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Published: 9 January 2025
Citation: Miloševi´c, N.; Rütter, M.;
Ventura, Y.; Feinshtein, V.; David, A.
Targeted Polymer–Peptide
Conjugates for E-Selectin Blockade in
Renal Injury. Pharmaceutics 2025,17,
82. https://doi.org/10.3390/
pharmaceutics17010082
Copyright: © 2025 by the authors.
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Article
Targeted Polymer–Peptide Conjugates for E-Selectin Blockade
in Renal Injury
Nenad Miloševi´c, Marie Rütter , Yvonne Ventura, Valeria Feinshtein and Ayelet David *
Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the
Negev, Beer-Sheva 84105, Israel
*Correspondence: ayeletda@bgu.ac.il; Tel: +972-8-6477364
Abstract:
Background/Objectives: Leukocytes play a significant role in both acute kidney
injury (AKI) and chronic kidney disease (CKD), contributing to pathogenesis and tissue
damage. The process of leukocyte infiltration into the inflamed tissues is mediated by the
interactions between the leukocytes and cell adhesion molecules (CAMs, i.e., E-selectin,
P-selectin, and VCAM-1) present on the inner surface of the inflamed vasculature. Di-
rectly interfering with these interactions is a viable strategy to limit the extent of excessive
inflammation; however, several small-molecule drug candidates failed during clinical
translation. We hypothesized that a synthetic polymer presenting multiple copies of the
high-affinity E-selecting binding peptide (P-Esbp) could block E-selectin-mediated func-
tions and decrease leukocytes infiltration, thus reducing the extent of inflammatory kidney
injury. Methods: P-Esbp was synthesized by conjugating E-selecting binding peptide (Esbp)
to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer with reactive ester groups
via aminolysis. The effects of P-Esbp treatment on kidney injury were investigated in
two different models: AKI model (renal ischemia—reperfusion injury—RIRI) and CKD
model (adenine-induced kidney injury). Results: We found that the mRNA levels of E-
selectin were up-regulated in the kidney following acute and chronic tissue injury. P-Esbp
demonstrated an extended half-life time in the bloodstream, and the polymer accumu-
lated significantly in the liver, lungs, and kidneys within 4 h post injection. Treatment
with P-Esbp suppressed the up-regulation of E-selectin in mice with RIRI and attenuated
the inflammatory process. In the adenine-induced CKD model, the use of the E-selectin
blocking copolymer had little impact on the progression of kidney injury, owing to the
compensating function of P-selectin and VCAM-1. Conclusion: Our findings provide
valuable insights into the interconnection between CAMs and compensatory mechanisms
in controlling leukocyte migration in AKI and CKD. The combination of multiple CAM
blockers, given simultaneously, may provide protective effects for preventing excessive
leukocyte infiltration and control renal injury.
Keywords:
cell adhesion molecules; E-selectin; VCAM-1; P-selectin; “drug-free”
macromolecular therapeutics; HPMA; kidney inflammation; nanomedicines
1. Introduction
The development of effective pharmacological treatments for acute kidney injury
(AKI) and chronic kidney disease (CKD) remains a significant challenge in global health [
1
].
Despite ongoing research efforts, there are currently no approved drugs specifically de-
signed to prevent, treat, or enhance recovery from kidney injury, and the current strategies
primarily focus on preventing the further deterioration of renal function [
2
]. Leukocytes
Pharmaceutics 2025,17, 82 https://doi.org/10.3390/pharmaceutics17010082
Pharmaceutics 2025,17, 82 2 of 14
play an important role in renal inflammation, contributing to both harmful and protective
effects [
3
]. Leukocyte trafficking into the inflamed tissue is a crucial component of the in-
flammatory response; however, when dysregulated or excessive, it can further contribute to
pathological development [
4
–
6
]. The entry of leukocytes into inflamed tissues is regulated
by their interactions with endothelial cells, specifically with the cell adhesion molecules
(CAMs) expressed on the endothelium surface. The major endothelial-expressed CAMs can
be divided into integrins, selectins (E-, L-, and P-selectin), and immunoglobulin superfam-
ily members (ICAM-1 and VCAM-1), each of them mediating different stages of leukocyte
trafficking [
7
]. Due to their role, CAMs have been recognized as viable targets for reducing
leukocyte influx and inhibiting excessive inflammation [
6
,
8
] with the aim of reducing tissue
injury. Several CAM inhibitors have been employed so far in preclinical development,
including recombinant ligands [
8
], mAbs against selectins [
9
,
10
] and small molecular in-
hibitors (glycomimetics) [
11
,
12
]. However, their clinical translation has stagnated due to a
combination of factors including insufficient binding affinity (glycomimetics), unfavorable
pharmacokinetics (PK), or, in most cases, due to a lack of efficacy.
Some of these challenges could be overcome by designing CAM-targeted nanomedicines
decorated with multiple high-affinity ligands for increased binding avidity to CAMs. Due
to their large size, targeted nanomedicines (such as nanoparticles, liposomes, and polymer–
drug conjugates) exhibit significantly longer half-life times in circulation compared to free
small-molecule drugs [
13
]. Although such nanomedicines are usually intended as drug
delivery platforms, a more recent approach aims to develop systems that exert biological
activity without the need to add low-molecular-weight drugs. These systems, termed
drug-free macromolecular therapeutics (DFMTs), exert their effects through strong binding
to their intended targets and blocking their function [14,15].
