Neuroligin-2-derived peptide-covered polyamidoamine-based (PAMAM) dendrimers enhance pancreatic β-cells' proliferation and functions

Article abstract of DOI:10.1039/c8md00419f

Pancreatic β-cell membranes and presynaptic areas of neurons contain analogous protein complexes that control the secretion of bioactive molecules. These complexes include the neuroligins (NLs) and their binding partners, the neurexins (NXs). It has been recently reported that both insulin secretion and the proliferation rates of β-cells increase when cells are co-cultured with full-length NL-2 clusters. The pharmacological use of full-length protein is always problematic due to its unfavorable pharmacokinetic properties. Thus, NL-2-derived short peptide was conjugated to the surface of polyamidoamine-based (PAMAM) dendrimers. This nanoscale composite improved β-cell functions in terms of the rate of proliferation, glucose-stimulated insulin secretion (GSIS), and functional maturation. This functionalized dendrimer also protected β-cells under cellular stress conditions. In addition, various novel peptidomimetic scaffolds of NL-2-derived peptide were designed, synthesized, and conjugated to the surface of PAMAM in order to increase the biostability of the conjugates. However, after being covered by peptidomimetics, PAMAM dendrimers were inactive. Thus, the original peptide-based PAMAM dendrimer is a leading compound for continued research that might provide a unique starting point for designing an innovative class of antidiabetic therapeutics that possess a unique mode of action.

Full text of DOI:10.1039/c8md00419f

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RESEARCH ARTICLE
Neuroligin-2-derived peptide-covered
polyamidoamine-based (PAMAM) dendrimers
enhance pancreatic β-cells' proliferation and
functions†
Cite this: DOI: 10.1039/c8md00419f
Anna Munder,a Yoni Moskovitz,a Aviv Meir,a Shirin Kahremany,ab Laura Levy,a
c
Michal Kolitz-Domb,a Guy Cohen,
Efrat Shtriker,a Olga Viskind,a
Jean-Paul Lellouche,ad Hanoch Senderowitz,
Steven D. Chessler,e
a
a
a
a and Arie Gruzman
*
*
Edward E. Korshin,
Sharon Ruthstein
Pancreatic β-cell membranes and presynaptic areas of neurons contain analogous protein complexes that
control the secretion of bioactive molecules. These complexes include the neuroligins (NLs) and their bind-
ing partners, the neurexins (NXs). It has been recently reported that both insulin secretion and the prolifera-
tion rates of β-cells increase when cells are co-cultured with full-length NL-2 clusters. The pharmacologi-
cal use of full-length protein is always problematic due to its unfavorable pharmacokinetic properties.
Thus, NL-2-derived short peptide was conjugated to the surface of polyamidoamine-based (PAMAM)
dendrimers. This nanoscale composite improved β-cell functions in terms of the rate of proliferation,
glucose-stimulated insulin secretion (GSIS), and functional maturation. This functionalized dendrimer also
protected β-cells under cellular stress conditions. In addition, various novel peptidomimetic scaffolds of
NL-2-derived peptide were designed, synthesized, and conjugated to the surface of PAMAM in order to in-
crease the biostability of the conjugates. However, after being covered by peptidomimetics, PAMAM
dendrimers were inactive. Thus, the original peptide-based PAMAM dendrimer is a leading compound for
continued research that might provide a unique starting point for designing an innovative class of anti-
diabetic therapeutics that possess a unique mode of action.
Received 28th August 2018,
Accepted 11th December 2018
DOI: 10.1039/c8md00419f
rsc.li/medchemcomm
β-cells occurs because of futile efforts to compensate for the
lack of insulin activity. This overstimulation, in turn, causes
Introduction
Type 2 diabetes mellitus (T2DM) is reaching catastrophic
numbers in human populations and causing high morbidity
and mortality due to its severe complications.1–3 One of the
most important pathophysiological components in T2DM is
the peripheral tissues' insulin resistance, which in turn leads
to increased efforts of β-cells to secrete more than physiologi-
cal amounts of insulin.4,5 This chronic overstimulation of
β-cell dysfunction. At the beginning of the disease, decreased
numbers of normal functioning β-cells are usually detected in
the pancreas.6 Thus, preservation or even augmentation of
functional pancreatic β-cell mass is one of the main goals of
therapeutic development for T2DM.7,8 In addition, successful
transplantation of β-cells/islets is dependent on a sufficient
amount of functioning β-cells.9,10 Thus, recent scientific ef-
forts have focused on identifying new strategies, which will be
able to positively influence β-cell function and/or proliferation
and thereby prevent or delay the onset of the disease and its
late complications.11–13 Several β-cell mass protective ap-
proaches have been recently developed. This includes, for ex-
ample, the use of H2S, which protects cells from apoptosis in-
duced by high glucose,14 targeting mitochondrial DNA,15
developing compounds that protect cells under ER stress-
induced autophagy,16 and many others.17,18
a Department of Chemistry, Faculty of Exact Sciences, Bar-Ilan University, Ramat-
Gan, Israel. E-mail: sharon.ruthstein@biu.ac.il, gruzmaa@biu.ac.il
b Department of Pharmacology, Cleveland Center for Membrane and Structural
Biology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA
c Skin Research Institute, Dead Sea and Arava Research Center, Masada, Israel
d Nanomaterials Research Center, Institute of Nanotechnology & Advanced
Materials (BINA), Bar-Ilan University, Ramat-Gan, Israel
e Division of Endocrinology, Diabetes & Metabolism, Department of Medicine,
University of California, Irvine, CA, USA
Regarding T1DM, which is also a life-threatening disease,
causing severe complications and increased mortality, there
is similarly a need for ways to protect β-cell mass.19–22 In this
† Electronic supplementary information (ESI) available: Experimental details of
the synthetic pathways, synthetic procedures, and analytical data of the synthe-
sized compounds are available. See DOI: 10.1039/c8md00419f
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autoimmune disease, patients lose the vast majority of their
β-cells owing to an aberrant immune attack.23 The main chal-
lenge in T1DM is to protect the remaining β-cells, or, in the
ideal case, to even induce β-cell regeneration. To date, only
immunosuppressive therapies that begin early after T1DM
onset have been able to slightly slow β-cell degeneration and
death.24,25 Together with immunosuppression, autoantigen
therapy and agents that can facilitate β-cell regeneration
might also be possible drug development options.26 Because
of the huge therapeutic potential in both types of diabetes,
the search for novel approaches for preserving or even in-
creasing the β-cell mass has always been very intense.
We recently reported that the HSA-28 peptide, which was
derived from NL-2 and covalently attached to the surface of
ytterbiumIJIII) cation-doped maghemite nanoparticles (NPs),
improved β-cell functionality.27 The peptide was selected
based on the interfacial residues that seem to dominate the
interactions between NL-2 and its binding partner neurexin
(NX), and which are also completely preserved in the corre-
sponding NL-2 sequence. The rationale to use NL-2 as a basis
for developing protective β-cell therapeutics was based on the
induced degradation).43–45 Although in vitro biological evalua-
tion of HSA-28D showed promising results, we tried to take
this project even further; various novel peptidomimetic scaf-
folds of HSA-28 were designed, synthesized, and conjugated
to the surface of PAMAM. Peptidomimetics are compounds
whose chemical moieties mimic the 3D structure of natural
peptides and retain the ability to bind to biological targets,
but without the disadvantages of peptides.46–49 However, cov-
ered by peptidomimetics, PAMAM dendrimers were inactive
in the proliferation screening assay. Thus, in this work, syn-
thesis of peptidomimetics was used only for developing new
organic scaffolds that might be used for the mimicking of
glutamic, phenylalanine, and leucine amino acids containing
peptides (as it was in our case). HSA-28-based PAMAM
dendrimers are leading compounds for upcoming work that
might reveal an innovative class of antidiabetic therapeutics
that possess a unique mode of action: increasing β-cell mass
and functionality via targeting its neuronal secretory and ad-
hesion machinery.
fact that the secretory/cell contact machinery of the β-cells is Methods
similar to that of neurons.28–31 One of the interacting protein
pairs (NLs and NXs) was targeted because of its role in
Materials
PAMAM dendrimer (ethylenediamine core, fifth generation), bo-
establishing the normal β-cell functioning brought about by
β-cell clustering.28–31 We hypothesized that β-cells treated
with NL-2 mimetics might be more functionalized and
protected after transplantation.
vine serum albumin (BSA), glucose oxidase (GO), thapsigargin
(Tg), protease inhibitor cocktail, radioimmunoprecipitation
assay buffer (RIPA), Rink amide resin, 2,2-diphenylethylamine,
benzhydrylamine, phenethylamine, benzylamine, glycinamide
hydrochloride, α- and β-naphthaldehyde, 1,2,3,4-tetra-
isoquinoline, n-bromoethylphthalimide, potassium carbonate,
potassium iodide, tert-butyl acrylate, isobutylamine, triethyl-
amine, 2-iminothiolane, and 3-maleimidopropionic acid were
purchased from Sigma-Aldrich (Merck) (Rehovot, Israel).
