Amphiphilic Endomorphin-1 derivative functions as self-assembling nanomedicine for effective brain delivery

Article abstract of DOI:10.1248/cpb.c19-00250

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂, EM-1), an endogenous μ-opioid receptor ligand with strong antinociceptive activity, is not in clinical use because of its limited metabolic stability and membrane permeability. In this study, we develop a short-peptide self-delivery system for brain targets with the capability to deliver EM-1 without vehicle. Two amphiphilic EM-1 derivatives, C₁₈-SS-EM1 and C₁₈-CONH-EM1, were synthesized by attaching a stearyl moiety to EM-1 via a disulfide and amide bond, respectively. The amphiphilicity of EM-1 derivatives enabled self-assembling into nanoparticles for brain delivery. The study assessed morphology, circular dichroism, and metabolic stability of the formulations, as well as their pharmacodynamics and in vivo distribution, directly monitored by near-IR fluorescence imaging in mouse brains. In aqueous solution, the C₁₈-SS-EM1 derivative self-assembled into spherical nanostructures with a diameter of 10–20nm. Near-IR fluorescence analysis visualized the accumulation of the peptides in the brain. Importantly, the analgesic effect of C₁₈-SS-EM1 nanoparticles was significantly stronger as compared to that of unmodified EM-1 or C₁₈-CONH-EM1 nanoparticles. An in vitro release study demonstrated that self-assembled C₁₈-SS-EM1 nanoparticles possessed reduction-responsive behavior. In summary, self-assembling C₁₈SS-EM1 nanoparticles, which integrate the advantages of lipidization, nanoscale characteristics and, labile disulfide bonds, represent a promising strategy for brain delivery of short peptides.

Full text of DOI:10.1248/cpb.c19-00250

Vol. 67, No. 9
Chem. Pharm. Bull. 67, 977–984 (2019)
977
Regular Article
Amphiphilic Endomorphin-1 Derivative Functions as Self-assembling
Nanomedicine for Effective Brain Delivery
a
a
a
b
a
a
Hui Liu, Xiaoning Zhao, Shan Liang, Linlan Fan, Zhaojun Li, Yun Zhang, and
,
a
Jingman Ni*
a
b
School of Pharmacy, Lanzhou University; Lanzhou, Gansu Province 730000, P. R. China: and Key Laboratory of
Preclinical Study for New Drugs of Gansu Province, School of Basic Medical, Lanzhou University; Lanzhou, Gansu
Province 730000, P. R. China.
Received March 18, 2019; accepted June 2, 2019
Endomorphin-1 (Tyr-Pro-Trp-Phe-NH , EM-1), an endogenous μ-opioid receptor ligand with strong
2
antinociceptive activity, is not in clinical use because of its limited metabolic stability and membrane per-
meability. In this study, we develop a short-peptide self-delivery system for brain targets with the capabil-
ity to deliver EM-1 without vehicle. Two amphiphilic EM-1 derivatives, C -SS-EM1 and C -CONH-EM1,
18
18
were synthesized by attaching a stearyl moiety to EM-1 via a disulfide and amide bond, respectively. The
amphiphilicity of EM-1 derivatives enabled self-assembling into nanoparticles for brain delivery. The study
assessed morphology, circular dichroism, and metabolic stability of the formulations, as well as their phar-
macodynamics and in vivo distribution, directly monitored by near-IR fluorescence imaging in mouse brains.
In aqueous solution, the C -SS-EM1 derivative self-assembled into spherical nanostructures with a diameter
18
of 10–20nm. Near-IR fluorescence analysis visualized the accumulation of the peptides in the brain. Impor-
tantly, the analgesic effect of C -SS-EM1 nanoparticles was significantly stronger as compared to that of
18
unmodified EM-1 or C -CONH-EM1 nanoparticles. An in vitro release study demonstrated that self-assem-
18
bled C -SS-EM1 nanoparticles possessed reduction-responsive behavior. In summary, self-assembling C -
18
18
SS-EM1 nanoparticles, which integrate the advantages of lipidization, nanoscale characteristics and, labile
disulfide bonds, represent a promising strategy for brain delivery of short peptides.
Key words peptide brain delivery; blood–brain barrier; endomorphin-1; self-assembling; disulfide bond
Introduction
to deliver drugs across the BBB due to their notable properties
4
,12,13)
Delivery of therapeutic peptides to the central nervous such as tiny size and an easily tailored surface.
However,
system (CNS) represents a challenge and is the subject of neu- there are still some challenges including low drug loading
ropharmaceuticals research. The most critical factor limiting capacity resulting from the polymers with high molecular
peptide delivery is the protective function of the blood–brain weight, poor reproducibility of dual- or multi-functional
barrier (BBB). The BBB consists of the endothelial cells in modifications and the potential long-term toxicity of synthetic
brain capillaries that form tight junctions, which control the nanomaterials.