We previously reported the synthesis of an HPMA-based polymer bearing multiple
copies of the high-affinity E-selectin binding peptide (Esbp; primary sequence DITWDQL-
WDLMK) intended to target E-selectin on activated endothelium. This polymer (designated
P-Esbp) was, at first, utilized as a drug delivery platform for a cytotoxic drug (doxoru-
bicin) payload to the tumor vasculature [
16
–
18
]. The “drug-free” version of the polymer,
without any drug attached, was shown to inhibit the metastatic spread of melanoma by
blocking E-selectin (in this case, the E-selectin blockade interfered with the attachment of
circulating cancer cells to the inflamed endothelium) [
17
]. Moreover, P-Esbp was shown
to inhibit leukocyte recruitment to inflamed vasculature and reduce atherosclerotic le-
sion development in atherosclerotic mice [
19
] and was further demonstrated to attenuate
neutrophil-mediated liver injury in a mice model of alcohol-related liver disease [20].
In this work, we investigated the ability of P-Esbp to attenuate kidney inflamma-
tion in mice models. Kidney inflammation was chosen as P-Esbp accumulates at high
concentration in the kidneys of mice (in addition to liver tissue) [
20
]. The renal ischemia–
reperfusion injury model (RIRI) is a surgical model representing a more acute type of
kidney inflammation, with pronounced neutrophil infiltration that is mediated, in part,
by E-selectin [
21
]. The second kidney inflammation model that we chose mimics several
aspects of CKD and is induced by feeding mice food that is high in adenine [
22
]. The
consumption of adenine-enriched food leads to the formation of an adenine metabolite—
2,8-dihydroxyadenine—which forms crystals within renal tubules and induces renal injury
and inflammation, followed by the loss of kidney function [23].
We tested whether the E-selectin blockade by P-Esbp could inhibit leukocyte infiltra-
tion into the inflamed kidneys, restrict inflammation, and exert beneficial effects on overall
kidney function in these mice models.
Pharmaceutics 2025,17, 82 3 of 14
2. Materials and Methods
2.1. Chemical Synthesis and Characterization of Polymers P-Esbp
All chemicals were of reagent grade and were obtained from Sigma-Aldrich
(Rehovot, Israel) unless stated otherwise. The N-terminal Lysine-harboring E-selectin-
binding peptide (Esbp, KDITWDQLWDLMR) and the control peptide with a scrambled
Esbp sequence (EsbpScrm, KRMIDWTWLQLDD) were purchased from GL Biochem Ltd.
(Shanghai, China). HPMA monomer was purchased from Polysciences (Warrington, PA,
USA). The monomers methacryloyl-glycylglycine p-nitrophenyl ester (MA-GG-ONp) and
methacryloyl-aminopropyl fluorescein-5-isothiocyanate (MAP–FITC) were synthesized as
described previously [16].
P-Esbp was synthesized by coupling the N-terminal lysine-harboring Esbp or Esbp-
Scrm to an HPMA-based precursor copolymer with reactive ester (O-nirtophenyl—ONp)
groups (P-(GG-ONp)-FITC) via ONp aminolysis, as described previously [
17
]. The conju-
gates were isolated and purified on a PD-10 column using double-distilled water as the
eluent. The content of conjugated peptides was estimated via
1
H-NMR, at 500 Hz, using
the Tryptophan (Typ, W) proton chemical shift (
δ
6.9–7.6, m, 10 H for Esbp/EsbpScrm).
P-Esbp-IR783 was synthesized as described previously [17].
2.2. Pharmacokinetic and Biodistribution Analysis of P-(Esbp)-IR783
The PK parameters of P-Esbp were analyzed in healthy BABL/c mice to provide a
baseline for understanding how the polymer distributes throughout the body without the
influence of kidney injury. P-(Esbp)-IR783 (Mw: 46.7 kDa; P
I
: 1.19; 3 mol% Esbp; 5 mol%
IR783) was administered to 8-week-old female BALB/c mice (1 mg polymer/mice; n = 3)
via tail vein injection. Animals were euthanized at designated time points (1 min, 5 min,
15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h). Blood and other major organs/tissues
including hearts, lungs, kidneys, livers, and spleens were isolated following euthanasia.
Serum sample fluorescent intensity was measured using the Infinite M-200 microplate
fluorescence reader (Tecan, Männedorf, Switzerland). Corresponding serum concentrations
were calculated using a previously constructed calibration curve prepared with known
concentrations of the same polymer in human plasma. Major organs were imaged individ-
ually, and regions of interest (ROIs) were analyzed using the IVIS-Lumina imaging system
without perfusion. The PK parameters, such as total clearance (CL), volume of distribution
(Vd), and biological half-life (t
1/2
), were determined using the bolus intravenous input
non-compartmental and two-compartmental analysis of WinNonlin. The area under the
curve (AUC) was calculated using the trapezoidal rule.
2.3. Animal Models of Kidney Inflammation
All animal experiments were approved and performed in compliance with the stan-
dards of the Ben-Gurion University of the Negev (BGU) Institutional Animal Care and
Use Committee (IACUC), protocol code IL-56–08-2019 (C), period of authorization from
09/12/2019 through 09/11/2022. Male C57BL/6 mice and female BALB/c mice were
obtained from Harlan Biotech Israel (HBI), (Rehovot, Israel) and housed in the animal
facility of the Ben-Gurion University of the Negev.
2.3.1. In Vivo Model of Acute Kidney Inflammation—Renal Ischemia–Reperfusion Injury
Model (RIRI)
The surgical procedure was based on the adapted protocol from a publication by
Singbartl and Ley [
24
,
25
]. During initial experiments, high mortality was observed with
32 min bilateral RIRI. In an attempt to reduce the mortality, 25 min of bilateral RIRI was
Pharmaceutics 2025,17, 82 4 of 14
performed in the following experiments. According to Wei et al. [
25
], the rise in the blood
urea nitrogen (BUN) levels is similar with both 25 and 30 min ischemia.