Fetal calf serum (FCS), phosphate buffered saline (PBS),
L-glutamine, trypan blue, Roswell Park Memorial Institute me-
dium (RPMI-1640), and antibiotics were purchased from Bio-
logical Industries (Beth-Haemek, Israel). Formaldehyde (4%
in PBS) was purchased from Bio-Lab (Jerusalem, Israel). Rink
amide resin, DIEA, and protected amino acids were obtained
from Chem Impex (Wood Dale, IL, USA). β-Mercaptoethanol
was purchased from Bio-Rad (Hercules, CA, USA). Alfa Aesar
(Ward Hill, MA, USA) supplied t-isopropanol-silane. Alexa
Fluor 633 Phalloidin was purchased from Life Technologies
(Carlsbad, CA, USA). Anti-glucagon, anti-C-peptide, and anti-
Pdx1 antibodies were supplied by Abcam (Cambridge, MA,
USA). The Mercodia Insulin ELISA kit was purchased from
Mercodia (Uppsala, Sweden). All organic solvents were pur-
chased from Carlo Erba Reagents (Val De Reuil, France).
Indeed, HSA-28-covered maghemite NPs increased prolif-
eration, resistance to oxidative stress, and insulin secretion
in both in vitro (INS-1E cells) and ex vivo (mouse islets)
models.27 However, HSA-28-covered ytterbiumIJIII) maghemite
(γ-Fe2O3)-based nanoparticles might be not suitable for use
in humans, because of genotoxicity32,33 and neurobehavioral
impairments.34 Herein, our aim was to improve peptide-
based cluster functions by using another core platform. Thus,
different types of functionalizing NPs were synthesized by
conjugation between HSA-28 and the surface amines of a
commercially available PAMAM dendrimer. Dendrimers have
three-dimensional, highly branched unique polymeric archi-
tectures (classically 5000–500 000 g mol−1) and have the bene-
fits of a low polydispersity index, controlled biodegradability,
biocompatibility, and the presence of various terminal multi-
valent functional groups on their surface.35 Dendrimers are
widely used in nanomedicine as potential carriers for various
drugs, bioactive molecules, and genes.36,37 The prepared
HSA-28D (HSA-28-coated PAMAM) improved β-cell functions
in terms of the rate of proliferation, glucose-stimulated insu-
lin secretion (GSIS), cell maturation, and insulin accumula-
tion. In addition, this functionalized dendrimer protected
β-cells under stressful conditions that exist in diabetes: oxida-
tive and ER stress.
Peptide synthesis
Peptides play a variety of important roles in biological sys-
tems and are also used as therapeutic agents.38–42 However,
the use of peptides as drugs is limited because of several
pharmacokinetic disadvantages (for example, protease-
The synthesis was performed as described in the literature.50
The obtained peptide was coupled with 3-maleimidopropionic
acid by the N-termini of the peptide according to the peptide
synthesis procedure.
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HPLC purification
Induction of ER stress
Preparative HPLC (Young Lin Instruments, Anyang, Korea)
was performed on a LUNA C18 preparative column (10 μm,
100 × 30 mm) from Phenomenex, Inc. (Torrance, CA, USA).
Compounds were purified using an increasing linear gradient
of acetonitrile in DDW at room temperature.
ER stress conditions were induced by supplying thapsigargin
(Tg, 0.75 μM) to the growing medium of the INS-1E cells.53 The
standard MTT test was performed after 24 h of incubation.
Immunofluorescence measurement of C-peptide, glucagon
and Pdx1 level
Peptide conjugation onto the surface of PAMAM dendrimer
Experiments were conducted in INS-1E cells that were seeded
on coverslips in 6-well plates. The cells were incubated with
HSA-28D for 72 h (control cells were untreated). Following in-
cubation, the slides were washed three times with pre-
warmed PBS and fixed with formaldehyde (4% in PBS). Sub-
sequently, the slides were washed three more times with pre-
warmed PBS. The level of C-peptide was determined by im-
munocytochemistry using anti-C-peptide antibody according
to the manufacturer's protocol. The membranes were stained
with Alexa Fluor 633 Phalloidin according to the manufac-
turer's protocol. The slides were washed three times with pre-
warmed PBS. Nuclei were stained with DAPI according to the
manufacturer's protocol. Fluorescent signals were visualized
with a confocal-Zeiss microscope equipped with a 60×/1.4 ob-
jective (Oberkochen, Germany). For the glucagon measure-
ment, cells were grown as described above. Slides were incu-
bated with HSA-28D (control cells were untreated) for 72 h.
The level of glucagon was determined by immunocyto-
chemistry using anti-glucagon antibody. Thereafter, the slides
were fixed with formaldehyde as described above. Cell mem-
branes and nuclei were stained as previously described. Visu-
alization of fluorescent signals proceeded as described above.
For Pdx1 level identification, cells were grown as described
above. Slides were incubated with HSA-28D for 72 h. The
level of PDX1 was determined by immunocytochemistry using
anti-PDX1 antibody. Thereafter, the slides were fixed with
formaldehyde as described above. Cell membranes and nu-
clei were stained as previously described. Visualization of
fluorescent signals proceeded as described above.
Two hundred μL of PAMAM dendrimer in methanol solution
(which corresponds to 0.01 g of PAMAM dendrimer) was used
for preparing each sample. First, the methanol was evapo-
rated under reduced pressure and the dendrimer was
dissolved in 0.5 ml of PBS. Then the solution of dendrimer
was treated with a 2-fold excess of 2-iminothiolane (from 0.1
M stock solution of 2-iminothiolane in PBS). After having
been shaken for 5 min, a 5-fold excess of HSA-28 peptide or
mimetics in PBS was added to the solution above, up to 2 ml
of the total volume. The reaction mixture was shaken over-
night at room temperature.
The coated NPs were purified using a centrifuge ultrafiltra-
tion system (3000 rpm, 20 min, 10 °C, 3 times) until the fil-
trate did not contain any traces of the compound (monitor-
ing by TLC under UV light or under vanillin stain).
Cell line
INS-1E β-cells were courtesy of Prof. Shlomo Sasson (Faculty
of Medicine, Hebrew University of Jerusalem, Jerusalem,
Israel). Cells were grown and maintained as described by
Pasternak et al.51
Cell counting (trypan blue exclusion assay)
Cells were detached by trypsin and mounted using trypan
blue (0.4%). Only uncolored cells were counted (live cells).
Cell counting proceeded according to the Abcam (Cambridge,
MA, USA) online protocol for work with a hemocytometer.
GSIS and insulin content assays
MTT cell viability assay
The assays were performed on INS-1E cells as described.54
Insulin quantification was performed using the Mercodia
Insulin ELISA kit according to the manufacturer's protocol.
Cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide reagent (MTT, 2 mg ml−1) in
growth medium for 2 h at 37 ° C. The medium was then aspi-
rated, and DMSO was added to solubilize the cells and col-
ored crystals. Absorbance at 570 nm was measured in a
SpectraMax M5 spectrophotometer (Sunnyvale, CA, USA).
The pharmacophore design
The pharmacophore was designed based on the structure of
HSA-28 peptide and NX-1 using LigandScout 4.0 software.55
Induction of oxidative stress
Oxidative stress conditions52 were induced by supplying glu-
cose oxidase (GO, 50 mU ml−1) to the growing medium of the
INS-1E cells. This resulted in an elevated H2O2 concentration
in the medium (reaching 29.0 9.6 μM after 4 h of incuba-
tion). The standard MTT cell viability test was performed af-
ter 4 h of incubation.
Synthesis of HSA-28 mimetics
The compounds were obtained by a reaction between com-
mercially available or synthesized in house aromatic amines
and tert-butyl acrylate as described by Ingallinella et al.56 The
detailed synthesis procedures are presented in the ESI.†
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HSA-28 mimetic conjugation onto the surface of the PAMAM
dendrimer
mM in 1 cm quartz cuvettes. The wavelength ranged from
250 to 500 nm.
3-Maleimidopropionic acid was coupled to all the synthesized
compounds above according to the procedure described by Results
Ruzza et al.57 The coupling reaction was followed by the treat-
Conjugation of HSA-28 peptide onto the surface of PAMAM
ment of all compounds with neat TFA for 1 h as described in
the literature. The conjugation of all compounds onto the
surface of PAMAM dendrimer was performed as described
before.
and estimation of the percentage of coating
The HSA-28 peptide was synthesized as described in the
Methods section with an amide moiety on its C-terminal and
a maleimide moiety on its N-terminal (Scheme 1). PAMAM
dendrimer (fifth generation) with an ethylene diamine core
and 128 amino groups available for reaction was chosen for
evaluating the peptide assembling because of its size similar-
ity to previously active maghemite nanoparticles (5.4 nm).27
The conjugation was performed using a known quick single-
step reaction involving maleimido-derivatized HSA-28 peptide
(Scheme 2).58 The coated dendrimer (HSA-28D) was purified
by centrifuge ultrafiltration until the filtrate did not show any
traces of starting materials (monitoring by TLC, followed by
vanillin staining).
For estimating the percentage of dendrimer coating, 6%
SDS PAGE was performed in TTS (Fig. 1A). As expected, the
coated nanoparticles appeared as a broad band at around 50
kDa, which corresponded to approximately 21 units of pep-
tide that was conjugated to the PAMAM. Based on this infor-
mation, the calculated percentage of the covering was approx-
imately 16%.
