University PAN facility).
Circular Dichroism Spectroscopy. Typical procedure for
the preparation of Cu-1−Cu-4 (for CD study) is as follows.
Compound 1 (1.0 mg) was dissolved in NaOAc buffer (0.1 M,
pH 5.5, 940 μL). To this was added 10 mM CuCl2 (60 μL,
approximately 2.0 mol equiv). The reaction mixture was
incubated at room temperature overnight with gentle agitation.
The reaction was monitored by analytical HPLC and stopped
when the starting material (compound 1) was consumed. D-Salt
polyacrylamide desalting column (Pierce, Rockford, IL, USA) was
used to purify the crude product. The purification was carried out
according to the manufacturer’s instruction. The column fractions
were monitored by measuring the absorbance at 220 nm using
Nanodrop spectrophotometer. Fractions containing Cu-1 were
combined and lyophilized.
Circular dichroism measurements were carried out at 20 °C
with a Jasco J-815 circular dichroism spectrometer (Jasco, Inc.,
Easton, MD, USA). CD spectra were obtained in an 1 mm path
length quartz cell and recorded from 190 to 260 nm in 1 nm
increments with a scanning speed of 20 nm/min and a response
time of 4 s. Three scans for each sample were averaged. Sample
concentrations were in 50 μM. Data are expressed in terms of
per-residue molar ellipticity (deg cm2/dmol) calculated per
number of amides in a molecule.
Radiolabeling. DOTA-conjugated compounds (10 μg)
were used for labeling. The bioconjugate was radiolabeled with
64Cu by addition of 64CuCl2 (111 MBq, 3 mCi) in NaOAc
buffer (pH 5.5, 0.1 N) followed by a 40 min incubation at
40 °C. EDTA (5 μL, 10 mM) was then added to quench free
64Cu2+. The radiolabeled complex was purified by analytical
radio-HPLC. The mobile phase was used as follows: [A, water +
0.1% TFA; B, CH3CN + 0.1% TFA] 3 min using 5% B, then a
linear gradient to 65% B over 30 min, then going to 85% B over
3 min, maintaining this solvent composition for another
3 min, and then a linear gradient back to 5% B over 3 min
(total 42 min). Flow rate of the radio-HPLC was 1.0 mL/min.
Fractions containing pure radiolabeled peptoid (or peptide) were
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Figure 3. CD spectra of 1−4 and Cu-1−Cu-4 in water (50 μM, 20 °C) were recorded as per-residue molar ellipticity (deg cm2/dmol).
combined and dried under reduced pressure using rotary
evaporator. The radiolabeled compound was reconstituted in
phosphate-buffered saline (PBS) and passed through a 0.22 μm
Millipore filter into a sterile vial for in vitro and animal
experiments.
obtained, and the images were reconstructed by the OSEM
algorithm. PET images were imported using ASIPro VM. ROIs
were drawn manually over the organ of interest on decay-
corrected whole-body coronal images. The mean counts per pixel
per minute were obtained from the ROIs and converted to counts
per milliliter per minute by using a calibration constant. No
attenuation correction was performed.
Statistical Methods. Statistical analysis was performed
using the Student’s t-test for unpaired data. A 95% confidence
level was chosen to determine the significance between groups,
with P < 0.05 being significantly different.
Biodistribution. 64Cu-DOTA-peptoids or peptides (30−
40 μCi) were injected into a tail vein in Balb/c mice (n = 4 for
each group), and then the mice were sacrificed at 2 and 24 h p.i.
For peptoid 64Cu-1, three administration routes including
intraperitoneal (i.p.), intravenous (i.v.) or gavages needle (p.o.)
injection were used to compare the in vivo profiles of the peptoid.
Major organs were excised and weighed, and their radioactivity
was measured in a Wallac 1480 automated γ-counter (Perkin-
Elmer, MA, USA). The radioactivity uptake in the organs was
expressed as a percentage of the injected radioactive dose per
gram of tissue (% ID/g).