1)
concentration and entry of drugs into the CNS. In addi-
Recently, scientists proposed a new concept for a drug self-
tion, low metabolic stability of peptides represents a serious delivery system that uses the drugs as materials to build a
liability that restricts their accumulation in the brain, which nanosystem. For example, hydrophobic anticancer drugs were
reduces their bioavailability or their ability to exert an ad- stimulated to self-assemble into diverse nanostructures by di-
2
)
14,15)
equate intrinsic activity. Many peptides are potential neuro- rect conjugation to a short peptide,
a short oligomeric eth-
16)
17)
pharmaceuticals including amyloid beta aggregation inhibitors ylene glycol or small organic molecules. This amphiphilic
3
)
4)
for Alzheimer’s disease, peptides for Parkinson’s disease,
self-assembling drug–drug conjugation strategy has attracted
5
)
and analgesic opioid peptides. Endomorphin-1 (Tyr-Pro-Trp- much attention as a promising approach to achieve improved
Phe-NH , EM-1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH , drug loading capacity, fewer side effects caused by carriers
2
2
EM-2), isolated from the bovine brain and, subsequently, from and passive target properties in drug delivery. Peptides, due
6
–8)
the human brain cortex,
agonists with improved affinity and selectivity. Both EM-1 to form self-assembling nanostructures by intermolecular hy-
are two μ-opioid receptor (MOR) to their unique secondary structures, have the intrinsic ability
18,19)
and EM-2 have a wide range of physiological and pharmaco- drogen bonding
as an advantage over conventional small
logical effects, among which the antinociceptive activity is the organic drugs in drug–drug conjugate delivery systems. In ad-
9,10)
most interesting.
However, similar to other neuropeptides, dition, the self-assembled short-peptide-based nanostructures
EMs remain undeveloped as clinically viable drugs for the have many beneficial properties, such as high synthesis yields,
treatment of pain because of the delivery challenges including stability, flexibility, inherent biocompatibility and biodegrad-
2
0)
exclusion from the brain and degradation within the blood.
ability. Mazza’ group reported the direct conjugation of
Over the last two decades, a considerable number of strate- dalargin, a leucine-enkephalin analog, to a palmitoyl moiety
21)
gies for the transport of therapeutic molecules across the BBB via an ester link. The resulting amphiphilic peptide could
have been proposed and tested in preclinical and clinical tri- form nanofibers with high drug loading capacity and remark-
4
,11)
als.
Among them, nanocarriers have shown their potential ably improved BBB permeability.
*
To whom correspondence should be addressed. e-mail: nijm@lzu.edu.cn
©
2019 The Pharmaceutical Society of Japan

9
78
Chem. Pharm. Bull.
Vol. 67, No. 9 (2019)
Disulfide bonds have been extensively used as reduction- SS-EM1 was cleaved in a mixture of trifluoroacetic acid–tri-
responsive linkers in brain targeted drug delivery because of isopropylsilane–water at a ratio of 95:2.5:2.5 for 3h at room
their susceptibility to a reductive environment and the rapid temperature.
2
2–24)
cleavage by reducing agents such as glutathione (GSH).
In our previous study, we also confirmed that, as compared
Synthesis of C -CONH-EM1
18
The amide-linked derivative C -CONH-EM1 was as-
18
to amide and maleimide, the disulfide bond was the most ef- sembled following the conventional Fmoc-based solid-phase
ficient linkage to promote endomorphin-1 translocation across method. Stearic acid was coupled to the N-terminus of EM-1
2
5)
the BBB due to its reductive effect. Importantly, disulfide as Fmoc amino acid.
bonds play a significant role in the folding and aggregation All crude peptide derivatives were purified and analyzed
which potentially can promote by reversed-phase (RP)-HPLC (Waters 1525, Waters) on a C18
2
6)
of peptides and proteins,
self-assembling. Wang et al. also proved that a disulfide bond column. The molecular mass was determined by electrospray
could balance the competition between intermolecular forces, ionization (ESI)-MS.
suggesting that its insertion into molecules could promote and
stabilize the self-assembling of nanomedicines.
In this report, we synthesized two amphiphilic EM-1 deriv- cipitation method. Briefly, the EM-1 derivatives were dis-
Self-assembly and Characterization Self-assembly of
2
7)
the nanomedicines was performed according to the nanopre-
2
7)
atives, C -SS-EM1 and C -CONH-EM1, by covalently link- solved in ethanol and added dropwise to deionized water over
18
18
ing a stearyl moiety to the opioid peptide EM-1 via a disulfide 30min, under mechanical stirring (approx. 600–800rpm) at
and amine bond, respectively, to stimulate self-assembling room temperature. The final ethanol concentration was main-
into nanoparticles (NPs). We profiled the in vitro properties of tained at 2–5% and the self-assembly occurred spontaneously
the novel EM-1 derivatives, studied their pharmacodynamics under these conditions.
in a mouse model, and directly monitored the drug distribu-
tion profile in the mouse brain.
The shape and morphology were visualized and imaged by
transmission electron microscopy (TEM, JEOL, Japan). The
particle sizes, zeta potential, and polydispersity index (PDI)
of self-assembled nanomedicines were measured by dynamic
Experimental
Materials All fluorenylmethyloxycarbonyl (Fmoc)-pro- light scattering (DLS) analysis using a Malvern Zetasizer
tected amino acids are of the L-form and all were obtained Nano ZS (Malvern, U.K.). The concentration of the solution
from GL Biochem Ltd. (Shanghai, China). The 1-hydroxy- was 10µmol/L and each sample analysis was carried out in
benzotriazole (HOBT) O-(1H-benzotriazole-1-yl)-N,N,N′,N′- triplicate.