In addition, the GPC analysis of HSA-28D revealed a single
peak at 47 kDa, compared to the calibration curve. This peak
has a PDI value of 1.0029, calculated by MW/Mn, where MW
is the weight's average molecular weight and Mn is the num-
ber's average molecular weight. This indicates the purity of
HSA-28D, due to the clear homogeneity seen in the GPC anal-
ysis. This result is also well correlated with the result pro-
vided by the Dendritech (the company that produced the
PAMAM dendrimer, 5 generations with an ethylenediamine
core) MS results for a specific lot that was used by our team
(please see Fig. S1†). The provided spectra indicate that the
“naked” PAMAM has a major peak approximately at 26 kDa.
Finally, the UV spectra of both dendrimers, the “naked” and
HSA-28D, clearly show a significant difference (Fig. S2†) in the
intensity of absorption in the UV range. HSA-28 has two aro-
matic rings (phenylalanine and tyrosine), which are responsible
Estimation of the dendrimer coating by HAS-28D and its
peptidomimetics
Polyacryl gel electrophoresis (PAGE). For estimating the
percentage of dendrimer coating the gel electrophoresis of
the peptide or peptidomimetics conjugated to dendrimers
was determined. In addition, as a negative control, we evalu-
ated the electrophoresis of PAMAM that was coated only by
3-maleimidopropionic acid and PAMAM alone. All samples
were loaded to glycine using 6% SDS PAGE gel and run
against Tris-Tricine-SDS (TTS) running buffer and stained
using silver stain.
Gel permeation chromatography (GPC). The molecular
weight and polydispersity index (PDI) were determined using
gel permeation chromatography (GPC) consisting of a Waters
Spectra Series P100 isocratic HPLC pump with an ERMA
ERC-7510 refractive index detector and a Rheodyne (Cotati,
CA) injection valve with a 20 μL loop (Waters, MA). The sam-
ples were eluted with super-pure HPLC water through a linear
BioSep SEC-s-3000 column (Phenomenex) at a flow rate of
1 mL min−1. The molecular weight was determined relative to
polyIJethylene glycol) standards (Polymer Standards Service-
USA, Silver Spring, MD, USA) with a molecular weight range
of 100–450 000 Da, human serum albumin (67 kDa, Sigma
Aldrich), and bovine plasma fibrinogen (340 kDa, Sigma
Aldrich), using Clarity chromatography software.
UV spectroscopy
An Agilent Cary 300 UV-vis spectrophotometer with a slit of 4
nm and a scan speed of 400 nm min−1 was used for the mea-
surements. The absorbance measurements were performed at
pH 7.4 by keeping the concentration of the samples at 0.175
Scheme 1 Chemical structure of modified HSA-28 peptide.
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Scheme 2 General procedure for one-step preparation of HSA-28D.
for the absorption in the aromatic wavelength range. Obvi-
ously, the “naked” dendrimer does not have aromatic rings;
hence, the absorption level in the UV range is very low com-
pared with HSA-28D. Based on the results obtained from all
these analytical techniques, the coupling of the peptide to the
“naked” dendrimer should be considered as validated.
Cells were incubated with HSA-28D and then exposed to
Tg or GO. Fig. 1C shows that the compound has excellent
cytoprotective characteristics by assisting cells in overcoming
both types of stress conditions.
HSA-28D's effect on C-peptide, glucagon, and the PDX
levels was investigated afterward. Determining the accumula-
tion of C-peptide in β-cells is a common method for indi-
rectly evaluating insulin production.63,64 This small peptide
links the two chains of insulin and assists in hormone matu-
ration. Direct measurement of insulin in vitro can be prob-
lematic because, in addition to INS-1E-produced insulin, even
the smallest amount of insulin from the growth medium
masks the actual results. In contrast, C-peptide in different
species is different, and C-peptide in FCS does not cross-
react with the C-peptide from rat INS-1E cells. The ratio be-
tween C-peptide and insulin levels is 1 : 1 in secretory gran-
ules and both are secreted simultaneously.65 INS-1E cells
were treated with HSA-28D. Slides were obtained as described
in “Methods” and the plasma membrane and nuclei were de-
noted by red and blue, respectively. Thereafter, slides were
exposed to a primary antibody against C-peptide (green) and
were evaluated by confocal microscopy. Cells treated with
HSA-28P expressed surprisingly more C-peptide (approxi-
mately 6-fold) compared with untreated cells. (Fig. 1D). We
normalized the intensity of the C-peptide level by the inten-
sity of the membrane signal. Peptide alone and naked
PAMAM did not have any effect on C-peptide accumulation
(data not shown).
In vitro evaluation of HSA-28D biological activity
The possible effect of HSA-28D on the rate of cell prolifera-
tion was investigated using the INS-1E cell line. Fig. 1B shows
that HSA-28D enhanced the rate of cell proliferation (around
40%) compared with untreated cells. The highest stimulatory
effect on the proliferation rate was obtained after 72 h of in-
cubation with HSA-28D (the concentration of the conjugated
HSA-28D with PAMAM HSA-28 was 3 μg ml−1). Interestingly,
the non-coated PAMAM dendrimer exhibited a highly toxic ef-
fect toward the cells (data not shown).
The next goal was to determine the possible HSA-28D cell
protective effect in vitro. To this end, two apoptosis-inducing
systems were used: ER and oxidative stress. Both types of
stress are involved in diabetes-related β-cell death.59,60 To
mimic ER stress in vitro, thapsigargin (Tg) was used.61 The
toxin dramatically elevates the intracellular levels of calcium
and depletes ER calcium stores, thereby inducing ER stress.
A glucose oxidase (GO)/glucose system was used as an in-
ducer of oxidative stress.62 We preferred to use this more
controllable system to generate H2O2 than to directly add it
to the medium at a high dose. The system continuously pro-
duces constant concentrations of H2O2. GO catalyzes the
transfer of two oxygen electrons to H2O2, using reducing
equivalents from the oxidation of glucose to glycolic acid.
Glucose is present in the medium at a high concentration (to
mimic the hyperglycemic conditions in vivo). Hence, the ROS
generation never runs out because of the accessibility of the
substrate to the enzymatic reaction.
It is a known phenomenon that an increased proliferation
rate of β-cell lines, such as INS-1E, leads to reduced differen-
tiation levels.66 Such a transformation might be manifested
by abnormally elevated levels that are not related to β-cell
hormones, for example, the production and secretion of the
insulin antagonist, glucagon.67 The basal levels of glucagon,
which is still produced and secreted by INS-1E, might rise be-
cause of losing the phenotype; this is characterized by high
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Fig. 1 (A) 6% SDS PAGE of HSA-28D. Samples (purified HSA-28D and loading buffer, 40 μl both) were boiled for 5 min, loaded to SDS PAGE,
and run against TTS buffer. Bands were visualized using silver stain. (B) The effect of HSA-28D on the proliferation rate of INS-1E cells. INS-1E
cells were seeded in 24-well plates and incubated for 72 h with the medium supplemented with HSA-28D ([HSA-28]-3 μg ml−1). Thereafter, the
cells were detached by trypsin, stained using trypan blue, and counted. The results are presented as a percentage compared to non-treated
cells. *p ≤ 0.05, n = 6. MEAN SE. (C) The effect of HSA-28D on cell viability under oxidative and ER stress conditions. INS-1E cells were incu-
bated for 72 h with medium supplemented with HSA-28D ([HSA-28]-3 μg ml−1). After incubation, 50 mU ml−1 glucose oxidase (GO) was added
for an additional 1.15 h and 0.113 μg ml−1 thapsigargin (Tg) was added for an additional 24 hours. Upon completion of both experiments, a stan-
dard MTT assay was performed. The cell viability of both experiments is presented as a percentage compared to non-treated cells. *,#p < 0.05,
n = 3. MEAN SE. (D) The effect of HSA-28D on C-peptide intracellular levels. Experiments were conducted on INS-1E cells that were seeded
on coverslips in 6-well plates. INS-1E cells were incubated for 72 h with HSA-28D ([HSA-28]-3 μg ml−1). Cells were fixed with formaldehyde,
permeabilized, and exposed to anti-C-peptide or anti-glucagon (E) or anti-Pdx1 (F) antibodies, followed by secondary antibody (green). Thereaf-
ter, the cell membranes were exposed to Alexa Fluor 633 Phalloidin (red) and the nuclei were stained with DAPI (blue). Fluorescent signals were
visualized with a confocal-Zeiss microscope. The experiment was run several times using the triplicate method, n = 6. Representative pictures
are shown. The effect of HSA-28D on GSIS (G) and insulin content (H) under low and high glucose concentrations. The INS-1E cells (blue col-
umns) were grown in 24-well plates and treated with HSA-28D ([HSA-28]-3 μg ml−1) (red columns). After 72 h, the GSIS was evaluated in the
presence of 3.3 mM (low) or 16.7 mM (high) glucose. H. Measurement of insulin content. The ELISA assay was performed for INS-1E lysates.
*p < 0.05, n = 3. MEAN SE.