C57BL/6 mice (age: 8–10 weeks; male; bodyweight more than 20 g) were anesthetized
with i.p. injection of ketamine and xylazine (100 mg/kg, 10 mg/kg). After confirming the
depth of surgical anesthesia, mice were shaved, and the operating area was disinfected.
Mice body temperature was measured using the thermostatic station with a rectal probe,
and mice were left to stabilize their body temperature to 37
°C
. During the surgery, body
temperature was monitored and kept at 37
±
0.4
°C
, as this is crucial for the reproducible
kidney injury [
26
]. Mice skin and muscles were cut to expose renal pedicle first on the left
and subsequently on the right side, as shown in Supplementary Figure S1A. The left renal
artery was clamped with a Micro Serrefines clamp (Fine Scientific Tools—Supplementary
Figure S1B); after confirmation of the color change in the kidney tissue following blood
occlusion, the muscle layer was closed with one suture. The right renal artery was also
clamped (the time difference between left and right clamping was less than 2 min) and
the skin was closed with one suture. Following 25 min of ischemia time, the sutures were
opened, the Micro Serrefines clips were removed, and the kidneys were inspected for
color change (from dark red to a physiological, light brown color), which is an indicator
of successful reperfusion. After closing the muscle layer and skin, mice received 1 mL of
heated saline and Buprenorphine (dose: 50
µ
g/kg subcutaneously). Mice were randomized
into groups, as follows: A—only RIRI; B—RIRI and i.p. 1 mg of P-Esbp in saline after
reperfusion and another dose of 1 mg of P-Esbp in saline the following morning; and
C—sham-operated mice (group C underwent all the procedures as A except for the clamp-
ing of the renal arteries). Animals were given thermal support by IR lamps and were
followed until regaining full consciousness. All the procedures and time points were
recorded for each mouse to exclude any mice where some deviations from the protocol
occurred (using surgery logbook—Supplementary Figure S1C). Mice were euthanized
exactly 24 h after reperfusion time. Upon euthanasia, blood samples and kidney tissue
samples were collected for biochemical analysis and tissue mRNA expression via qRT-PCR,
as described below.
2.3.2. In Vivo Model of Adenine-Induced Chronic Kidney Disease (CKD)
Experimental Design of Adenine-Induced CKD Murine Model
A casein-based diet with adenine (TD.130900–Adenine Diet (0.2% adenine, total phos-
phate content 0.9%, and total calcium content 0.6%), purchased from Harlan Biotech Israel
(HBI), Rehovot, Israel) was formulated to have similarities to the diet described by Jia
et al. [
22
]. The vitamin mix was increased by 50% and 2 ppm additional vitamin K and
10 ppm additional thiamin HCl were added to make the diet more suitable for irradiation
sterilization. The control diet was also purchased from the same vendor and was identical
in formulation to the adenine diet, apart from lacking adenine.
For the calibration experiments, 8-week-old male C57BL/6 mice were randomized into
groups receiving either the adenine diet 0.2% for 5 days (n = 4), 14 days (n = 3), and 25 days
(n = 6) or the control diet (n = 9; euthanized on days 14 and 25). Three mice were housed
per cage. Every second day, mice body weight (BW) was determined as well as average
food intake. On the designated days, mice were euthanized, and serum and kidney tissue
samples were obtained and processed for further biochemical, histological, and mRNA
expression analyses.
In the interventional experiment, C57BL/6 male mice were randomized into groups
receiving the 0.2% adenine diet for 25 days and i.p. injections of either P-Esbp, P-EsbpScrm,
or saline; the control group received the control diet and saline injections. Treatments
Pharmaceutics 2025,17, 82 5 of 14
were administered every second day from day 6 in the form of 1 mg of the corresponding
polymer dissolved in 200 µL of saline.
Tissue and Serum Processing and Analysis
Total RNA was extracted from kidney samples with TRIzol reagent and was tran-
scribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit. RT-PCR was
performed using Taqman probes for E-selectin, TNF-
α
, and IL-1
β
, which were normalized
to the expression levels of GAPDH (Supplementary Table S1). The results are expressed
as fold induction relative to the control-fed groups. Upon blood collection, full blood was
let to clot for 15–30 min at room temperature. Following centrifugation at 2000
×
gfor
10 min, serum was collected and kept at 4
◦
C. Serum creatinine and serum urea levels were
determined using a clinical chemistry analyzer Beckman Coulter AU5800 (Soroka Medical
Center, Beer Sheva, Israel).
3. Results
3.1. Targeted Polymer Synthesis
A polymer precursor for Esbp conjugation (P-(GGONp)-FITC) was synthesized, pu-
rified, and characterized as previously reported [
16
,
17
,
20
]. The molecular weight of P-
(GGONp)-FITC was 34 kDa (determined via size-exclusion chromatography on an ACTA-
FPLC), which is below the renal glomerular filtration threshold, allowing for the polymer to
be renally excreted. The content of the reactive group for peptide conjugation—ONp—was
7.5 mol% (determined spectrophotometrically). P-Esbp was synthesized and characterized
as previously described [
20
]. The structure of the FITC-labeled, E-selectin-binding polymer
(P-(Esbp)-FITC) is shown in Figure 1. The characteristics of the synthesized polymer and
control (P-(EsbpScrm)-FITC) are shown in Table 1. The content of conjugated peptides
was estimated via NMR using the ratio of signal intensities of tryptophan present in the
peptide sequence (
δ
6.9–7.6, m, 10H) and the signal of HPMA (
δ
0.9–1.1, m, 6H). The
content of Esbp/EsbpScrm in P-Esbp/P-EsbpScrm was estimated close to the theoretical
maximum (the content of ONp groups), indicating the full conversion of reactive ONp
groups (Table 1).