RESULTS
■
Synthesis and Radiolabeling. Five DOTA conjugated
compounds (three different cationic amphipathic peptoids 1−3
and two peptide controls 4−5 in Figure 1) were successfully
synthesized by solid-phase synthesis followed by N-terminal
DOTA conjugation. Both peptoid and peptide sequences were
synthesized by automated peptide synthesizer according to the
submonomer protocol20 and standard Fmoc/tBu method,54
respectively (Figure 2A). Then, the DOTA chelator was
manually conjugated at the N-terminus of the pepoids or
peptides by activating the carboxylic acid of DOTA-tris(t-Bu
ester) with HATU (Figure 2B).55 After deprotection and
cleavage from the resin with trifluoroacetic acid, the DOTA
conjugates were purified by preparative HPLC. It should be
noted that less than 2 h of cleavage/deprotection resulted in
incomplete tert-butyl ester deprotection of DOTA-tris(t-Bu
ester); therefore, 2−3 h of cleavage reaction were used for all
five compounds. The identity of each compound was confirmed
by mass spectrometry (Supporting Information, Table S1).
Subsequently, the conjugates were radiolabeled with 64Cu in
order to monitor their in vivo behaviors (Figure 2C). The
purification of radiolabeled complex, using a PD-10 column,
afforded 64Cu-DOTA-peptoid/peptide (64Cu-1−64Cu-5) with
>95% radiochemical purity and modest specific activity of
3.98−6.83 MBq/nmol. Further mouse serum stability test
Small Animal PET Imaging. Small animal PET imaging of
Balb/c mice was performed on a microPET R4 rodent model
scanner (Siemens Medical Solutions USA, Inc., Knoxville, TN,
USA). The mice were injected with 64Cu-DOTA-peptoids or
peptides (∼40 μCi) through the tail vein. A dynamic image of
the first 35 min and a series of static images were acquired.
During the scan, anesthesia was maintained with 2% isofluorane
in 1 L/min oxygen, and imaging was performed in a prone
position. For dynamic scan, image acquisition was initiated
once the mice were centered in the field of view. Radiolabeled
peptoid was administered with immediate data collection and
image acquisition. Acquisition lasted about 35 min. Image re-
construction was performed by a two-dimensional OSEM
(ordered subsets expectation maximum) algorithm with a spatial
resolution of 1.66−1.85 mm. No attenuation or partial volume
corrections were performed. Regions of interest (ROIs) were
selected using ASIPro VM software (Siemens Medical Solutions
USA Inc., Knoxville, TN, USA). For static scan at different times
p.i. (0.25, 1, 2, 4, and 24 h), the mice were anesthetized with 2%
isofluorane and placed in the prone position and in the center of
the field of view of microPET. The 5 min static scans were
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demonstrated that over 95% radiolabeled complexes remained
intact after 1 h incubation at 37 °C (Supporting Information,
Figure S5).
Circular Dichroism Studies. Conformational information
of unmetalated 1−4 as well as copper-incorporated conjugates
Cu-1−Cu-4 was obtained by circular dichroism (CD) spec-
troscopy (Figure 3). CD study of pexiganan was previously
reported (Table 1);56 therefore, compound 5 was not included
in this study. For the CD study, nonradioactive copper-bound
DOTA conjugates, namely, Cu-1−Cu-4, was prepared, and the
physicochemical properties of radioactive and nonradioactive
copper compounds are assumed to be the same.
For helical peptoid conjugates 1 and 3, the maintenance of
helical folds was observed after copper incorporation (Cu-1 and
Cu-3). In addition, conformations of 1 and Cu-1 at body
temperature (37 °C) were examined and indicated minimal
temperature dependence at 20 and 37 °C (Supporting Information,
Figure S2). As predicted, compounds 2 and Cu-2 indicated an
almost unstructured conformation due to the lack of structure-
inducing residue, Nspe or (S)-N-(1-phenylethyl)glycine, in the
peptoid sequence. Uncharacteristic, but α-helix-like, CD signatures
were obtained for 4 and Cu-4, and the handedness was the
opposite of the peptoid conjugates’ handedness. The CD spectra in
this study clearly showed that the influence of copper incorporation
on the overall conformation was minimal.