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levels of differentiated β-cells.68 Thus, the extent of intracellu-
lar accumulation of glucagon was investigated in INS-1E cells;
as shown in Fig. 1E, HSA-28D did not lead to glucagon
accumulation.
use of peptides is very restricted because of their substantial
problematic pharmacokinetic and pharmacodynamic proper-
ties, such as rapid proteolytic degradation, non-specific bio-
logical activity due to binding to “off target” macromolecules,
low oral and even systemic bioavailability, the intense rate of
elimination in the liver, and finally, also the high possibility
of activating the immune system. To circumvent these chal-
lenging issues, several modern pharmacological approaches
are in use: smart oral pharmaceutical formulations, slow re-
lease techniques, conjugates with organic molecules (usually
polymers) that are targeted to specific tissues, peptides and
peptidomimetics.71–73 Peptidomimetics are small organic
compounds that can mimic the peptide arrangement
(pharmacophores) by replacing peptide functional groups
and amide bonds by very similar structural domains and
moieties. The 3D structures of peptides are closely
reconstructed by peptidomimetics with the ability to interact
and affect the biological targets. In contrast, the pharmaco-
logical and pharmacokinetic disadvantages of peptides are
not present, due to changes in the covalent structures of
these molecules.
To further investigate the biological activity of HSA-28D, the
possible effect of the compound on Pdx1 (pancreatic and duo-
denal homeobox 1) production was tested. Pdx1 is a transcrip-
tion factor needed for pancreatic development and β-cell matu-
ration.69 Type 2 diabetes is characterized by the presence of a
subpopulation of Pdx1-deficient β-cells.70 The data presented
in Fig. 1F clearly show that HSA-28D significantly elevated the
basal level of the transcription factor (around 12 times).
Finally, the effect of HSA-28D on glucose-stimulated insu-
lin secretion (GSIS) and insulin content in INS-1E cells was
evaluated. HSA-28D significantly (around 2-fold) enhanced
GSIS in comparison with non-treated cells (Fig. 1G). The total
insulin content was also profoundly elevated upon treatment
with HSA-28D in INS-1E cells, which were maintained at high
glucose concentrations (Fig. 1H).
Pharmacophore-based design and synthesis of HSA-28
peptide mimetics
Usually, computer modeling is used for designing a set of
peptidomimetics, based on the structure of a known biologi-
cally active peptide. However, mimicking the structure of a
long peptide such as HSA-28 (9 amino acids) was very
Peptides are one of the most abundant biomolecules in the
physiology of any organism. However, the pharmacological
Fig. 2 (A). Pharmacophore design. Structure-based pharmacophore model generated with LigandScout from the structure of HSA-28 and NX-1.
Pharmacophore features are color-coded: red represents negative charge features, yellow represents the hydrophobic feature, and gray represents
excluded volumes. Excluded volumes are points in space occupied by protein atoms, which represent steric hindrances in the binding site. Such
points cannot be occupied by ligand atoms. (B). The structures of the synthesized HSA-28 mimetics.
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challenging. Thus, only side chains critical for the binding of
glutamic, phenylalanine, and leucine amino acids were mim-
icked, as shown in Fig. 2A. The pharmacophore model was
created, based on the structure of the complex between HSA-
28 and NX-1 using LigandScout 4.0 software.74
markers, the average size (50 kDa) and the covering efficiency
(around 16%) of the modified PAMAM dendrimers were iden-
tified (Fig. 1A).
It was shown that adult pancreatic β-cells still have a high
ability to proliferate in vivo and that this self-duplication pro-
cess is still important together with the generation of new
β-cells from stem cells.87,88 The observed ability of HSA-28D
to increase the rate of INS-1E cell proliferation (see Fig. 1B)
may be very important; further work is necessary to deter-
mine if the proliferative effect seen here with the INS-1 β-cell
line would be seen also with primary β-cells. Prior results,
however, demonstrate that NL-2 strongly influences the β-cell
number in vivo.29 Interestingly, cells treated with the com-
pound were almost 50% more proliferative.
Ten compounds (Fig. 2B) that fitted the pharmacophore
model were synthesized: tert-butyl 3-((2,2-diphenylethyl)-
amino)propanoate
(1);
tert-butyl
3-(benzhydrylamino)-
propanoate (2); tert-butyl 3-(phenethylamino)propanoate (3);
tert-butyl 3-(benzylamino)propanoate (4); tert-butyl 3-((2-
methoxy-1-(naphthalen-2-yl)-2-oxoethyl)amino)propanoate (5);
tert-butyl 3-((2-methoxy-1-(naphthalen-1-yl)-2-oxoethyl)amino)-
propanoate (6); methyl 4-(((3-(tert-butoxy)-3-oxopropyl)amino)-
methyl)benzoate (7); tert-butyl 3-((2-(3,4-dihydroisoquinolin-
2IJ1H)-yl)ethyl)amino)propanoate
(8);
tert-butyl
3-((2-(3-
The most important observation here was related to the
fact that HSA-28D was able to protect INS-1E cells against
oxidative and ER stress, as shown in Fig. 1C. The β-cells are
very sensitive towards oxidative stress because of their rela-
tively weak molecular antioxidative defense system.89 One of
the major pathophysiological causes of diabetes is oxidative
stress.90 Lack of effective antioxidant protection triggers
β-cell dysfunction in the early stage of diabetes followed by
cell apoptosis in the progressive stages of the disease.91 The
combination of a low replication rate of the β-cells and the
absence of a sufficient antioxidative response leads to cell
death during diabetes.92 Classical antioxidant therapy was
tested in many clinical antidiabetic trials.93 Unfortunately,
direct antioxidative stress protection of β-cells and mainte-
nance of β-cell mass were never achieved.94 In addition to
its own direct toxicity, oxidative stress causes total cellular
dysfunction, which appears as cytosolic, mitochondrial, per-
oxisomal, or endoplasmic reticulum stress.94 The latter plays
a crucial role in diabetic β-cell apoptosis.95 In general, ER
stress is caused by a disproportion between the rate of
proper protein folding and misfolding.96 Misfolded proteins
are unable to undergo degradation in proteasome or lyso-
some and accumulate in the ER lumen. In order to restore
ER functionality, cells activate an emergency chain of ac-
tions termed unfolded protein response (UPR). There is sub-
stantial evidence for the presence of ER stress in diabetic
β-cells.97 Chronic ER stress leads to apoptosis.98 ER stress
was also targeted as a possible site for a pharmacological
intervention to protect β-cells under diabetic condi-
tions.99,100 The use of chemical and more specific pharma-
ceutical chaperones, prevention of eIF2B inhibition, calcium
leak blockers, stimulators of ER folding capacities, and
other groups of compounds was proposed.95 However, using
this approach, substantial progress in β-cell protection has
not yet been achieved.
(isobutylcarbamoyl)-3,4-dihydroisoquinolin-2IJ1H)-yl)ethyl)-
amino)propanoate (9, novel compound); and tert-butyl 3-((2-
amino-2-oxoethyl)amino)propanoate (10).
All compounds were obtained by a reaction between com-
mercially available or synthesized in-house aromatic amines
and tert-butyl acrylate (all synthetic procedures and analytical
characterization studies are described in detail in the ESI†).
All the synthesized final compounds were coupled with
3-maleimidopropionic acid and treated with TFA to remove
the tert-butyl group. The obtained molecules were used for
further conjugation to PAMAM NPs. The percentage of NPs
used for covering was evaluated similarly to HSA-28D (the re-
sults are presented in the ESI,† Fig. S3).
The possible effect of all INS-1E cells being coated by
peptidomimetic dendrimers was tested according to their
proliferation rate. However, none of the compounds signifi-
cantly affected the rate of cell proliferation (Fig. S4†).
Discussion
PAMAM dendrimers bring a unique possibility to create tree-
like homogenous structures around one central core. The
presence of free primary amine groups on the PAMAM sur-
face allows a large spectrum of conjugational reactions. Re-
garding the attachment of biogenic peptides, it was shown
that non-direct conjugation of a peptide to PAMAM via a PEG
linker or in combination with an additional short linker is
the most useful approach.75–79 Different linkers besides PEG
were also used for the biogenic peptide conjugation to
PAMAM.80–84 In addition, direct covering of PAMAM
dendrimers by peptide was also reported.85 However, to our
knowledge we are the first to show the successful binding of
a peptide to PAMAM using the 2-iminothiolane/malonic anhy-
dride system. Indeed, A. Harada reported the conjugation of
PAMAM via a 2-iminothiolane linker to polylysine; however,
the S–S bond was used for coupling by the authors and not
the peptide-3-(alkylthio)pyrrolidine-2,5-dione moiety, as we
used.86 To prove that HSA-28 conjugates with the PAMAM
HSA-28D displayed a significant protective effect in β-cells
that were maintained under both main apoptotic triggers (ER
and oxidative stress). In light of the failure of conventional
anti-oxidative stress and anti-ER stress therapeutic ap-
proaches for diabetes, such a dual effect might play a very
important role in the future development of NL-2 mimetic-
based β-cells for protective therapy.
dendrimers to
a sufficient extent, we applied a non-
traditional method for the conjugation chemistry technique:
SDS-PAGE. Based on the positions of the molecular weight
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It is known that non-covered PAMAM dendrimers (espe-
cially cationic ones) are extremely toxic for cells.101–104
According to our experience, INS-1E cells died at a 34.6 μM
concentration of a “naked” PAMAM dendrimer. This observa-
tion was in agreement with recently published articles about
the toxicity of PAMAMs.105,106 In addition, it is also known
that in general, high concentrations of positively charged
polyamines can destroy and damage the plasma membrane
and kill animal cells.107,108 However, by covering PAMAM
nanoparticles with peptides (even at 16%), positively charged
amines can be eliminated by creating a neutral amide bond.
Thus, covered PAMAM (even partially) was not toxic to the
cells in biologically active concentrations.