tetramethyluronium hexafluorophosphate (HBTU) and N,N-
Circular Dichroism (CD) Measurements The CD mea-
diisopropylethylamine (DIEA) were also purchased from surements of the C -CONH-EM1 and C -SS-EM1 NPs were
18
18
GL Biochem Ltd. (Shanghai, China). Rink Amide MBHA performed on a CD spectropolarimeter (JASCO, J-810) using
resin (0.45mmol/g) were obtained from Nankai Hecheng a quartz cell with a 1.0mm path length. The concentration of
S&T Co., Ltd. (Tianjin, China). A near-infrared (NIR) dye, the solution was 50µmol/L for CD measurements. The CD
1
(
,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide spectra were obtained with solvent subtraction from 190 to
DiR), was purchased from Fanbo Biochemicals Ltd. (Beijing, 260nm at a 10nm·min− scan rate and were averaged from
China). All other reagents and chemicals were obtained from five repeat scans per sample. The obtained CD spectral in-
J&K Scientific Ltd. (Beijing, China).
tensity is presented as the mean molar ellipticity per residue
1
2
−1
−1
Animals Male Kunming mice (18–22g) supplied by the (deg·cm ·dmol ·residue ). For secondary structure determi-
Animal Center at the Medical College of Lanzhou Univer- nation, samples were analyzed in the range of 190–240nm to
sity (Gansu, China) were used in the analgesia studies. Adult derive the content of α-helix (%) and β-strand (%) using k2d3
Balb/c male nude mice (8 weeks of age) purchased from the software, a package freely available online (http://www.ogic.
Vital River Laboratory Animal Technology Co., Ltd. (Beijing, ca/projects/k2d3).
China) were used in the NIR fluorescence studies. All animal
Metabolic Stability Studies Metabolic stability of EM-1
experiments were performed according to the principles and and its self-assembled nanomedicines was assessed in mouse
guidelines of the Ethics Committee at Lanzhou University.
serum and brain homogenate. Fresh plasma and 15% mouse
Synthesis of EM-1 Conjugates Two different EM-1 con- brain homogenate were prepared as described previously.
2
8)
jugates were synthesized by forming covalent linkages via an The amounts of remaining intact peptides were determined
amide or a disulfide bond.
by RP-HPLC. Briefly, approximately 10µL of self-assembled
nanomedicine solution (2mmol/L) was incubated in 190µL
Synthesis of C -SS-EM1
18
The synthesis of stearic acid containing a disulfide was test or control solution at 37°C. At designated time points
2
7)
performed as described previously. Briefly, dithiodiglycolic (0, 10, 30, 60, 120, 240, 480min), 20µL aliquots were re-
acid was induced to form the corresponding anhydride using moved from the samples and immediately mixed with 90µL
acetic anhydride as a dehydration agent. Excessive acetic of acetonitrile to precipitate proteins. Enzymatic activities
anhydride was removed using a rotary evaporation apparatus were stopped on ice for 5min and terminated by acidifying
at 30°C. The residue was reacted with stearyl alcohol, using with 90µL of a 0.5% acetic acid solution. Then, aliquots were
4
-dimethylaminopyridine (DMAP) as a catalyst to expedite centrifuged at 10000×g for 15min at 4°C. The supernatants
product formation.
were collected and analyzed by RP-HPLC.
The EM-1 peptide was synthesized by standard solid-phase
Assessment of Antinociception The antinociceptive
peptide synthesis using Fmoc chemistry. The stearic acid con- responses were assessed in Kunming mice using the warm
taining disulfide was used as Fmoc amino acid and attached to water tail-flick test. Test groups (n=6–8) were treated by
the N-terminus of EM-1. The resulting EM-1 derivative C - intravenous (i.v.) injection (via tail vein) with EM-1, C18-
18

Vol. 67, No. 9 (2019)
Chem. Pharm. Bull.
979
CONH-EM1 NPs or C -SS-EM1 NPs dispersed in water was dialyzed for 16h to remove free dye molecules (molecular
18
(
10 µmol/kg). To present the locus of antinociception, animals weight cutoff=1000g/mol). The dialysate was freeze-dried
were pretreated peripherally with opioid antagonists naloxone and dissolved in distilled water for the experiment in male
50mg/kg, intraperitoneal (i.p.)) and naloxone methiodide BALB/c nude mice. In vivo imaging was performed using
50mg/kg, i.p.) before i.v. challenge with peptide derivatives. the IVIS Lumina II imaging system (Caliper Life Science).
The warm water tail-flick test was performed as described 15 µmol/L DiR-loaded C -SS-EM1 NPs or DiR alone was
(
(
18
earlier. The mouse tail was immersed in hot water maintained injected into the tail vein of the mice (n=3 for each group).
at 50± 0.2°C and the latencies to flicking, bending, and swing Before optical imaging, the mice were anesthetized by inha-
were measured. Before treatment, baseline latency times lation of isoflurane mixed with medical air using a gaseous
[control latency (CL)] were recorded and selected as 3–5s; the anesthesia system. Whole body fluorescence images were re-
maximum cut-off time was set at 10s. The tail-flick responses corded at 10, 30, 60, 90, 120, 240, and 360min after the injec-
were measured at designated times points after i.v. injection of tion. Images were captured using a CCD camera and analyzed
drugs. The latency to tail-flick was defined as the test latency with the Lumina II Living Imaging 4 software. After comple-
(
TL) and 0.9% sodium chloride was used as control. The tion of the in vivo fluorescence imaging studies, we performed
antinociceptive response was expressed by converting anal- the ex vivo NIR fluorescence imaging analysis. Animals were
gesic tail-flick times to percentage of maximal possible effect sacrificed within 1h after i.v. administration and perfused
(
%MPE): %MPE=100 ×(TL−CL)/(10 −CL).