Biodistribution. Radiolabeled peptoids and peptides 64Cu-
1−64Cu-5 were injected intravenously into a tail vein in Balb/c
mice. After 2 and 24 h postinjection (p.i.), the mice were
euthanized, and the radioactivity of the organs and the blood
was determined. The activity in the selected organs was
expressed as a percentage of the injected radioactivity per gram
of tissue (%ID/g). The results of the biodistribution studies are
summarized in Figure 4.
Figure 4. Biodistribution of 64Cu-DOTA-peptoids/peptides (64Cu-
1−64Cu-5) in Balb/c mice at 2 h (A) and 24 h (B). Data were
expressed as mean
SD, indicating the percentage administered
activity (injected dose) per gram of tissue (%ID/g) after intravenous
injection of about 40 μCi (1.48 MBq) tracers (n = 4).
Peptoid conjugates 64Cu-1 and 64Cu-2 exhibited prominent
uptake in the liver, with lower liver uptake for 64Cu-2 than
64Cu-1. The detected activity in the liver was 137.6 13.4%ID/
g for 64Cu-1 and 125.3 16.8%ID/g for 64Cu-2 at 2 h p.i., and
than 1%ID/g), suggesting their incapability of crossing the
blood-brain-barrier.
the activity remained at 95.6
10.5%ID/g for 64Cu-1 and
72.2 11.3%ID/g for 64Cu-2 at 24 h p.i. Compared with 64Cu-
The effects of different administration routes (oral, intra-
venous, and intraperitoneal) on the in vivo biodistribution were
then examined using 64Cu-1 (Supporting Information, Figure
S3). When administered intraperitoneally, predominant disposi-
tion of 64Cu-1 was in the abdomen region. After intraperitoneal
injection, activity was detected in most abdomen organs including
intestine, colon, stomach, liver, spleen, and pancreas, and the
activity persisted through 24 h. Significant kidney and thymus
uptake was also observed after the intraperitoneal injection. When
administered orally, the activity in stomach reached up to 4.82
1.44%ID/g at 2 h and then dropped to 0.54 0.22%ID/g at
24 h. The uptake of 64Cu-1 could only be measured in the
digestive system (i.e., stomach, small intestine, colon), suggesting
that 64Cu-1 cannot be absorbed into blood circulations when
administered orally.
Small-Animal PET Imaging. Imaging studies were
conducted at 0.25, 1, 2, 4, and 24 h after tail vein injection
of 64Cu-1−64Cu-5. Decay-corrected coronal microPET images
are shown in Figure 5. Three peptoids and two peptides
displayed different in vivo distribution patterns; particularly,
uptakes in liver and in kidney were noticeable. For 64Cu-1 and
64Cu-2, the main excretion route is likely hepatic; little kidney
activity was observed. 64Cu-3 demonstrated the highest kidney
activity of all three peptoids. Compared to peptoids, higher
kidney activity and lower liver activity were observed for peptides.
1 and 64Cu-2, 64Cu-3 exhibited lower liver uptake at both 2 and
24 h p.i. with 56.7
1.8%ID/g and 47.3
4.4%ID/g,
respectively. Unlike the liver uptake, 64Cu-3 showed the highest
kidney uptake, which was around 31.2 3.6%ID/g at 2 h, while
it was 14.4 0.9%ID/g and 12.55 2.3%ID/g for 64Cu-1 and
64Cu-2, respectively. At 24 h p.i., the activity of 64Cu-3 in the
kidney remained 19.3 2.6%ID/g, which was the highest level
of all the conjugates tested in this study.