The test compound dramatically increased C-peptide (in-
sulin) accumulation in INS-1E cells (Fig. 1D). Cells incu-
bated with HSA-28D in the presence of 22.5 mM glucose for
72 h enhanced the intracellular insulin content almost
6-fold. These data support our hypothesis that mimicking
NL-2/NX interaction between β-cells leads to high levels of
maturation of those cells and not only to increased prolifer-
ation. The stability of the β-cell phenotype after treatment
with HSA-28D was proved by the absence of glucagon in the
cytosol (Fig. 1E). Moreover, the high maturation of the
treated cells was also indicated by the increased intracellu-
lar levels of PDX1. PDX1 is a master transcription factor
that tightly regulates pancreatic development, the β-cell pro-
liferation, and most importantly, sufficient structural and
functional maturation of β-cells.109 Normal expression of
PDX1 in β-cells blocks glucagon expression and selectively
promotes the expression of β-cell-specific genes.110 It is im-
portant to mention that T2DM is characterized not only by
apoptosis of the β-cells but also by dematuration of β-cells
and their transdifferentiation to different types of cells (usu-
ally to α-cells). PDX1-deficient β-cells are found in progres-
sive T2DM.111 Apoptosis, low rates of proliferation, and
transdifferentiation lead to a constant decline in β-cell mass
during the progression of T2D and these result in a con-
stant failure to generate enough new β-cells. It was very en-
couraging that under HSA-28D treatment, PDX1 levels in
INS-1E were significantly elevated (Fig. 1F), suggesting that
the increased insulin accumulation in treated cells is attrib-
uted to increased maturation.
by ELISA, the cell insulin content was also markedly high in
the treated HSA-28D cells (Fig. 1H) and these results were
correlated with the results that we obtained by fluorescence
microscopy (Fig. 1G). However, the difference was consider-
ably lower than that obtained fluorescently (6-fold in the
fluorescence method versus 2.1-fold in the colorimetric
method), which might be explained by the higher sensitivity
of fluorescence detection. Alternatively, the maturation of
insulin granules in HSA-28D treated cells might also explain
the difference.
To overcome possible low bioavailability and other
pharmacokinetically related disadvantages, our next step in
the project was to try to develop peptidomimetics based on
the HSA-28 structure. Such molecules (conjugated to
PAMAM), on the one hand, might mimic the 3D structure of
the native peptide, but on the other hand, non-peptide
bonds and non-natural amino acid moieties should create
more biologically stable molecules with identical or even
upgraded activity. Based on the in silico pharmacophore
model, feasible structures for the organic synthetic com-
pounds were created and fitted to the model. After evalua-
tion of the in silico binding score, the first ten molecules
were synthesized. We tried to mimic the three most impor-
tant interactions/natural amino acid moieties in HSA-28,
such as the branched lipophilic moiety of leucine, the
charged carboxylic acid of glutamic acid, and finally, the
phenyl moiety of phenylalanine.
Compound 9 is a novel peptidomimetic obtained in 7-step
synthesis with a linear yield of 24%. Such a molecule might
be used for general glutamic, phenylalanine, and leucine
amino acid mimetics in the development of mimetics abso-
lutely non-relevant to NL-2 peptidomimetics.
A proliferation assay was used for evaluating molecules
covered by ten peptidomimetic PAMAMs. Unfortunately, none
of the synthesized molecules had a positive effect on the INS-
1E proliferation rate (Fig. S4†). This disappointing result can
be explained by the fact that the peptide conformations cho-
sen for mimicking the peptide interactions in silico might not
be reproducible by peptidomimetics when they are conju-
gated to PAMAM. In addition, it is also possible that for suffi-
cient mimicking of NL-2 interactions with NXs, an entire
scaffold of nine amino acids is essential. In this case, the syn-
thesis of a nine amino acid peptidomimetic sequence can be
very challenging.
The most important test for the functionality of the ob-
served effects of the test compound on INS-1E was GSIS. As
shown in Fig. 1G, three days of administration of HSA-28D
led to a significant augmentation of the rate of insulin secre-
tion under physiological conditions (medium glucose level 13
mM). Interestingly, INS-1E cells treated with the test com-
pound slightly decreased basal insulin secretion. These re-
sults underscored two very important issues related to the
HSA-28D mode of action: the insulin secretion occurred only
as a physiological response to elevated glucose levels and not
by some non-specific leaking of insulin from damaged
plasma membranes. Second, large quantities of insulin that
accumulated during three days of exposing the cell to the test
compound were able to be secreted physiologically. Measured
In summary, the ability of the functionalized NPs reported
here to prevent β-cells's substantial apoptosis, an increase in
their mass, maturation, and most importantly, their ability to
secrete insulin in a glucose-dependent manner might be used
for developing novel β-cell protective therapeutic agents and
β-cell transplantation supporters, especially considering that
a PAMAM-based core for such NPs is biocompatible.
Conflicts of interest
The authors declare no conflict of interest.
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Acknowledgements
cell therapy in type 1 diabetes, Acta Diabetol., 2016, 53,
683–691.
This study was supported by a Bar-Ilan University new faculty
grant, a D-cure (Diabetes Care in Israel) Young Investigator
Award, and a NOFAR program (Israel Ministry of Industry) for
A. G. The Israel Science Foundation (application number
117/2014) provided a grant for A. G. and J.-P. L. In addition,
S. K. is thankful for the support of her work by the Wolf
Foundation. S. D. C. was supported by NIH/NIDDK grant
R01DK080971. G. C. is partially supported by the Israel Min-
istry of Science and Technology. This work was also funded
by the 7th Framework RTD European Project (FP7-530 NMP-
2010-LARGE-4 area) – Large Collaborative Projects – Project
531 SaveMe (grant agreement no. 263307) for J.-P. L. and
finally, by The Bar-University Rector's Grant for Interdisci-
plinary Research (2017) for A. G., J.-P. L. and H. S. We would
like to thank Steve Manch for the English editing of the
manuscript.
10 Y. Evron, C. K. Colton, B. Ludwig, G. C. Weir, B. Zimermann,
S. Maimon, T. Neufeld, N. Shalev, T. Goldman, A. Leon, K.
Yavriyants, N. Shabtay, T. Rozenshtein, D. Azarov, A. R.
DiIenno, A. Steffen, P. de Vos, S. R. Bornstein, U. Barkai and
A. Rotem, Long-term viability and function of transplanted
islets macroencapsulated at high density are achieved by
enhanced oxygen supply, Sci. Rep., 2018, 8, 6508.
11 P. Mondal, A. Prasad and K. Girdhar, Interventions to
improve beta-cell mass and function, Ann. Endocrinol.,
2017, 78, 469–477.
12 S. Tritschler, F. J. Theis, H. Lickert and A. Bottcher,
Systematic single-cell analysis provides new insights into
heterogeneity and plasticity of the pancreas, Mol. Metab.,
2017, 6, 974–990.
13 J. Shirakawa, D. F. De Jesus and R. N. Kulkarni, Exploring inter-
organ crosstalk to uncover mechanisms that regulate beta-cell
function and mass, Eur. J. Clin. Nutr., 2017, 71, 896–903.
14 M. Okamoto, T. Ishizaki and T. Kimura, Protective effect of
hydrogen sulfide on pancreatic beta-cells, Nitric Oxide,
2015, 46, 32–36.
15 J. S. Danobeitia, P. J. Chlebeck, I. Shokolenko, X. Ma, G.
Wilson and L. A. Fernandez, Novel Fusion Protein
Targeting Mitochondrial DNA Improves Pancreatic Islet
Functional Potency and Islet Transplantation Outcomes,
Cell Transplant., 2017, 26, 1742–1754.
16 J. J. Yin, Y. B. Li, Y. Wang, G. D. Liu, J. Wang, X. O. Zhu
and S. H. Pan, The role of autophagy in endoplasmic
reticulum stress-induced pancreatic beta cell death,
Autophagy, 2012, 8, 158–164.
References
1 A. Weisman, G. S. Fazli, A. Johns and G. L. Booth, Evolving
Trends in the Epidemiology, Risk Factors, and Prevention
of Type 2 Diabetes: A Review, Can. J. Cardiol., 2018, 34,
552–564.
2 W. D. Strain, S. V. Hope, A. Green, P. Kar, J. Valabhji and
A. J. Sinclair, Type 2 diabetes mellitus in older people: a
brief statement of key principles of modern day
management including the assessment of frailty. A national
collaborative stakeholder initiative, Diabet. Med., 2018, 35,
838–845.
3 J. R. Petrie, T. J. Guzik and R. M. Touyz, Diabetes,
Hypertension, and Cardiovascular Disease: Clinical Insights
and Vascular Mechanisms, Can. J. Cardiol., 2018, 34,
575–584.
4 H. Mulder and C. Ling, Mitochondrial dysfunction in
pancreatic beta-cells in Type 2 diabetes, Mol. Cell. Endo-
crinol., 2009, 297, 34–40.
5 E. U. Alejandro, B. Gregg, M. Blandino-Rosano, C. Cras-
Meneur and E. Bernal-Mizrachi, Natural history of beta-cell
adaptation and failure in type 2 diabetes, Mol. Aspects Med.,
2015, 42, 19–41.