NIR Fluorescence Imaging The NIR fluorescent dye DiR the whole brains were removed and placed into the imaging
was employed as a probe for fluorescent imaging by loading system as described above.
it onto C -SS-EM1 NPs. An aliquot of 2mg C -SS-EM1 was Reduction Release Study of Self-assembled C -SS-EM1
with heparinized saline to remove the blood from the brain;
18
18
18
dissolved in 1mL of ethanol and 0.1mL of a DiR solution NPs in Vitro The in vitro release characteristics of the self-
0.5mg/mL) was added under stirring for 10min at room tem- assembled C -SS-EM1 NPs were evaluated in the presence
(
18
perature; this mixture was slowly added dropwise to deionized or absence of dithiothreitol (DTT) by HPLC analysis. Briefly,
water and stirred moderately for 30min. Then, the solution 1mmol/L self-assembled C -SS-EM1 NPs was prepared in
18
Fig. 1. Molecular Structure and Self-assembly Characterization of EM-1 Derivatives
(
A, D) Molecular structures of C -SS-EM1 and C -CONH-EM1. (B, E) TEM images of self-assembled C -SS-EM1 and C -CONH-EM1 NPs after dispersion in water.
1
8
1
8
1
8
1
8
Scale bar: 20nm. (C, F) DLS curve of C -SS-EM1 and C -CONH-EM1 NPs. Inset in Fig. C: a photograph of C -SS-EM1 solution of nanomolecular. (The concentration
18
18
18
was 10µmol/L.) (Color figure can be accessed in the online version.)

9
80
Chem. Pharm. Bull.
Vol. 67, No. 9 (2019)
water. Equal volumes aqueous solutions of C -SS-EM1 NPs ly different CD spectral features but shared the same trends.
18
and DTT were mixed to reach a final DTT concentration of For C -CONH-EM1, the most prominent features were an
18
10mmol/L. Then, the solutions were continuously mixed by intense maximum at 205nm and a minimum at 220nm. While
horizontal shaking (100rpm) at 37°C with or without DTT. At C -SS-EM1 exhibited a similar minimum at 220nm, it had a
18
the designated time (0.5, 1, 2, 4, 6h), samples were collected much stronger maximum at 195nm and the molar ellipticity
and replaced with an equal volume of water. The intact pro- increased significantly. According to the α-helix and β-strand
drug content and release rate of C -SS-EM1 NPs was deter- content prediction using the k2d3 software, the percentages
18
mined by RP-HPLC.
β-strand in C -CONH-EM1 and C -SS-EM1 were 17.07 and
18 18
Statistical Analysis Each group were performed at least 43.97, respectively. Thus, the CD analysis showed that the C -
18
three independent experiments. All data were expressed as the SS-EM1 had a strong tendency to form β-strands.
mean± standard deviation (S.D.) except for the assessment of Metabolic Stability Studies Figure 3 illustrates the
antinociception test using mean± standard error (S.E.M.). Stu- metabolic stability of EM-1 and the two self-assembled EM-1
dent’s t-test was used to compare the difference between two derivative NPs. Whether in the serum or the brain homog-
groups. p<0.05 was considered to demonstrate a statistically enate, unmodified EM-1 was rapidly degraded within minutes,
2
5)
significant difference (*p<0.05).
which corroborated previous results.
The self-assembled
C -CONH-EM1 NPs displayed an almost equipotent meta-
18
Results
bolic stability, maintaining the original peptide level for up
Preparation and Characterization of Self-assembled to 120min in the serum as well as in the brain homogenate.
Amphiphilic EM-1 NPs Two amphiphilic derivatives of For the self-assembled C -SS-EM1 NPs, the degradation rate
18
EM-1, C -CONH-EM1 and C -SS-EM1, were synthesized in serum was slightly lower than that of C -CONH-EM1. In
18
18
18
using Fmoc-based chemistry, in which synthesis of the peptide contrast, the cumulative degradation curve in the brain ho-
was followed by the linking a stearic moiety to the tyrosine mogenate indicated a significant decrease of C -SS-EM1 NPs
18
via an amide or disulfide bond as shown in Figs. 1A and D.
over time with approximately 50% peptide remaining after
The self-assembled nanoparticles were prepared according 120min, which may be caused by the cleavage of the disulfide
to the nanoprecipitation method. Figures 1B and E show TEM bond.
images of C -SS-EM1 and C -CONH-EM1 NPs, respectively.
Assessment of Antinociception The analgesic effects of
18
18
The C -SS-EM1 can self-assemble into well-dispersed spheri- unmodified EM-1 or self-assembled EM-1 derivatives were
18
cal nanostructures with uniform size (close to 15nm), whereas assessed performing the tail-flick test on Kunming mice after
the C -CONH-EM1 generated non-uniform nanostructures
18
under the same conditions (Fig. 1E). The consistent results
were presented in the DLS curve. Compared with the self-
assembled C -CONH-EM1 NPs (Fig. 1F), the DLS curve of
18
self-assembled C -SS-EM1 NPs in aqueous solution indicated
18
a narrow particle size distribution range of 10–20nm with an
average diameter of approximately 16.09nm (Fig. 1C). The
PDI value was 0.12, further confirming a narrow particle
diameter distribution. The zeta potential, also determined by
DLS, was negative (−14.6mV). The inset in Fig. 1C showed
a photograph of self-assembled C -SS-EM1 NP in aqueous
18
solution at a concentration of 10µmol/L, maintaining a stable
and transparent solution.