As for the peptide controls, both 64Cu-4 and 64Cu-5 showed
lower liver uptake and faster liver clearances compared to all
three peptoid conjugates (P < 0.05). 64Cu-4 has a direct
sequence homology to 64Cu-1 and a similar hydrophobicity
to 64Cu-2 (Table 1). Compared to both peptoids, 64Cu-4
showed lower liver uptake and higher kidney uptake at 2 h p.i.
(Figure 4A), reminiscent of the different hydrodynamic radii
coming from the lack of hydrogen-bond donors in the peptoid
backbone. Then, the kidney activity of 64Cu-4 decreases faster
and becomes similar to that of 64Cu-2 at 24 h p.i. (Figure 4B),
which can be explained by the higher in vivo stability of
peptoids over peptides. Between the two peptides, less
hydrophobic 64Cu-5 exhibited lower liver uptake than 64Cu-4
did. In addition, 64Cu-5 had a much lower spleen uptake than
the other four conjugates tested at both 2 and 24 h. All three
peptoids and two peptides showed minimal brain uptake (lower
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Figure 5. Representative decay-corrected coronal small-animal PET images of Balb/c mice at different time points after intravenous (iv) injection of
64Cu-1−64Cu-5 (n = 4 for each group).
Figure 6. Representative decay-corrected coronal small-animal PET images of Balb/c mice at different time points after administration of 64Cu-1
through peritoneal (A), oral (B), and intravenous (C) injections (n = 4 for each group).
Among the tested conjugates, 64Cu-5 showed fastest clearance in
both hepatic and renal pathways. Activity in the gastrointestinal
(GI) tract could also be observed for all tested compounds, but
markedly lower for 64Cu-1.
(Figure 6). Radioactivity was found throughout the whole
abdominal cavity when 64Cu-1 was administered intra-
peritoneally. The permissive activity in abdomen clears rather
slowly; therefore, it was complicated to differentiate organs in
24 h based upon PET imaging. Interestingly, a symmetrical
anatomical structure was distinctly visible inside the upper body
The distribution patterns of 64Cu-1 with different admin-
istration routes were compared and shown by PET imaging
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of the mice after the intraperitoneal administration of 64Cu-1,
and biopsy confirmed the symmetrical organ as thymus.
Thymic uptake has been reported for several radioactive
pharmaceuticals such as 18F-fluorodeoxyglucose (18F-FDG),
radioiodine, and gallium-67 citrate; however, the mechanism
through which it occurs is not completely understood.57,58
When 64Cu-1 was administered through the tail vein, the
compound essentially accumulated in the liver through 24 h as
described above. Very low GI activity and kidney activity were
observed in 24 h. In mice with orally administrated 64Cu-1,
only GI activity was observed initially, and the peptoid con-
jugate was eliminated after 24 h as seen from PET imaging at
24 h p.i. A similar distribution pattern was observed for the oral
administration of 64Cu-3 (Supporting Information, Figure S4).
the CD data prove useful as a preliminary indicator of intact
peptoid conformation after radiolabeling.
The hepatobiliary system and the kidneys are the major routes
by which drugs and their metabolites are eliminated from the
body. Generally, small and hydrophilic organic compounds are
preferentially excreted via the kidneys. In contrast, compounds
with relatively high molecular weight (>500 Da) and amphipathic
structure are mainly excreted via the liver (and then into bile).60,61
As large and amphipathic compounds, 64Cu-1−64Cu-5 all
showed that major uptake and excretion occurred in the liver;
however, different uptake profiles in the liver and in the kidney
are observed for each conjugate by the combined effect of the
structure, hydrophobicity, and resistance to proteolysis (Figures 4
and 5).