6 C. Chen, C. M. Cohrs, J. Stertmann, R. Bozsak and S.
Speier, Human beta cell mass and function in diabetes:
Recent advances in knowledge and technologies to
understand disease pathogenesis, Mol. Metab., 2017, 6,
943–957.
17 S. Z. Safi, R. Qvist, G. Ong, H. Karimian, M. Imran and I.
Shah, Stimulation of beta-adrenergic receptors plays a pro-
tective role via increased expression of RAF-1 and PDX-1 in
hyperglycemic rat pancreatic islet (RIN-m5F) cells, Arch.
Med. Sci., 2017, 13, 470–480.
18 D. A. Cunha, M. Cito, P. O. Carlsson, J. M. Vanderwinden,
J. D. Molkentin, M. Bugliani, P. Marchetti, D. L. Eizirik and
M. Cnop, Thrombospondin 1 protects pancreatic beta-cells
from lipotoxicity via the PERK-NRF2 pathway, Cell Death
Differ., 2016, 23, 1995–2006.
19 J. Chen, S. T. Hui, F. M. Couto, I. N. Mungrue, D. B. Davis,
A. D. Attie, A. J. Lusis, R. A. Davis and A. Shalev,
Thioredoxin-interacting protein deficiency induces Akt/Bcl-
xL signaling and pancreatic beta-cell mass and protects
against diabetes, FASEB J., 2008, 22, 3581–3594.
20 A. Kaminitz, S. Ash and N. Askenasy, Neutralization Versus
Reinforcement of Proinflammatory Cytokines to Arrest
7 I. Song, C. Muller, J. Louw and L. Bouwens, Regulating the
beta cell mass as a strategy for type-2 diabetes treatment,
Curr. Drug Targets, 2015, 16, 516–524.
Autoimmunity in Type
1 Diabetes, Clin. Rev. Allergy
Immunol., 2017, 52, 460–472.
8 R. Scharfmann, M. Didiesheim, P. Richards, V. Chandra,
M. Oshima and O. Albagli, Mass production of functional
human pancreatic beta-cells: why and how?, Diabetes, Obes.
Metab., 2016, 18(Suppl 1), 128–136.
21 C. S. Hampe, Protective role of anti-idiotypic antibodies in
autoimmunity–lessons for type 1 diabetes, Autoimmunity,
2012, 45, 320–331.
22 A. Fierabracci, Peptide immunotherapies in Type
1
9 S. Pellegrini, E. Cantarelli, V. Sordi, R. Nano and L.
Piemonti, The state of the art of islet transplantation and
diabetes: lessons from animal models, Curr. Med. Chem.,
2011, 18, 577–586.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2018

View Article Online
MedChemComm
Research Article
23 N. S. Wilcox, J. Rui, M. Hebrok and K. C. Herold, Life and
death of beta cells in Type 1 diabetes: A comprehensive
review, J. Autoimmun., 2016, 71, 51–58.
37 Y. Kim, E. J. Park and D. H. Na, Recent progress in
dendrimer-based
nanomedicine
development,
Arch.
Pharmacal Res., 2018, 41, 571–582.
24 C. M. Hull, M. Peakman and T. I. M. Tree, Regulatory T cell
dysfunction in type 1 diabetes: what's broken and how can
we fix it?, Diabetologia, 2017, 60, 1839–1850.
38 B. Gomes, M. T. Augusto, M. R. Felicio, A. Hollmann, O. L.
Franco, S. Goncalves and N. C. Santos, Designing improved
active peptides for therapeutic approaches against
infectious diseases, Biotechnol. Adv., 2018, 36, 415–429.
39 R. Nuti, N. S. Goud, A. P. Saraswati, R. Alvala and M. Alvala,
Antimicrobial Peptides: A Promising Therapeutic Strategy
in Tackling Antimicrobial Resistance, Curr. Med. Chem.,
2017, 24, 4303–4314.
25 A. Itoh and W. M. Ridgway, Targeting innate immunity to
downmodulate adaptive immunity and reverse type
1
diabetes, ImmunoTargets Ther., 2017, 6, 31–38.
26 J. Ludvigsson, Therapies to Preserve beta-Cell Function in
Type 1 Diabetes, Drugs, 2016, 76, 169–185.
27 A. Munder, L. L. Israel, S. Kahremany, R. Ben-Shabat-
Binyamini, C. Zhang, M. Kolitz-Domb, O. Viskind, A.
Levine, H. Senderowitz, S. Chessler, J. P. Lellouche and A.
Gruzman, Mimicking Neuroligin-2 Functions in beta-Cells
by Functionalized Nanoparticles as a Novel Approach for
Antidiabetic Therapy, ACS Appl. Mater. Interfaces, 2017, 9,
1189–1206.
28 C. Zhang, A. T. Suckow and S. D. Chessler, Coculture
analysis of extracellular protein interactions affecting
insulin secretion by pancreatic beta cells, J. Visualized Exp.,
2013, e50365.
29 C. Zhang, A. T. Suckow and S. D. Chessler, Altered
pancreatic islet function and morphology in mice lacking
the Beta-cell surface protein neuroligin-2, PLoS One,
2013, 8, e65711.
30 A. T. Suckow, C. Zhang, S. Egodage, D. Comoletti, P. Taylor,
M. T. Miller, I. R. Sweet and S. D. Chessler, Transcellular
neuroligin-2 interactions enhance insulin secretion and are
integral to pancreatic beta cell function, J. Biol. Chem.,
2012, 287, 19816–19826.
31 A. T. Suckow, D. Comoletti, M. A. Waldrop, M. Mosedale, S.
Egodage, P. Taylor and S. D. Chessler, Expression of
neurexin, neuroligin, and their cytoplasmic binding
partners in the pancreatic beta-cells and the involvement of
neuroligin in insulin secretion, Endocrinology, 2008, 149,
6006–6017.
32 R. A. R. Villacis, J. S. Filho, B. Pina, R. B. Azevedo, A. Pic-
Taylor, J. F. Mazzeu and C. K. Grisolia, Integrated assessment
of toxic effects of maghemite (gamma-Fe2O3) nanoparticles
in zebrafish, Aquat. Toxicol., 2017, 191, 219–225.
33 G. Qualhato, T. L. Rocha, E. C. de Oliveira Lima, E. S. DM,
J. R. Cardoso, C. Koppe Grisolia and S. M. T. de Saboia-
Morais, Genotoxic and mutagenic assessment of iron oxide
(maghemite-gamma-Fe2O3) nanoparticle in the guppy
Poecilia reticulata, Chemosphere, 2017, 183, 305–314.
34 V. Dhakshinamoorthy, V. Manickam and E. Perumal,
Neurobehavioural Toxicity of Iron Oxide Nanoparticles in
Mice, Neurotoxic. Res., 2017, 32, 187–203.
35 D. Astruc, C. Deraedt, R. Djeda, C. Ornelas, X. Liu, A.
Rapakousiou, J. Ruiz, Y. Wang and Q. Wang, Dentromers, a
Family of Super Dendrimers with Specific Properties and
Applications, Molecules, 2018, 23, 966.
40 G. Trapani, C. Satriano and D. La Mendola, Peptides and
their Metal Complexes in Neurodegenerative Diseases: from
Structural Studies to Nanomedicine Prospects, Curr. Med.
Chem., 2018, 25, 715–747.
41 M. Wang, K. P. Rakesh, J. Leng, W. Y. Fang, L. Ravindar, D.
Channe Gowda and H. L. Qin, Amino acids/peptides
conjugated heterocycles: A tool for the recent development
of novel therapeutic agents, Bioorg. Chem., 2018, 76,
113–129.
42 H. Bruzzoni-Giovanelli, V. Alezra, N. Wolff, C. Z. Dong, P.
Tuffery and A. Rebollo, Interfering peptides targeting
protein-protein interactions: the next generation of drugs?,
Drug Discovery Today, 2018, 23, 272–285.
43 W. Shen and T. Matsui, Current knowledge of intestinal
absorption of bioactive peptides, Food Funct., 2017, 8,
4306–4314.
44 J. Richard, Challenges in oral peptide delivery: lessons
learnt from the clinic and future prospects, Ther. Delivery,
2017, 8, 663–684.
45 T. Mavromoustakos, S. Durdagi, C. Koukoulitsa, M. Simcic,
M. G. Papadopoulos, M. Hodoscek and S. G. Grdadolnik,
Strategies in the rational drug design, Curr. Med. Chem.,
2011, 18, 2517–2530.
46 A. Mizuno, K. Matsui and S. Shuto, From Peptides to
Peptidomimetics:
Features of Cyclopropane,
14394–14409.
A
Strategy Based on the Structural
Chemistry, 2017, 23,
47 N. Qvit, S. J. S. Rubin, T. J. Urban, D. Mochly-Rosen and
E. R. Gross, Peptidomimetic therapeutics: scientific
approaches and opportunities, Drug Discovery Today,
2017, 22, 454–462.
48 M. A. Blaskovich, Unusual Amino Acids in Medicinal
Chemistry, J. Med. Chem., 2016, 59, 10807–10836.
49 P. Zer-Aviv, M. Shubely, Y. Moskovits, O. Viskind, A. Albeck,
D. Vertommen, S. Ruthstein, M. Shokhen and A. Gruzman,
A new oxopiperazin-based peptidomimetic molecule in-
hibits prostatic acid phosphatase secretion and induces
prostate cancer cells apoptosis, ChemistrySelect, 2016, 1,
4658–4667.