Pharmaceutics 2024, 16, x FOR PEER REVIEW 5 of 14
3. Results
3.1. Targeted Polymer Synthesis
A polymer precursor for Esbp conjugation (P-(GGONp)-FITC) was synthesized, pu-
rified, and characterized as previously reported [16,17,20]. The molecular weight of P-
(GGONp)-FITC was 34 kDa (determined via size-exclusion chromatography on an ACTA-
FPLC), which is below the renal glomerular filtration threshold, allowing for the polymer
to be renally excreted. The content of the reactive group for peptide conjugation—ONp—
was 7.5 mol% (determined spectrophotometrically). P-Esbp was synthesized and charac-
terized as previously described [20]. The structure of the FITC-labeled, E-selectin-binding
polymer (P-(Esbp)-FITC) is shown in Figure 1. The characteristics of the synthesized pol-
ymer and control (P-(EsbpScrm)-FITC) are shown in Table 1. The content of conjugated
peptides was estimated via NMR using the ratio of signal intensities of tryptophan present
in the peptide sequence (δ 6.9–7.6, m, 10H) and the signal of HPMA (δ 0.9–1.1, m, 6H). The
content of Esbp/EsbpScrm in P-Esbp/P-EsbpScrm was estimated close to the theoretical
maximum (the content of ONp groups), indicating the full conversion of reactive ONp
groups (Table 1).
Table 1. Characteristics of synthesized polymers and precursor copolymers.
HPMA Copolymer Mw [kDa]
a
Polydispersity
b
%mol FITC
or IR783
c
%mol ONp/Pep-
tide/Scrm
d
P-(GGONp)-FITC 34.0 1.42 1.8 7.50
P-(Esbp)-FITC 34.1 1.35 1.8 7.49
P-(EsbpScrm)-FITC 33.2 1.2 1.8 7.35
a,b
Weight-average molecular weight (Mw) and polydispersity (PI) of precursors and copolymer
peptide conjugates were estimated via size-exclusion chromatography on an ACTA-FPLC system,
using a Sephacryl 16/60 S-400 column (GE Healthcare) calibrated with fractions of known-molecu-
lar-weight HPMA homopolymers or by using the GPC/ HPLC Shimadzu system equipped with
UV-VIS, refractive index, and multiangle light scaering DAWN 8 EOS (Wya Technology Corp.,
Santa Barbara, CA) detectors using a TSK 3000 SWXL column (Tosoh Bioscience, Japan).
c
The con-
tents of FITC residues were determined by measuring the UV absorbance at 492 nm (ε = 82,000 M
−1
cm
−1
).
d
The contents of peptide-targeting moieties were estimated via
1
H-NMR at 500 Hz using the
Tryptophan (Typ, W) chemical shift of aromatic amino acids (δ 6.9–7.6, m, 10H).
Figure 1.
Structure of the FITC-labeled E-selectin-binding polymer (P-(Esbp)-FITC). HPMA indi-
cates N–(2–hydroxypropyl)methacrylamide; Esbp—E-selectin-binding peptide. FITC—fluorescein
isothiocyanate.
Pharmaceutics 2025,17, 82 6 of 14
Table 1. Characteristics of synthesized polymers and precursor copolymers.
HPMA Copolymer Mw [kDa] aPolydispersity b%mol FITC or
IR783 c
%mol
ONp/Peptide/Scrm d
P-(GGONp)-FITC 34.0 1.42 1.8 7.50
P-(Esbp)-FITC 34.1 1.35 1.8 7.49
P-(EsbpScrm)-FITC 33.2 1.2 1.8 7.35
a,b
Weight-average molecular weight (Mw)and polydispersity (PI) of precursors and copolymer peptide conjugates
were estimated via size-exclusion chromatography on an ACTA-FPLC system, using a Sephacryl 16/60 S-400
column (GE Healthcare) calibrated with fractions of known-molecular-weight HPMA homopolymers or by using
the GPC/ HPLC Shimadzu system equipped with UV-VIS, refractive index, and multiangle light scattering
DAWN 8 EOS (Wyatt Technology Corp., Santa Barbara, CA) detectors using a TSK 3000 SWXL column (Tosoh
Bioscience, Japan).
c
The contents of FITC residues were determined by measuring the UV absorbance at 492 nm
(
ε
= 82,000 M
−1
cm
−1
).
d
The contents of peptide-targeting moieties were estimated via
1
H-NMR at 500 Hz using
the Tryptophan (Typ, W) chemical shift of aromatic amino acids (δ6.9–7.6, m, 10H).
3.2. Pharmacokinetics (PK) and Biodistribution (BD) Analysis of P-(Esbp)-IR783
We first analyzed the PK and BD characteristics of P-Esbp in healthy BALB/c female 8-
week-old mice using near-infrared (NIR) optical imaging. The half-life time of HPMA-based
copolymers in circulation can vary significantly, depending on the molecular weight and
the specific composition of the polymer [
27
]. P-Esbp-IR783 (Mw~47 kDa) has a distribution
(t
1/2
alpha) of approximately 1 minute and an elimination half-life (t
1/2
beta) of 8.94 h. The
blood data were fitted with a two-compartmental model (Figure 2), consistent with other
examples of HPMA-based copolymer conjugates.
Pharmaceutics 2024, 16, x FOR PEER REVIEW 6 of 14
Figure 1. Structure of the FITC-labeled E-selectin-binding polymer (P-(Esbp)-FITC). HPMA indi-
cates N–(2–hydroxypropyl)methacrylamide; Esbp—E-selectin-binding peptide. FITC—fluorescein
isothiocyanate.