After intravenous administration, biodistribution studies of
64Cu-1 demonstrated highest liver accumulations at both 2 and
24 h (Figure 4). A slow elimination from the liver region was
observed by PET images (Figure 5). Although the secondary
structures of 64Cu-1 and 64Cu-2 are very distinct, they showed
similar in vivo pharmacokinetics based on biodistribution and
microPET studies, suggesting their overall hydrophobicity and
amphipathicity. At both 2 and 24 h p.i., 64Cu-3 showed the
highest level of kidney uptake among all three peptoids and two
peptides tested (Figure 4). With only one shorter hydrophobic
residue than 64Cu-1, 64Cu-3 showed more than 50% lower liver
uptake and noticeably higher kidney uptake; and this result
exemplifies the strong influence of structural modification on
in vivo biodistribution. Both biodistribution and PET results
indicated faster clearance of peptide controls compared to
peptoids (Figures 4 and 5). 64Cu-5 indicated lowest uptake in
both the liver and the kidney and fastest elimination. Along
with the hydrophilic property of 64Cu-5, the random coil
structure, therefore vulnerability to proteolysis, can explain the
result.
DISCUSSION
■
Designed to mimic naturally occurring proteins and peptides,
peptoids are interesting materials whose properties lie in
between natural biopolymers and non-natural synthetic
polymers. The structural similarity to peptides enabled peptoids
to exhibit an impressive diversity of biological activity;28−30
however, the slight variation of backbone structure provides
peptoids with distinct pharmacological properties such as
resistance to proteolysis.21−23
Previously, a library of cationic amphipathic peptoids were
synthesized based upon defined sequences and variations of
secondary structures. It was demonstrated that the main chain
length, helicity, charge, and hydrophobicity of peptoids could
alter the in vitro antimicrobial activity.31,32 Similarly, we
envisioned that the modulation of peptoid structure could
influence the in vivo pharmacokinetic profiles and biodistribu-
tion, which is critical information for further development of
antimicrobial peptoids as therapeutic agents.
Previous in vivo data on biodistribution of peptoids are
limited. Wang et al. used a H-labeled tripeptoid to test the
Different administration routes were investigated with 64Cu-
1 and 64Cu-3. When administered orally, the peptoids showed
poor bioavailability. The minimal absorption was indicated by
high activity in the GI tract and little activity in the liver or in
the kidney system (Supporting Information, Figure S3 and S4).
For peptides, poor oral bioavailability has been widely accepted,62
and our result suggests the similarity between peptides and
peptoids in terms of the poor absorption after oral administration.
However, it should be noted that peptides usually have rapid
elimination kinetics in the range of minutes due to the extensive
hydrolysis in the GI tract by peptidases.63,64 Therefore, the 24 h
passage through the GI tract without rapid digestion constitutes a
unique aspect of peptoids, which originates from the protease
resistance and the property as a non-natural biopolymer. The high
in vivo stability of peptoids can be advantageous for some
applications. If the site of action of a designed peptoid is in the GI
tract, such as peptoid-based bacterial toxin sequestrants developed
by Kodadek et al.65 or antimicrobial peptoids for bacterial
digestive infections, oral administration may provide a better
therapeutic effect. Prolonged localized exposure to the pathogens
and fewer side effects are expected due to the longer retention in
the GI tract and the poor absorption to the systemic circulation,
respectively.
3
absorption and disposition in rats and compared the labeled
tripeptoid to a 3H-labeled tetrapeptide.59 The tripeptoid
appeared to be more metabolically stable, but showed lower
oral absorption and more rapid biliary excretion. Although the
in vivo pharmacokinetic properties of peptoid were investigated,
the peptoids tested by Wang et al. can be classified as a small
molecule with molecular weight less than 500 Da. In our
present study, we examined antimicrobial peptoids that have
secondary structures and molecular weight higher than 1500 Da,
which render the peptoids similar to small proteins rather than to
small molecules. Since distinct pharmacological properties
between small and large molecules are reported,60 biodistribution
study of the antimicrobial peptoids is important for further in vivo
applications.