50 R. Behrendt, P. White and J. Offer, Advances in Fmoc solid-
phase peptide synthesis, J. Pept. Sci., 2016, 22, 4–27.
51 L. Pasternak, E. Meltzer-Mats, G. Babai-Shani, G. Cohen, O.
Viskind, J. Eckel, E. Cerasi, S. Sasson and A. Gruzman,
Benzothiazole derivatives augment glucose uptake in
skeletal muscle cells and stimulate insulin secretion from
36 J. Li, H. Liang, J. Liu and Z. Wang, Poly (amidoamine)
(PAMAM) dendrimer mediated delivery of drug and pDNA/
siRNA for cancer therapy, Int. J. Pharm., 2018, 546, 215–225.
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View Article Online
Research Article
pancreatic beta-cells via AMPK activation, Chem. Commun.,
MedChemComm
amino)phenyl)-5-oxopyrrolidine-3-carboxylic acid) as a novel
Nrf2 enhancer, ChemPlusChem, 2018, 83, 320–333.
63 A. Castell-Auvi, L. Cedo, V. Pallares, M. Blay, M. Pinent and
A. Ardevol, Grape seed procyanidins improve beta-cell func-
tionality under lipotoxic conditions due to their lipid-
lowering effect, J. Nutr. Biochem., 2013, 24, 948–953.
64 M. Fukaya, C. A. Brorsson, K. Meyerovich, L. Catrysse, D.
Delaroche, E. C. Vanzela, F. Ortis, R. Beyaert, L. B. Nielsen,
M. L. Andersen, H. B. Mortensen, F. Pociot, G. van Loo, J.
Storling and A. K. Cardozo, A20 Inhibits beta-Cell Apoptosis
by Multiple Mechanisms and Predicts Residual beta-Cell
Function in Type 1 Diabetes, Mol. Endocrinol., 2016, 30,
48–61.
2014, 50, 11222–11225.
52 R. Shapira, S. Rudnick, B. Daniel, O. Viskind, V. Aisha, M.
Richman, K. R. Ayasolla, A. Perelman, J. H. Chill, A.
Gruzman and S. Rahimipour, Multifunctional cyclic D,L-
alpha-peptide architectures stimulate non-insulin depen-
dent glucose uptake in skeletal muscle cells and protect
them against oxidative stress, J. Med. Chem., 2013, 56,
6709–6718.
53 V. Rosengren, H. Johansson, J. Lehtio, L. Fransson, A.
Sjoholm and H. Ortsater, Thapsigargin down-regulates pro-
tein levels of GRP78/BiP in INS-1E cells, J. Cell. Biochem.,
2012, 113, 1635–1644.
54 G. Cohen, O. Shamni, Y. Avrahami, O. Cohen, E. C. Broner,
N. Filippov-Levy, C. Chatgilialoglu, C. Ferreri, N. Kaiser and
S. Sasson, Beta cell response to nutrient overload involves
phospholipid remodelling and lipid peroxidation,
Diabetologia, 2015, 58, 1333–1343.
55 M. Muchtaridi, H. N. Syahidah, A. Subarnas, M. Yusuf,
S. D. Bryant and T. Langer, Molecular Docking and 3D-
Pharmacophore Modeling to Study the Interactions of
Chalcone Derivatives with Estrogen Receptor Alpha, Phar-
maceuticals, 2017, 10, 81.
65 A. H. Rubenstein, D. F. Steiner, D. L. Horwitz, M. E. Mako,
M. B. Block, J. I. Starr, H. Kuzuya and F. Melani, Clinical
significance of circulating proinsulin and C-peptide, Recent
Prog. Horm. Res., 1976, 33, 435–475.
66 M. Vincent, Y. Guz, M. Rozenberg, G. Webb, M. Furuta, D.
Steiner and G. Teitelman, Abrogation of protein convertase
2 activity results in delayed islet cell differentiation and
maturation, increased alpha-cell proliferation, and islet
neogenesis, Endocrinology, 2003, 144, 4061–4069.
67 J. C. Schisler, P. B. Jensen, D. G. Taylor, T. C. Becker, F. K.
Knop, S. Takekawa, M. German, G. C. Weir, D. Lu, R. G.
Mirmira and C. B. Newgard, The Nkx6.1 homeodomain
transcription factor suppresses glucagon expression and
regulates glucose-stimulated insulin secretion in islet beta
cells, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 7297–7302.
68 L. C. Bollheimer, C. E. Wrede, F. Rockmann, I. Ottinger, J.
Scholmerich and R. Buettner, Glucagon production of the
rat insulinoma cell line INS-1-A quantitative comparison
with primary rat pancreatic islets, Biochem. Biophys. Res.
Commun., 2005, 330, 327–332.
69 K. A. D'Amour, A. G. Bang, S. Eliazer, O. G. Kelly, A. D.
Agulnick, N. G. Smart, M. A. Moorman, E. Kroon, M. K.
Carpenter and E. E. Baetge, Production of pancreatic
hormone-expressing endocrine cells from human embry-
onic stem cells, Nat. Biotechnol., 2006, 24, 1392–1401.
70 K. Fujimoto and K. S. Polonsky, Pdx1 and other factors that
regulate pancreatic beta-cell survival, Diabetes, Obes.
Metab., 2009, 11(Suppl 4), 30–37.
56 P. Ingallinella, D. Fattori, S. Altamura, C. Steinkuhler, U.
Koch, D. Cicero, R. Bazzo, R. Cortese, E. Bianchi and A.
Pessi, Prime site binding inhibitors of a serine protease:
NS3/4A of hepatitis
C virus, Biochemistry, 2002, 41,
5483–5492.
57 P. Ruzza, A. Calderan, A. Donella-Deana, B. Biondi, L.
Cesaro, A. Osler, S. Elardo, A. Guiotto, L. A. Pinna and G.
Borin, Conformational constraints of tyrosine in protein
tyrosine kinase substrates: Information about preferred
bioactive side-chain orientation, Biopolymers, 2003, 71,
478–488.
58 P. Chaumet-Riffaud, I. Martinez-Duncker, A. L. Marty, C.
Richard, A. Prigent, F. Moati, L. Sarda-Mantel, D.
Scherman, M. Bessodes and N. Mignet, Synthesis and
application of lactosylated, 99mTc chelating albumin for
measurement of liver function, Bioconjugate Chem.,
2010, 21, 589–596.
59 J. Wang, X. Yang and J. Zhang, Bridges between
mitochondrial oxidative stress, ER stress and mTOR
signaling in pancreatic beta cells, Cell. Signalling, 2016, 28,
1099–1104.
60 S. Z. Hasnain, J. B. Prins and M. A. McGuckin, Oxidative
and endoplasmic reticulum stress in beta-cell dysfunction
in diabetes, J. Mol. Endocrinol., 2016, 56, R33–R54.
61 I. Yoshida, A. Monji, K. Tashiro, K. Nakamura, R. Inoue
and S. Kanba, Depletion of intracellular Ca2+ store itself
may be a major factor in thapsigargin-induced ER stress
and apoptosis in PC12 cells, Neurochem. Int., 2006, 48,
696–702.
71 A. M. Wagner, M. P. Gran and N. A. Peppas, Designing the
new generation of intelligent biocompatible carriers for
protein and peptide delivery, Acta Pharm. Sin. B, 2018, 8,
147–164.
72 P. Batista, P. M. Castro, A. R. Madureira, B. Sarmento and
M. Pintado, Recent insights in the use of nanocarriers for
the oral delivery of bioactive proteins and peptides,
Peptides, 2018, 101, 112–123.
73 S. H. Wang and J. Yu, Structure-based design for binding
peptides in anti-cancer therapy, Biomaterials, 2018, 156,
1–15.
62 S. Kahremany, I. Babaev, P. Hasin, T. Tamir, T. Ben-Zur, G.
Cohen, Z. Jiang, S. Weintraub, D. Offen, S. Rahimipour, B.
Major, H. Senderowitz and A. Gruzman, Computer-aided
design and synthesis of (1-(4-((3,4-dihydroxybenzylidene)-
74 G.
Wolber
and
T.
Langer,
LigandScout:
3-D
pharmacophores derived from protein-bound ligands and
their use as virtual screening filters, J. Chem. Inf. Model.,
2005, 45, 160–169.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2018

View Article Online
MedChemComm
Research Article
75 S. V. K. Rompicharla, P. Kumari, B. Ghosh and S. Biswas,
Octa-arginine modified polyIJamidoamine) dendrimers for
improved delivery and cytotoxic effect of paclitaxel in
cancer, Artif. Cells, Nanomed., Biotechnol., 2018, 1–13.
76 P. Ma, J. Chen, X. Bi, Z. Li, X. Gao, H. Li, H. Zhu, Y. Huang,
J. Qi and Y. Zhang, Overcoming Multidrug Resistance
through the GLUT1-Mediated and Enzyme-Triggered Mito-
chondrial Targeting Conjugate with Redox-Sensitive Pacli-
taxel Release, ACS Appl. Mater. Interfaces, 2018, 10,
12351–12363.
87 X. Xu, J. D'Hoker, G. Stange, S. Bonne, N. De Leu, X. Xiao,
M. Van de Casteele, G. Mellitzer, Z. Ling, D. Pipeleers, L.
Bouwens, R. Scharfmann, G. Gradwohl and H. Heimberg,
Beta cells can be generated from endogenous progenitors
in injured adult mouse pancreas, Cell, 2008, 132, 197–207.