CD Measurements CD studies were conducted using the
peptide derivatives at a 50µmol/L concentration to investigate
changes in the secondary structure. As shown in Fig. 2, the EM1 in Water
Fig. 2. Circular Dichroism Spectra of the C -CONH-EM1 and C -SS-
18
18
two amphiphilic EM-1 derivatives were associated with slight-
(Color figure can be accessed in the online version.)
Fig. 3. The Degradation Profile of the EM-1 and Its Self-assembled Derivatives in (A) Mouse Serum, (B) 15% Brain Homogenate at 37°C
Data are given as mean± S.D. (n=3). (Color figure can be accessed in the online version.)

Vol. 67, No. 9 (2019)
Chem. Pharm. Bull.
981
Fig. 4. (A) Tail-Flick Test versus Time Curves of the Antinociceptive Effect after i.v. Administration of Sodium Chloride (0.9% w/v, ○), EM-1 Solu-
tion (10µmol/kg, ▲), C -CONH-EM1 NPs Solution (10µmol/kg, ◆) and C -CONH-EM1 NPs Solution (10µmol/kg, ■)
18
18
Data are given as mean± S.E.M. (n=6–8). (B) Antagonism of antinociception elicited by 10µmol/kg C -CONH-EM1 and C -CONH-EM1 NPs in the tail-flick assay
18
18
by naloxone (50mg/kg, i.p., administered 10min before drugs) and by naloxone methiodide (50mg/kg, i.p., administered 10min before drugs). The error bar indicates the
S.E.M. of the mean (n=6–8), and the asterisk indicates that the response is significantly different from NP group (p<0.05). (Color figure can be accessed in the online
version.)
Fig. 5. NIR Fluorescence Imaging of DiR-Load C -SS-EM1 NPs
18
2
(
A) Optical in vivo imaging of nude mouse after i.v. administrated DiR-load C -SS-EM1 NPs and free DiR. (B) Time course of fluorescence intensity (p/s/cm /sr) after
18
i.v. injection. (n=3) (C) Ex vivo imaging of perfused mouse brains 30min after i.v. injection of the DiR-load C -SS-EM1 NPs and free DiR and saline as controls. (Color
18
figure can be accessed in the online version.)

9
82
Chem. Pharm. Bull.
Vol. 67, No. 9 (2019)
i.v. administration. The results presented in Fig. 4A show that
the i.v. injection of either C -CONH-EM1 or C -SS-EM1 NPs
18
18
at a dose of 10µmol/kg led to an enhanced analgesic activity.
Especially the C -SS-EM1 NPs displayed a strong analgesic
18
activity, approximately 6.2-times more antinociception than
unmodified EM-1 and 2-times more than C -CONH-EM1
18
NP. The maximum analgesic effect of C -SS-EM1 NPs was
18
observed at 30min after injection and longer durations of ac-
tion were recorded up to 90min. Furthermore, the mice were
then pretreated with naloxone and naloxone methiodide to test
whether the opioid antagonists were able to suppress the anti-
nociceptive activity. Naloxone and naloxone methiodide both
act on opioid receptors but naloxone methiodide has limited
access to the brain. As shown in Fig. 4B, the antinociceptive
effects of the EM-1 derivatives were effectively blocked by
naloxone, suggesting that MOR was involved in producing
analgesia. In contrast, naloxone methiodide did not alter the
analgesic effect of i.v. administrated EM-1 derivatives, sug-
gesting the site of action is only in the central.
Fig. 6. Reduction-Responsive Behavior of C -SS-EM1 NP
18
Solutions were incubated in the presence or absence of 10mM DTT. Data are
given as mean± S.D. (n=3). (Color figure can be accessed in the online version.)
NIR Fluorescence Imaging of DiR-Labeled C -SS-EM1 EM-1 was employed not only as cargo but also as an integral
18
NPs To demonstrate the delivery of self-assembled C -SS- component of the drug delivery carrier. We synthesized two
18
EM1 NPs into the mouse brain, fluorescence imaging experi- peptide derivatives composed of a stearyl moiety linked to
ments based on monitoring the fluorescence intensity of DiR the EM-1 by a disulfide bond (C -SS-EM1) or an amide bond
18
were performed. Real-time in vivo images of the distribution (C -CONH-EM1). Based on the intrinsic amphiphilicity of
18
of free DiR or DiR-labeled C -SS-EM1 NPs in a living mouse the EM-1 derivatives, we predicted their self-assembly into
18
are shown in Fig. 5A. Visible fluorescence accumulation of nanovesicles in water. TEM was used to detect the potential
DiR-labeled C -SS-EM1 NPs happened rapidly in the brain at nanostructures of self-assembled derivatives. We found that
18
10min after injection and reached the highest levels at 60min. both EM-1 derivatives could self-assemble into a nanostruc-
In contrast, the fluorescence signal from free DiR was elimi- ture, but the self-assembled C -SS-EM1 had a more uniform
18
nated quickly from the body and it only remained at very low morphology than the C -CONH-EM1 and formed a homo-
18
levels in the brain.