When designing radiolabeled conjugates, we chose 64Cu-DOTA
labeling at the N-terminus of peptoids because we expected (1)
more straightforward synthesis and (2) less impact on the peptoid
secondary structure. The latter was confirmed by CD study. The
comparison of CD spectra before and after N-terminal DOTA
conjugation indicated nearly identical CD signatures: the intensity
of spectral maxima at 190 nm and minima at 220 nm did not
change much.32 If the labeling was made on the side chain, the
impact on the peptoid conformation could be larger. CD spectra
in Figure 3 clearly demonstrate minimal conformational change
after Cu2+ binding. Although our observation proves neither
DOTA conjugation nor metalation changed the CD signature of
peptoid, this does not prove that the peptoid with complexed
Cu2+ has the same pharmacokinetic characteristics. Nonetheless,
Our data indicated that peptoids, compared to peptides, had
a general in vivo property of higher tissue accumulation and
slower elimination as well as higher stability. These observations
can be rationalized in two ways: membrane permeability and
protease resistance. Studies have shown that the cellular uptake
could be improved by directly translating peptides into peptoids
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due to the lipophilic nature and the stability of peptoids com-
pared to those of peptides.24,37,66 In addition, peptoids used in
this study are cationic and amphipathic, which are recently found
to be effective in cell membrane penetration.44 As the first step for
the tissue uptake is transport across the cell membrane (i.e.,
proximal tubular cells for the kidney uptake or hepatocyte cells for
the liver uptake),60,61 the efficient membrane permeability of the
cationic amphipathic peptoids appears to play an important role
in the higher tissue uptake. Second, the in vivo stability of
peptoids against proteolysis provides longer circulation time,
which leads to the higher tissue uptake and slower elimination.
Unique in vivo properties of cationic amphipathic peptoids
are demonstrated in our study. Applications of the peptoids, for
example, as a long-lasting liver targeting carrier or as an
antimicrobial therapeutic agent against digestive infections or
against urinary tract infections, now seem to be reasonable.
In conclusion, our results suggest that the in vivo pharma-
cokinetics of peptoids can be tuned by varying the structure and
modifying the physicochemical properties of peptoids. In
comparison to peptides, peptoids exhibited higher in vivo
stability, higher tissue uptake, and a slower elimination rate.
The in vivo biodistribution of peptoids can be confirmed by
real-time small animal PET imaging. Using the noninvasive
in vivo imaging approaches described here, pharmacokinetic
properties of various peptoids can be predicted, which will
provide useful information for suitable application and
development of the peptoids.
(S)-N-(1-phenylethyl)glycine; MIC, minimum inhibitory con-
centration
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ASSOCIATED CONTENT
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S
* Supporting Information
Supplementary figures and tables including HPLC, ESI-MS,
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AUTHOR INFORMATION
■
Corresponding Author
*Zhen Cheng, Ph.D.: 650-723-7866 (Phone), 650-736-7925
(Fax), E-mail: zcheng@stanford.edu. Annelise E. Barron, Ph.D.:
650-721-1151 (Phone), E-mail: aebarron@stanford.edu.
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the DOD-PCRP-NIA (PC094646
to Z. C.), the National Institutes of Health (RO1 AI072666 to
A. E. B.), and the GIST specialized research program (GIST-
K02360 to J. S.).
ABBREVIATIONS:
■
AMP, antimicrobial peptide; PET, positron emission tomog-
raphy; CD, circular dichroism; MBq, megabecquerel; Ci, curie;
I.P., intraperitoneal; I.V., intravenous; P.O., oral; P.I., post-
injection; % ID/g, percentage of injected dose per gram of
tissue; GI, gastrointestinal; DOTA, 1,4,7,10-tetraazacyclodode-
cane-1,4,7,10-tetraacetic acid; TFA, trifluoroacetic acid; TIS,
triisopropylsilane; HATU, 2-(7-aza-1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; DIEA, N,N-
diisopropylethylamine; DMF, N,N-dimethylformamide; Nspe,
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