3.2. Pharmacokinetics (PK) and Biodistribution (BD) Analysis of P-(Esbp)-IR783
We first analyzed the PK and BD characteristics of P-Esbp in healthy BALB/c female
8-week-old mice using near-infrared (NIR) optical imaging. The half-life time of HPMA-
based copolymers in circulation can vary significantly, depending on the molecular
weight and the specific composition of the polymer [27]. P-Esbp-IR783 (Mw ~ 47 kDa) has
a distribution (t1/2 alpha) of approximately 1 minute and an elimination half-life (t1/2 beta)
of 8.94 h. The blood data were fied with a two-compartmental model (Figure 2), con-
sistent with other examples of HPMA-based copolymer conjugates.
Figure 2. Compartmental analysis and pharmacokinetic data of P-(Esbp)-IR783 in healthy mice
after intravenous bolus injection.
P-(Esbp)-IR783 accumulated significantly in the liver of healthy mice, and a substan-
tial amount of polymer was also detected at the first 4 h in the lungs and kidneys (Figure
3). Detectable levels were observed in the liver and kidneys even 48 hours post-injection.
The liver, lungs, and kidneys are characterized by high tissue perfusion and discontinu-
ous vascular walls (these fenestrae are generally between 50 and 100 nanometers in diam-
eter) that allow substances circulating in the plasma to extravasate. These results align
with other examples of polymer–drug conjugates or nano-sized formulations, such as lip-
osomes, polymeric micelles, and nanoparticles [28]. The significant perfusion and accu-
mulation of P-Esbp in the kidneys highlight its potential use for treating kidney diseases
by targeting the E-selectin that is present at the luminal aspect of inflamed blood vessels.
Since E-selectin expression levels are significantly up-regulated in response to inflamma-
tory stimuli, P-Esbp may substantially accumulate in the blood vessels of the injured kid-
ney and the renal localization is expected to be higher.
Figure 2.
Compartmental analysis and pharmacokinetic data of P-(Esbp)-IR783 in healthy mice after
intravenous bolus injection.
P-(Esbp)-IR783 accumulated significantly in the liver of healthy mice, and a substantial
amount of polymer was also detected at the first 4 h in the lungs and kidneys (Figure 3).
Detectable levels were observed in the liver and kidneys even 48 hours post-injection. The
liver, lungs, and kidneys are characterized by high tissue perfusion and discontinuous
vascular walls (these fenestrae are generally between 50 and 100 nanometers in diameter)
that allow substances circulating in the plasma to extravasate. These results align with
other examples of polymer–drug conjugates or nano-sized formulations, such as liposomes,
polymeric micelles, and nanoparticles [
28
]. The significant perfusion and accumulation
of P-Esbp in the kidneys highlight its potential use for treating kidney diseases by target-
ing the E-selectin that is present at the luminal aspect of inflamed blood vessels. Since
E-selectin expression levels are significantly up-regulated in response to inflammatory
stimuli, P-Esbp may substantially accumulate in the blood vessels of the injured kidney
and the renal localization is expected to be higher.
Pharmaceutics 2025,17, 82 7 of 14
Pharmaceutics 2024, 16, x FOR PEER REVIEW 7 of 14
Figure 3. The biodistribution profile of P-Esbp-IR783 in heathy mice.
3.3. P-Esbp Reduced Kidney Damage in Acute Kidney Inflammation—Renal Ischemia–
Reperfusion Injury (RIRI) Model
Renal ischemia–reperfusion injury (RIRI) is a model of acute renal damage, and some
reports have suggested the important role of E-selectin in mediating the inflammatory
response following reperfusion [24]. In a series of experiments, we investigated the effects
of P-Esbp on renal injury parameters and the renal expression of E-selectin to determine
if it can influence AKI.
In this experimental seing, kidney injury parameters (i.e., urea and creatinine) were
elevated after the procedure compared to sham-operated mice. P-Esbp treatment reduced
the level of kidney damage, in accordance with Singbartl [24], yet the effects were not
statistically significant (Figure 4A,B). Further analysis of renal tissue expression of E-se-
lectin revealed that E-selectin mRNA levels were up-regulated by about 8-fold in the kid-
ney of RIRI mice relative to sham-control mice. This marked up-regulation of E-selectin is
crucial in initiating the inflammatory cascade and plays a pivotal role in facilitating neu-
trophil recruitment to the injured tissue. P-Esbp treatment significantly suppressed the
up-regulation of E-selectin in RIRI mice (Figure 4C). The expression levels of pro-inflam-
matory cytokines—TNFα and IL-1β (Figure 4D,E)—were lower in the P-Esbp-treated
group relative to untreated RIRI group, indicating that P-Esbp therapy aenuates inflam-
matory processes in the acute experimental seing of renal injury.
Figure 3. The biodistribution profile of P-Esbp-IR783 in heathy mice.
3.3. P-Esbp Reduced Kidney Damage in Acute Kidney Inflammation—Renal Ischemia–Reperfusion
Injury (RIRI) Model
Renal ischemia–reperfusion injury (RIRI) is a model of acute renal damage, and some
reports have suggested the important role of E-selectin in mediating the inflammatory
response following reperfusion [
24
]. In a series of experiments, we investigated the effects
of P-Esbp on renal injury parameters and the renal expression of E-selectin to determine if
it can influence AKI.
In this experimental setting, kidney injury parameters (i.e., urea and creatinine) were
elevated after the procedure compared to sham-operated mice. P-Esbp treatment reduced
the level of kidney damage, in accordance with Singbartl [
24
], yet the effects were not sta-
tistically significant (Figure 4A,B). Further analysis of renal tissue expression of E-selectin
revealed that E-selectin mRNA levels were up-regulated by about 8-fold in the kidney
of RIRI mice relative to sham-control mice. This marked up-regulation of E-selectin is
crucial in initiating the inflammatory cascade and plays a pivotal role in facilitating neu-
trophil recruitment to the injured tissue. P-Esbp treatment significantly suppressed the up-
regulation of E-selectin in RIRI mice (Figure 4C). The expression levels of pro-inflammatory
cytokines—TNF
α
and IL-1
β
(Figure 4D,E)—were lower in the P-Esbp-treated group rel-
ative to untreated RIRI group, indicating that P-Esbp therapy attenuates inflammatory
processes in the acute experimental setting of renal injury.