geneous and monodisperse population according to the PDI
To confirm that the observed fluorescence signal was truly value (Fig. 1C, insets). As shown in Fig. 1A, the chemical
from the brain parenchyma and not from the vascular system, structure of the C -SS-EM1 contains three essential elements:
18
an ex vivo fluorescence imaging experiment was performed the stearic moiety, the active peptide EM-1, and the disulfide
after whole-body optical imaging at 1h. As shown in Fig. 5C, bond. First, the stearic moiety as the hydrophobic segment
the strongest signal was observed in the brain of mice treated induces hydrophobic interactions, which are responsible for
with C -SS-EM1 NPs. Conversely, no significant fluorescence driving self-assembly and exposing the functional peptide
1
8
19)
signal was detected in the brains of saline-treated mice and group on the surface of the nanostructure. Second, the EM-1
only a relatively weak signal was detected in the brains of (YPWF) is the active drug that serves as the hydrophilic
DiR-treated mice.
segment of the amphiphilic derivative. This design dramati-
Reduction Release Study of Self-assembled C -SS-EM1 cally increases the drug-loading capacity and diminishes po-
18
NPs in Vitro The release of the active peptide represents a tential side effects due to excipients. Third, a disulfide bond
critical step for self-assembled prodrugs to exert their func- was chosen as a biodegradable linker to covalently attach
tion. Therefore, the reduction-responsive release behavior of the stearic moiety to the peptide. The disulfide bond can be
C -SS-EM1 NPs was tested in vitro in the presence or ab- rapidly cleaved in the presence of reducing agents such as
18
sence of DTT at 37°C. As shown in Fig. 6, the C -SS-EM1 GSH and has been extensively used as a reduction-responsive
1
8
2
7,33,34)
NPs treated with 10mM DTT showed faster release rates than linker.
Here, the only structural difference between the
those without DTT. For example, after 2h of incubation, the C -SS-EM1 and C -CONH-EM1 is the internal chemical
18
18
release of C -SS-EM1 NPs in the presence of 10mM DTT bond, but their nanomorphology differed significantly. We hy-
18
was 70%, whereas only 10% was released from the C -SS- pothesize that the disulfide bond plays a wider role in the self-
18
EM1 NPs without a reducing agent. The above result indicated assembly. In general, disulfide bond formation is associated
that the in vitro release behavior of C -SS-EM1 NPs was with protein folding and the S–S bonds show a preference for
18
2
7)
reduction-responsive.
dihedral angles approaching 90°. This may play an essential
role in balancing the intermolecular forces in peptide amphi-
philes to control the self-assembly. Self-assembled C -CONH-
Discussion
18
Self-assembling peptide-based nanostructures and materials, EM1 exhibited a poor nanomorphology possibly because of its
2
9,30)
21,31)
such as cell-penetrating peptides,
peptide amphiphiles
strongly polar amide group. The CD spectra further supported
32)
or hydrogels, have been tested as delivery vectors for thera- a role of the disulfide bonds during the self-assembly process.
peutic molecules due to their inherent biocompatibility and The disulfide conjugate C -SS-EM1 exhibited much stronger
18
biodegradable properties.
change at 195nm (Fig. 2) and its β-strand content was ap-
In this study, the pharmacologically active neuropeptide proximately 2.5-fold higher than that of the amide conjugate

Vol. 67, No. 9 (2019)
Chem. Pharm. Bull.
983
C -CONH-EM1, which demonstrated that the disulfide bond CNS, the opioid peptides including endomorphins should be
1
8
37,38)
dramatically affected the CD spectra.
able to cross the BBB intact.
Van Dorpe et al. described
39)
To study the resistance of the amphiphilic derivatives to the transport properties of EMs. Both EM-1 and EM-2
enzymatic hydrolysis, fresh serum and 15% brain homogenate display high influx constant rates (K =1.06 and 1.14µL/
in
were chosen to simulate the conditions of the peripheral blood (g×min), respectively), which were consistent with their
circulation and CNS environment. Like other small peptides, logp values (2.04 and 1.94, respectively). Additionally, the
the EMs exhibited very poor metabolic stability and low mem- capillary depletion method showed that EMs entered the
brane permeability. Lipidation has been shown as the most brain parenchyma for about 90%. It is hence possible that the
robust and effective approach to improve the metabolic stabil- EMs penetrate the BBB by passive transmembrane diffusion.
35,36)
ity of a peptide and its BBB transport.
In this study, the However, there are two factors influencing EM brain delivery.
stearyl moiety was introduced as the lipidic group, which im- One factor is the presence of the efflux system at the BBB.
39)
40,41)
proved the metabolic stability profile (Fig. 3). Having the same In some studies Van Dorpe et al. and Kastin et al.,
it is
lipidation, the C -CONH-EM1 displayed a similar degrada- demonstrated that EM-1 and EM-2 entering the brain could be
18
tion rate in the serum and in the brain homogenate, whereas pumped out again by a saturable efflux system. The half-time
the self-assembled C -SS-EM1 NPs displayed a significantly disappearance is 15 and 22min, respectively. Another factor is
18
faster degradation in the brain as shown in Fig. 3B. A possible their fast degradation by peptidases. The study showed both
interpretation is that C -SS-EM1 was stable in the peripheral EM-1 and EM-2 showed the poorest brain and plasma stabil-
18
plasma until crossing the BBB but was subsequently cleaved ity among the eight investigated opioid peptides. Compared
in the reductive environment of the brain tissue to release its to the brain tissue, the plasma showed half-life stabilities of a
biologically active form from the self-assembled NPs. These few minutes only. In present study, the prodrug strategy and
results were further verified by the reduction-release in vitro nanocarriers were combined to enhance penetration of EM-1
study of self-assembled C -SS-EM1 NPs (Fig. 6). After the across the BBB after systemic administration. The introduc-
18
addition of the reducing agent DTT, the C -SS-EM1 NPs dis- tion of fatty acid is able to increase the lipophilic character of
18
played a faster release rate than those without DTT.