88 Y. Dor, J. Brown, O. I. Martinez and D. A. Melton, Adult
pancreatic beta-cells are formed by self-duplication rather
than stem-cell differentiation, Nature, 2004, 429, 41–46.
89 S. Lenzen, Oxidative stress: the vulnerable beta-cell,
Biochem. Soc. Trans., 2008, 36, 343–347.
77 Q. Hu, Q. Chen, X. Yan, B. Ding, D. Chen and L. Cheng,
Chondrocyte affinity peptide modified PAMAM conjugate
as a nanoplatform for targeting and retention in cartilage,
Nanomedicine, 2018, 13, 749–767.
90 C. M. O. Volpe, P. H. Villar-Delfino, P. M. F. Dos Anjos and
J. A. Nogueira-Machado, Cellular death, reactive oxygen
species (ROS) and diabetic complications, Cell Death Dis.,
2018, 9, 119.
78 Y. Wang, L. Wang, G. Chen and S. Gong, Carboplatin-
Complexed and cRGD-Conjugated Unimolecular Nano-
particles for Targeted Ovarian Cancer Therapy, Macromol.
Biosci., 2017, 17, 1600292.
91 M. Tiedge, S. Lortz, J. Drinkgern and S. Lenzen, Relation
between antioxidant enzyme gene expression and
antioxidative defense status of insulin-producing cells, Dia-
betes, 1997, 46, 1733–1742.
79 Z. Chen, F. Lian, X. Wang, Y. Chen and N. Tang, Arginine-
glycine-aspartic acid-polyethylene glycol-polyamidoamine
dendrimer conjugate improves liver-cell aggregation and
function in 3-D spheroid culture, Int. J. Nanomed., 2016, 11,
4247–4259.
80 E. Orocio-Rodriguez, G. Ferro-Flores, C. L. Santos-Cuevas,
M. Ramirez Fde, B. E. Ocampo-Garcia, E. Azorin-Vega and
F. M. Sanchez-Garcia, Two Novel Nanosized Radiolabeled
Analogues of Somatostatin for Neuroendocrine Tumor
Imaging, J. Nanosci. Nanotechnol., 2015, 15, 4159–4169.
81 A. Ghai, B. Singh, P. Panwar Hazari, M. K. Schultz, A.
Parmar, P. Kumar, S. Sharma, D. Dhawan and A. Kumar
Mishra, Radiolabeling optimization and characterization of
(68)Ga labeled DOTA-polyamido-amine dendrimer conju-
gate - Animal biodistribution and PET imaging results,
Appl. Radiat. Isot., 2015, 105, 40–46.
82 S. P. Kambhampati, M. K. Mishra, P. Mastorakos, Y. Oh,
G. A. Lutty and R. M. Kannan, Intracellular delivery of
dendrimer triamcinolone acetonide conjugates into
microglial and human retinal pigment epithelial cells, Eur.
J. Pharm. Biopharm., 2015, 95, 239–249.
83 A. Satsangi, S. S. Roy, R. K. Satsangi, R. K. Vadlamudi and
J. L. Ong, Design of a paclitaxel prodrug conjugate for
active targeting of an enzyme upregulated in breast cancer
cells, Mol. Pharmaceutics, 2014, 11, 1906–1918.
92 W. Wang, C. Liu, M. Jimenez-Gonzalez, W. J. Song and
M. A. Hussain, The undoing and redoing of the diabetic
beta-cell, J. Diabetes Complications, 2017, 31, 912–917.
93 R. Rahimi, S. Nikfar, B. Larijani and M. Abdollahi, A review
on the role of antioxidants in the management of diabetes
and its complications, Biomed. Pharmacother., 2005, 59,
365–373.
94 S. Lenzen, Chemistry and biology of reactive species with
special reference to the antioxidative defence status in
pancreatic beta-cells, Biochim. Biophys. Acta, 2017, 1861,
1929–1942.
95 M. Cnop, S. Toivonen, M. Igoillo-Esteve and P. Salpea,
Endoplasmic
reticulum
stress
and
eIF2alpha
phosphorylation: The Achilles heel of pancreatic beta cells,
Mol. Metab., 2017, 6, 1024–1039.
96 R. Y. Hampton, ER stress response: getting the UPR hand
on misfolded proteins, Curr. Biol., 2000, 10, R518–521.
97 D. L. Eizirik, A. K. Cardozo and M. Cnop, The role for
endoplasmic reticulum stress in diabetes mellitus, Endocr.
Rev., 2008, 29, 42–61.
98 D. T. Rutkowski and R. J. Kaufman, A trip to the ER: coping
with stress, Trends Cell Biol., 2004, 14, 20–28.
99 A. L. Clark and F. Urano, Endoplasmic reticulum stress in
beta cells and autoimmune diabetes, Curr. Opin. Immunol.,
2016, 43, 60–66.
84 L. Y. Jiang, B. Lv and Y. Luo, The effects of an RGD-PAMAM
dendrimer conjugate in 3D spheroid culture on cell prolif-
eration, expression and aggregation, Biomaterials, 2013, 34,
2665–2673.
100 C. Evans-Molina, M. Hatanaka and R. G. Mirmira, Lost in
translation: endoplasmic reticulum stress and the decline
of beta-cell health in diabetes mellitus, Diabetes, Obes.
Metab., 2013, 15(Suppl 3), 159–169.
85 S. L. Mekuria and H. C. Tsai, Preparation of self-assembled
core-shell nano structure of conjugated generation 4.5 poly
(amidoamine) dendrimer and monoclonal Anti-IL-6 antibody
as bioimaging probe, Colloids Surf., B, 2015, 135, 253–260.
86 A. Harada, R. Matsuki, S. Ichimura, E. Yuba and K. Kono,
Intracellular environment-responsive stabilization of poly-
mer vesicles formed from head-tail type polycations com-
posed of a polyamidoamine dendron and polyIJL-lysine),
Molecules, 2013, 18, 12168–12179.
101 R. Duncan and L. Izzo, Dendrimer biocompatibility and
toxicity, Adv. Drug Delivery Rev., 2005, 57, 2215–2237.
102 F. P. Seib, A. T. Jones and R. Duncan, Comparison of the
endocytic properties of linear and branched PEIs, and
cationic PAMAM dendrimers in B16f10 melanoma cells,
J. Controlled Release, 2007, 117, 291–300.
103 W. Lesniak, A. U. Bielinska, K. Sun, K. W. Janczak, X. Shi,
J. R. Baker, Jr and L. P. Balogh, Silver/dendrimer
nanocomposites as biomarkers: fabrication, characterization,
This journal is © The Royal Society of Chemistry 2018
Med. Chem. Commun.

View Article Online
Research Article
in vitro toxicity, and intracellular detection, Nano Lett.,
MedChemComm
skin tumorigenesis by stimulating the recruitment of bulge
stem cells but not via toxic polyamine catabolic metabolites,
Amino Acids, 2014, 46, 543–552.
2005, 5, 2123–2130.
104 J. F. Kukowska-Latallo, K. A. Candido, Z. Cao, S. S. Nigavekar,
I. J. Majoros, T. P. Thomas, L. P. Balogh, M. K. Khan and
J. R. Baker, Jr, Nanoparticle targeting of anticancer drug
improves therapeutic response in animal model of human
epithelial cancer, Cancer Res., 2005, 65, 5317–5324.
108 M. Li, Q. Li, Y. H. Zhang, Z. Y. Tian, H. X. Ma, J. Zhao, S. Q.
Xie and C. J. Wang, Antitumor effects and preliminary
systemic toxicity of ANISpm in vivo and in vitro, Anti-Cancer
Drugs, 2013, 24, 32–42.
105 L. Albertazzi, L. Gherardini, M. Brondi, S. Sulis Sato, A.
Bifone, T. Pizzorusso, G. M. Ratto and G. Bardi, In vivo
distribution and toxicity of PAMAM dendrimers in the
central nervous system depend on their surface chemistry,
Mol. Pharmaceutics, 2013, 10, 249–260.
106 P. C. Naha and H. J. Byrne, Generation of intracellular
reactive oxygen species and genotoxicity effect to exposure
of nanosized polyamidoamine (PAMAM) dendrimers in
PLHC-1 cells in vitro, Aquat. Toxicol., 2013, 132–133, 61–72.
107 C. S. Hayes, K. DeFeo-Mattox, P. M. Woster and S. K.
Gilmour, Elevated ornithine decarboxylase activity promotes
109 M. L. Stitzel, I. Kycia, R. Kursawe and D. Ucar,
Transcriptional Regulation of the Pancreatic Islet:
Implications for Islet Function, Curr. Diabetes Rep.,
2015, 15, 66.
110 W. Wang, S. Jin and K. Ye, Development of Islet Organoids
from H9 Human Embryonic Stem Cells in Biomimetic 3D
Scaffolds, Stem Cells Dev., 2017, 26, 394–404.
111 J. Sun, L. Q. Mao, K. S. Polonsky and D. C. Ren, Pancreatic
beta-Cell Death due to Pdx-1 Deficiency Requires Multi-BH
Domain Protein Bax but Not Bak, J. Biol. Chem., 2016, 291,
13529–13534.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2018