Pharmaceutics 2025,17, 82 8 of 14
Pharmaceutics 2024, 16, x FOR PEER REVIEW 8 of 14
Figure 4. Effects of P-Esbp treatment (1 mg in 200 µL of saline at the time of reperfusion and 1 mg
the following morning) on kidney function biochemical parameters—creatinine (A) and urea (B) 24
h after 25 min RIRI. Renal tissue expression levels of E-selectin (C), TNFα (D), and IL-1β (E) 24 h
after 25 min RIRI. #—statistically significant difference compared to the sham-control group, p <
0.05; *—statistically significant difference compared to the RIRI group, p < 0.05.
3.4. Continuous P-Esbp Treatment Did Not Affect Chronic Kidney Injury in Adenine-Induced
CKD Model
Previous studies indicated the development of chronic kidney injury in mice fed with
an adenine-rich 0.2% diet [29–31]. In the first experiment, we investigated the effects of an
adenine-rich diet on biochemical, and inflammatory cytokine parameters, especially on
the expression paerns of CAMs in renal tissues on days 5, 14, and 25 after starting the
diet.
Adenine-fed C57BL/6 mice experienced a reduction in BW, which was more pro-
nounced in the first week of feeding and stabilized in the following weeks (Figure 5A,B—
individual profiles). This reduction in BW was comparable with that reported in the liter-
ature, and, on average, it was around 10% of the original weight [29,32]. The average food
intake was 3.2 g of food per mouse per day in the control group and 2.2 g per mouse per
day in the adenine group. Kidney injury was confirmed by elevated serum levels of creat-
inine and serum urea (Figure 5C,D) and their levels gradually increased throughout the
course of the experiment.
The inflammatory cytokines TNFα and IL-1β also showed a rising trend in mRNA
expression (Figure 6A,B), with a gradual increase in their levels from day 5 to day 25.
Figure 4.
Effects of P-Esbp treatment (1 mg in 200
µ
L of saline at the time of reperfusion and
1 mg the following morning) on kidney function biochemical parameters—creatinine (
A
) and urea
(
B
) 24 h after 25 min RIRI. Renal tissue expression levels of E-selectin (
C
), TNF
α
(
D
), and IL-1
β
(
E
) 24 h after 25 min RIRI. #—statistically significant difference compared to the sham-control group,
p< 0.05; *—statistically significant difference compared to the RIRI group, p< 0.05.
3.4. Continuous P-Esbp Treatment Did Not Affect Chronic Kidney Injury in Adenine-Induced
CKD Model
Previous studies indicated the development of chronic kidney injury in mice fed with
an adenine-rich 0.2% diet [
29
–
31
]. In the first experiment, we investigated the effects of an
adenine-rich diet on biochemical, and inflammatory cytokine parameters, especially on the
expression patterns of CAMs in renal tissues on days 5, 14, and 25 after starting the diet.
Adenine-fed C57BL/6 mice experienced a reduction in BW, which was more pro-
nounced in the first week of feeding and stabilized in the following weeks (Figure 5A,B—
individual profiles). This reduction in BW was comparable with that reported in the
literature, and, on average, it was around 10% of the original weight [
29
,
32
]. The average
food intake was 3.2 g of food per mouse per day in the control group and 2.2 g per mouse
per day in the adenine group. Kidney injury was confirmed by elevated serum levels of
creatinine and serum urea (Figure 5C,D) and their levels gradually increased throughout
the course of the experiment.
Pharmaceutics 2025,17, 82 9 of 14
Pharmaceutics 2024, 16, x FOR PEER REVIEW 9 of 14
Endothelial CAMs are overexpressed in response to inflammation and signaling by cyto-
kines. Our results showed that the CAMs E-selectin, VCAM-1, and P-selectin were up-
regulated in kidney tissues of adenine-fed mice. The E-selectin levels were elevated at the
earliest time point of 5 days and this trend continued for time points at 14 days and 25
days (Figure 6C), demonstrating the highest folds at the last time point (4.6-fold average).
Both P-selectin (Figure 6D) and VCAM-1 (Figure 6E) expression levels were enhanced
during the course of the experiment, and the fold increase in their mRNA levels was about
5–10-times higher than that of E-selectin.
To investigate the effects of E-selectin blockage with P-Esbp, adenine-fed mice re-
ceived 10 i.p. injections of P-Esbp or the polymer with the scrambled version of the pep-
tide—P-EsbpScrm. Since the half-life time of P-Esbp is approximately 9 h in circulation,
and it takes about 4 to 5 half-lives for an almost complete clearance from the body, P-Esbp
was injected once every two days to ensure a continuous dose of the polymer conjugate
in circulation, and to ensure that it was available for endothelial E-selectin blockade under
chronic conditions. In line with previously described results, biochemistry parameters,
serum creatinine, and serum urea (Figure 7A,B) were profoundly higher in mice fed with
an adenine diet throughout the experiment (from day 5 to day 25). However, treatment
with P-Esbp did not inhibit the rise in serum creatinine and urea levels. The lack of thera-
peutic efficacy may be aributed to the complementary roles of CAMs. The level of P-
selectin was significantly up-regulated in adenine-fed mice. E- and P-selectin function co-
operatively and can compensate each other in various biological processes. We thus as-
sume that targeting and blocking all the three CAMs (E-selectin, P-selectin, and VCAM-
1), simultaneously, might provide therapeutic benefits. A suboptimal dosing regimen of
P-Esbp can also explain the results. Overall, in the chosen animal model, feeding protocol,
and dosing regimen, E-selectin blockade cannot substantially inhibit chronic kidney in-
jury and inflammation caused by adenine diet.