EM-1. The self-assembled nanoparticles can effectively avoid
The potent antinociceptive effect after peripheral admin- the enzymatic hydrolysis and readily cross BBB by small size
istration indicates the ability of the peptide derivatives to mediated endocytotic mechanisms.
cross the BBB. Compared with the free EM-1, the two self-
assembled EM-1 derivatives with an internal amide or disul- Conclusion
fide linkage produced an enhanced antinociceptive effect (Fig.
In this study, we successfully synthesized the reduction-
4
A). That was probably related to the fatty acid modification. responsive EM-1 derivative C -SS-EM1. In an aqueous
18
As described earlier, the lipidation as a modification increases medium, it self-assembled into NPs. We concluded from our
the uptake by the brain by modulating peptide lipophilic- profiling experiments that the self-assembled C -SS-EM1 NPs
18
ity, which promotes translocation across the BBB by passive consisted of a hydrophobic core that was surface-decorated
diffusion. Interestingly, although the stearic acid was incor- with peptide β-sheets. Based on the structural similarity to
porated into both peptide derivatives, the disulfide conjugate micelles, we concluded that the tightly arranged peptide moi-
C -SS-EM1 NPs exhibited approximately 2-fold stronger eties provide protection against peptide-degrading activities in
18
antinociception as compared to that of the amide-containing the plasma and at the BBB. As compared with the unmodified
derivative C -CONH-EM1. This is linked to the formation of EM-1 or the amide-linked C -CONH-EM1, the self-assembled
18
18
self-assembled nanostructures of C -SS-EM1. It is clear that C -SS-EM1 NPs exhibited significantly increased antinoci-
18
18
the self-assembled nanoparticle arrangement prevents the ac- ceptive activities. The NIR fluorescent experiments further
cess of peptidases to the EM-1 and protects the integrity of confirmed our hypothesis that the C -SS-EM1 NPs could
18
the peptide in the plasma. In addition, as shown in Fig. 4B, cross the BBB and accumulate in the mouse brain. In sum-
the antinociceptive effects after i.v. administration of the self- mary, the C -SS-EM1 NP design integrated the advantages
18
assembled EM1 derivative NPs were significantly reversed by of lipidization, nanocarriers and, a labile disulfide bond that
naloxone but not by naloxone methiodide. Naloxone is known resulted in improved brain targeting, which indicated the great
to potentially antagonize the antinociceptive effect induced by potential of the self-assembled prodrug NPs in brain drug de-
μ-opioid agonists on central and peripheral receptors. Nalox- livery and therapy.
one methiodide, its quaternary derivative, is thought act only
in the periphery and is used to distinguish between central
Acknowledgments This work was supported by the Na-
and peripheral sites of action for drugs acting on opioid recep- tional Natural Science Foundation of China (No. 81773564);
tor. These results further revealed that the observed analgesic the Key Science and Technology Program of the Gansu
effects may involve central μ-opioid receptors.
Province (No. 17YF1FA125); the Natural Science Foundation
To directly demonstrate that the peptide NPs crossed the of Gansu Province (No. 17JR5RA204); and the Fundamen-
BBB, the NIR dye DiR was employed as a fluorescent marker tal Research Funds for the Central Universities (No. lzu-
to monitor the distribution of C -SS-EM1 NP in the brain. jbky-2016-144, lzujbky-2017-204).
18
The in vivo and ex vivo studies showed that the fluorescence
intensity of DiR-labeled C -SS-EM1 NPs was significantly
Conflict of Interest The authors declare no conflict of
18
higher than that of the free DiR, indicating that the brain interest.
targeting of C -SS-EM1 NPs was successful, confirming the
18
results of the pharmacodynamics analysis.
As opioid-based pain treatment occurs mainly within the
References
1) Pardridge W. M., Expert Opin. Drug Deliv., 13, 963–975 (2016).

9
84
Chem. Pharm. Bull.
Vol. 67, No. 9 (2019)
2)
McGowan J. W., Bidwell G. L. 3rd, Vig P. J., Ther. Deliv., 6, 841–
53 (2015).
24) Goyal K., Konar A., Kumar B. S. H., Koul V., Nanoscale, 10,
8
17781–17798 (2018).
3)
Carter M. D., Simms G. A., Weaver D. F., Clin. Pharmacol. Ther., 25) Liu H., Zhang W., Ma L., Fan L., Gao F., Ni J., Wang R., Int. J.
8
8, 475–486 (2010).
Pharm., 476, 1–8 (2014).
4
)
Brasnjevic I., Steinbusch H. W., Schmitz C., Martinez-Martinez P.,
Prog. Neurobiol., 87, 212–251 (2009).
Bodnar R. J., Peptides, 62, 67–136 (2014).
26) Hidaka Y., Shimamoto S., Biomol. Concepts, 4, 597–604 (2013).
27) Wang Y., Liu D., Zheng Q., Zhao Q., Zhang H., Ma Y., Fallon J. K.,
Fu Q., Haynes M. T., Lin G., Zhang R., Wang D., Yang X., Zhao L.,
He Z., Liu F., Nano Lett., 14, 5577–5583 (2014).
5
6
)
)
Hackler L., Zadina J. E., Ge L. J., Kastin A. J., Peptides, 18, 1635–
1
639 (1997).
28) Mallareddy J. R., Borics A., Keresztes A., Kover K. E., Tourwe D.,
Toth G., J. Med. Chem., 54, 1462–1472 (2011).