Figure 5.
Induction of CKD by adenine diet (0.2%). Mice body weight as group average (
A
) and
individual weight (
B
). Biochemical parameters of serum creatinine (
C
) and serum urea (
D
) at different
time points from the initiation of the adenine diet.
The inflammatory cytokines TNF
α
and IL-1
β
also showed a rising trend in mRNA
expression (Figure 6A,B), with a gradual increase in their levels from day 5 to day 25. En-
dothelial CAMs are overexpressed in response to inflammation and signaling by cytokines.
Our results showed that the CAMs E-selectin, VCAM-1, and P-selectin were up-regulated in
kidney tissues of adenine-fed mice. The E-selectin levels were elevated at the earliest time
point of 5 days and this trend continued for time points at 14 days and 25 days (Figure 6C),
demonstrating the highest folds at the last time point (4.6-fold average). Both P-selectin
(Figure 6D) and VCAM-1 (Figure 6E) expression levels were enhanced during the course of
the experiment, and the fold increase in their mRNA levels was about 5–10-times higher
than that of E-selectin.
To investigate the effects of E-selectin blockage with P-Esbp, adenine-fed mice received
10 i.p. injections of P-Esbp or the polymer with the scrambled version of the peptide—P-
EsbpScrm. Since the half-life time of P-Esbp is approximately 9 h in circulation, and it
takes about 4 to 5 half-lives for an almost complete clearance from the body, P-Esbp was
injected once every two days to ensure a continuous dose of the polymer conjugate in
circulation, and to ensure that it was available for endothelial E-selectin blockade under
chronic conditions. In line with previously described results, biochemistry parameters,
serum creatinine, and serum urea (Figure 7A,B) were profoundly higher in mice fed with
an adenine diet throughout the experiment (from day 5 to day 25). However, treatment
with P-Esbp did not inhibit the rise in serum creatinine and urea levels. The lack of
therapeutic efficacy may be attributed to the complementary roles of CAMs. The level of
P-selectin was significantly up-regulated in adenine-fed mice. E- and P-selectin function
Pharmaceutics 2025,17, 82 10 of 14
cooperatively and can compensate each other in various biological processes. We thus
assume that targeting and blocking all the three CAMs (E-selectin, P-selectin, and VCAM-
1), simultaneously, might provide therapeutic benefits. A suboptimal dosing regimen of
P-Esbp can also explain the results. Overall, in the chosen animal model, feeding protocol,
and dosing regimen, E-selectin blockade cannot substantially inhibit chronic kidney injury
and inflammation caused by adenine diet.
Pharmaceutics 2024, 16, x FOR PEER REVIEW 10 of 14
Figure 5. Induction of CKD by adenine diet (0.2%). Mice body weight as group average (A) and
individual weight (B). Biochemical parameters of serum creatinine (C) and serum urea (D) at dif-
ferent time points from the initiation of the adenine diet.
Figure 6. mRNA expression paerns of inflammatory markers in renal tissues of adenine-rich diet-
fed mice. TNFα (A); IL-1β (B); E-selectin (C); P-selectin (D); and VCAM-1 (E).
Figure 7. Effects of P-Esbp treatment (1 mg in 200 µL of saline i.p. every second day from day 6 of
the experiment; total of ten doses) on kidney function biochemical parameters from the interven-
tional experiment. Serum creatinine (A) and serum urea (B) upon animal euthanasia on day 28.
4. Discussion
In this study, we investigated different models of kidney inflammation and identified
those with a clear involvement of CAMs. In the RIRI model of rapid and acute inflamma-
tion, E-selectin mRNA was about eight times higher 24 after ischemia and reperfusion,
indicating that E-selectin plays a significant role in the early inflammatory response fol-
lowing AKI. Treatment with P-Esbp aenuated inflammatory processes in RIRI mice by
significantly suppressing the up-regulation of E-selectin expression. This is in line with
Figure 6.
mRNA expression patterns of inflammatory markers in renal tissues of adenine-rich diet-fed
mice. TNFα(A); IL-1β(B); E-selectin (C); P-selectin (D); and VCAM-1 (E).
Pharmaceutics 2024, 16, x FOR PEER REVIEW 10 of 14
Figure 5. Induction of CKD by adenine diet (0.2%). Mice body weight as group average (A) and
individual weight (B). Biochemical parameters of serum creatinine (C) and serum urea (D) at dif-
ferent time points from the initiation of the adenine diet.
Figure 6. mRNA expression paerns of inflammatory markers in renal tissues of adenine-rich diet-
fed mice. TNFα (A); IL-1β (B); E-selectin (C); P-selectin (D); and VCAM-1 (E).
Figure 7. Effects of P-Esbp treatment (1 mg in 200 µL of saline i.p. every second day from day 6 of
the experiment; total of ten doses) on kidney function biochemical parameters from the interven-
tional experiment. Serum creatinine (A) and serum urea (B) upon animal euthanasia on day 28.
4. Discussion
In this study, we investigated different models of kidney inflammation and identified
those with a clear involvement of CAMs. In the RIRI model of rapid and acute inflamma-
tion, E-selectin mRNA was about eight times higher 24 after ischemia and reperfusion,
indicating that E-selectin plays a significant role in the early inflammatory response fol-
lowing AKI. Treatment with P-Esbp aenuated inflammatory processes in RIRI mice by
significantly suppressing the up-regulation of E-selectin expression. This is in line with
Figure 7.