29) Pan Z. Z., Wang H. Y., Zhang M., Lin T. T., Zhang W. Y., Zhao P.
F., Tang Y. S., Xiong Y., Zeng Y. E., Huang Y. Z., Acta Pharmacol.
Sin., 37, 1110–1120 (2016).
30) Zhang P., Cheetham A. G., Lin Y. A., Cui H., ACS Nano, 7, 5965–
5977 (2013).
31) Fatouros D. G., Lamprou D. A., Urquhart A. J., Yannopoulos S. N.,
Vizirianakis I. S., Zhang S., Koutsopoulos S., ACS Appl. Mater.
Interfaces, 6, 8184–8189 (2014).
7
8)
)
Vaccarino A. L., Kastin A. J., Peptides, 22, 2257–2328 (2001).
Zadina J. E., Hackler L., Ge L. J., Kastin A. J., Nature (London),
3
86, 499–502 (1997).
Liu W. X., Wang R., Med. Res. Rev., 32, 536–580 (2012).
0) Keresztes A., Borics A., Toth G., ChemMedChem, 5, 1176–1196
2010).
1) Alam M. I., Beg S., Samad A., Baboota S., Kohli K., Ali J., Ahuja
9)
1
(
1
A., Akbar M., Eur. J. Pharm. Sci., 40, 385–403 (2010).
2) Li X., Tsibouklis J., Weng T., Zhang B., Yin G., Feng G., Cui Y.,
Savina I. N., Mikhalovska L. I., Sandeman S. R., Howel C. A.,
Mikhalovsky S. V., J. Drug Target., 25, 17–28 (2017).
1
32) Shah R. N., Shah N. A., Del Rosario Lim M. M., Hsieh C., Nuber
G., Stupp S. I., Proc. Natl. Acad. Sci. U.S.A., 107, 3293–3298
(2010).
1
3) Saraiva C., Praca C., Ferreira R., Santos T., Ferreira L., Bernardino
L., J. Control. Release, 235, 34–47 (2016).
4) Cheetham A. G., Zhang P., Lin Y. A., Lock L. L., Cui H., J. Am.
33) Song J., Zhang W., Kai M., Chen J., Liang R., Zheng X., Li G.,
Zhang B., Wang K., Zhang Y., Yang Z., Ni J., Wang R., Mol.
Pharm., 10, 2934–2941 (2013).
1
Chem. Soc., 135, 2907–2910 (2013).
1
5) Lock L. L., LaComb M., Schwarz K., Cheetham A. G., Lin Y. A.,
34) Song Q., Wang X., Wang Y., Liang Y., Zhou Y., Song X., He B.,
Zhang H., Dai W., Wang X., Zhang Q., Mol. Pharm., 13, 190–201
(2016).
Zhang P., Cui H., Faraday Discuss., 166, 285–301 (2013).
16) Shen Y., Jin E., Zhang B., Murphy C. J., Sui M., Zhao J., Wang J.,
Tang J., Fan M., Van Kirk E., Murdoch W. J., J. Am. Chem. Soc.,
35) Pignatello R., Puleo A., Guccione S., Raciti G., Acquaviva R.,
Campisi A., Ventura C. A., Puglisi G., Eur. J. Med. Chem., 40,
1074–1079 (2005).
36) Varamini P., Toth I., Front. Pharmacol., 4, 155 (2013).
37) Weksler B. B., Subileau E. A., Perriere N., Charneau P., Holloway
K., Leveque M., Tricoire-Leignel H., Nicotra A., Bourdoulous S.,
Turowski P., Male D. K., Roux F., Greenwood J., Romero I. A.,
Couraud P. O., FASEB J., 19, 1872–1874 (2005).
38) Kastin A. J., Pan W., Curr. Pharm. Des., 14, 1615–1619 (2008).
39) Van Dorpe S., Adriaens A., Polis I., Peremans K., Van Bocxlaer J.,
De Spiegeleer B., Peptides, 31, 1390–1399 (2010).
40) Kastin A. J., Fasold M., Smith R., Horner K., Zadina J., Exp. Brain
Res., 139, 70–75 (2001).
1
32, 4259–4265 (2010).
7) Huang P., Wang D., Su Y., Huang W., Zhou Y., Cui D., Zhu X., Yan
D., J. Am. Chem. Soc., 136, 11748–11756 (2014).
8) Cui H., Webber M. J., Stupp S. I., Biopolymers, 94, 1–18 (2010).
9) Dehsorkhi A., Castelletto V., Hamley I. W., J. Pept. Sci., 20, 453–
67 (2014).
0) Gentilucci L., Tolomelli A., Squassabia F., Curr. Med. Chem., 13,
449–2466 (2006).
1) Mazza M., Notman R., Anwar J., Rodger A., Hicks M., Parkinson
1
1
1
4
2
2
2
G., McCarthy D., Daviter T., Moger J., Garrett N., Mead T., Briggs
M., Schatzlein A. G., Uchegbu I. F., ACS Nano, 7, 1016–1026 (2013).
2) Bohn K., Lange A., Chmielewski J., Hrycyna C. A., Mol. Pharm.,
2
2
14, 1107–1119 (2017).
41) Kastin A. J., Akerstrom V., Pan W., J. Mol. Neurosci., 18, 7–14
(2002).
3) Zhu Y., Zhang J., Meng F., Deng C., Cheng R., Feijen J., Zhong Z.,
J. Control. Release, 233, 29–38 (2016).