Effects of P-Esbp treatment (1 mg in 200
µ
L of saline i.p. every second day from day 6 of the
experiment; total of ten doses) on kidney function biochemical parameters from the interventional
experiment. Serum creatinine (A) and serum urea (B) upon animal euthanasia on day 28.
4. Discussion
In this study, we investigated different models of kidney inflammation and identified
those with a clear involvement of CAMs. In the RIRI model of rapid and acute inflammation,
E-selectin mRNA was about eight times higher 24 after ischemia and reperfusion, indicating
that E-selectin plays a significant role in the early inflammatory response following AKI.
Pharmaceutics 2025,17, 82 11 of 14
Treatment with P-Esbp attenuated inflammatory processes in RIRI mice by significantly
suppressing the up-regulation of E-selectin expression. This is in line with previous studies
showing that the blockade of E-selectin or P-selectin (by a monoclonal antibody or small-
molecule selectin ligand) decreases neutrophil recruitment into the kidney and preserves
organ morphology and function and in sepsis-induced AKI [
33
–
35
]. Yet, polymer treatment
only mildly influenced kidney injury parameters (i.e., urea and creatinine). In a model of
more gradual, chronic kidney inflammation, we confirmed the up-regulation of all three
CAMs (E-selectin, P-selectin, and VCAM-1) in kidney samples from adenine-fed animals.
While E-selectin expression was up-regulated approximately 8-fold in RIRI after 24 h, its
levels increased only 2-fold in CKD at the initial time point (day 5) and reached 4–5-fold on
day 25. P-selectin and VCAM-1 have reached higher folds of up-regulation compared to
E-selectin (8-fold and 20-fold, respectively, on day 5, and 20–35 fold of increase on day 25).
This indicates that P-selectin and VCAM-1 play a more substantial role in the progression
of CKD than E-selectin. Multiple i.p. injections of P-Esbp in the adenine-diet-induced
CKD model did not protect mice from kidney injury. The physiological role of P-selectin
is to work synergistically with E-selectin in the mediation of initial leukocyte adhesion to
activated endothelium during acute and chronic inflammation. It is possible that due to
the overlapping and mutually compensating functions of selectins, the blockade of only
one selectin family member was not sufficient for inhibiting chronic inflammation and
renal injury. For comparison, in an inflammatory model of alcohol-induced liver injury (the
NIAAA model), where E-selectin is the sole CAM that was up-regulated to a significant
extent [
20
,
36
], E-selectin blockade by P-Esbp showed a profound anti-inflammatory efficacy.
The increase in P-selectin and VCAM-1 expression in RIRI was less pronounced after 24 h
when compared to CKD after 5 days. Specifically, the upregulation was approximately six
times higher for P-selectin and twelve times higher for VCAM-1 in RIRI [
37
–
39
], which is ~
three times lower relative to their up-regulation in CKD. This might explain the beneficial
effects of P-Esbp observed in RIRI but not in CKD. Future experiments with the combination
of E-selectin, P-selectin, and VCAM-1 blockers given simultaneously might attenuate the
manifestation of adenine-diet-induced kidney injury. Our results show that therapeutic
success in treating one disease is not a guarantee for benefit across different pathologies.
This is evident in several other drug candidates (i.e., Inclacumab, a monoclonal antibody
against P-selectin, was dropped for cardiovascular diseases treatment and is now in trials
for the treatment of sickle cell disease [
40
]). Effectively blocking a single CAM might be
beneficial in one inflammatory setting, but might provide limited efficacy in others [
41
,
42
].
Taken together, the results from this study give several insights on the process of devel-
oping a polymer–peptide conjugate specifically designed to target and block E-selectin to
prevent renal injury. E-selectin has a more significant and immediate role in the inflamma-
tory processes in acute compared to chronic renal injury. The upregulation of E-selectin in
response to inflammatory stimuli was more pronounced in AKI than CKD. Treatment with
P-Esbp suppressed the up-regulation of E-selectin in mice with AKI. The mild protective
effects in the models of kidney inflammation highlight the interconnected nature of CAMs
and their different individual contributions to the specific pathological process. Careful
monitoring of potential compensatory increases in other CAMs is crucial when targeting
E-selectin. Overall, more effort should be invested in precisely characterizing different
inflammatory diseases and/or animal models to pinpoint those where the blockage of one
or several CAMs would provide the most therapeutic benefits.
Supplementary Materials:
The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/pharmaceutics17010082/s1, Figure S1: The incision lo-
cations for left and right kidney clamping-adapted from Ref. [
25
]; Table S1: Primer sequences for
real-time PCR.
Pharmaceutics 2025,17, 82 12 of 14
Author Contributions:
N.M. synthesized the polymers, designed the experiments, developed the
animal models, performed the experiments, and analyzed the data. M.R. assisted with polymer
synthesis and characterization, as well as manuscript preparation. M.R., Y.V. and V.F. helped to plan
and carry out the experiments on mice. A.D. and N.M. wrote the manuscript. AD was involved in
planning and supervised the work. All authors have read and agreed to the published version of
the manuscript.
Funding:
This research was funded by the Israel Science Foundation (grant # 1115/19) and by the
LEDUCQ Foundation, as part of the PRIMA (Preventing Rheumatic Injury BioMarker Alliance)
Network (grant #22ARF02).
Institutional Review Board Statement:
The study protocol was approved by the IACUC of the
Ben-Gurion University of the Negev, Authorization number: IL-56-08-2019 (C).
Data Availability Statement: Data will be made available upon reasonable request.
Acknowledgments:
We would like to acknowledge Kreitman School of Advanced Graduate Studies
(Ben-Gurion University of the Negev, Israel) for supporting N.M and M.R. with Ph.D. and short term
post-doctoral scholarship.
Conflicts of Interest: The authors declare no conflict of interest.
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