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Acta Biomaterialia 169 (2023) 517–529
Contents lists available at ScienceDirect
Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio
Full length article
L-Arginine self-delivery supramolecular nanodrug for NO gas therapy
Mengsi Zhang
a
, Hao Jin
a
, Yi Liu
a , b
, Lanlan Wan
c , ∗, Shuwei Liu
b , ∗, Hao Zhang
a , b , d , ∗∗
a
State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , PR China
b
Joint Laboratory of Optical Functional Theranostics in Medicine and Chemistry, The First Hospital of Jilin University, Changchun 13 00 21, PR China
c
Department of Anesthesia, The Second Hospital of Jilin University, Changchun 13 00 41, PR China
d
Green Catalysis Center, College of Chemistry, Zhengzhou University, Zhengzhou 450 0 01, PR China
a r t i c l e i n f o
Article history:
Received 19 April 2023
Revised 3 July 2023
Accepted 27 July 2023
Available online 1 August 2023
Keywo rds:
Gas therapy
L-Arginine
Supramolecular nanodrugs
Polyphenol
Antitumor
a b s t r a c t
NO gas therapy is a supplementary approach for tumor treatment due to the advantages of minimal
invasion, little drug resistance, low side effect and amplified efficacy. l -Arginine (L-Arg), a natural NO
source with good biocompatibility, can release NO under the stimulation of H
2
O
2
in tumor microenviron-
ment. However, the conventional l -Arg delivery systems via noncovalent loading usually lead to inevitable
premature leakage of nano-cargos during blood circulation. In this work, an efficient l -Arg self-delivery
supramolecular nanodrug (SDSND) for tumor treatment is demonstrated by combining Mannich reac-
tion and π- πstacking. l -Arg links to (-)-epigallocatechin gallate (EGCG) with the assistance of formalde-
hyde through Mannich reaction, and then assembles into nanometer-sized particles via π- πstacking. The
guanidine group of l -Arg and the phenolic hydroxyl groups of EGCG are preserved in the SDSNDs, which
allows for accomplishing gas therapy by provoking tumor cell apoptosis and combining with EGCG to
amplify apoptosis, respectively. In addition, the SDSNDs exhibit high biocompatibility and avoid the pre-
mature leakage of l -Arg in blood circulation, providing an alternative l -Arg delivery system for NO gas
therapy.
Statement of significance
NO gas therapy has attracted emerging interest in tumor treatment. However, the controlled NO release
and the avoidance of premature leakage of NO donors remain challenging. In this work, L-Arginine (L-Arg)
self-delivery supramolecular nanodrug for efficient tumor therapy is demonstrated through the Mannich
reaction of L-Arg, (-)-epigallocatechin gallate (EGCG) and formaldehyde. Stimulated by tumor microenvi-
ronment, the guanidine groups of L-Arg allow for accomplishing NO release and thus provoking tumor
cell apoptosis. The nanodrug also avoids the premature leakage of L-Arg in blood circulation. Moreover,
the preserved phenolic hydroxyl groups of EGCG combine with L-Arg to amplify apoptosis. The nanodrug
exhibits high biocompatibility and good therapeutic effect, providing an alternative L-Arg delivery system
for NO gas therapy.
©2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Gas therapy, which utilizes gaseous molecules accumulation to
accomplish diseases therapy, has attracted emerging interest in the
past decade [1–4] . As the most competitive candidate among gas
signaling molecules, such as carbon monoxide, hydrogen sulfide
and oxygen, nitric oxide (NO) is prominent in neural communi-
∗Corresponding authors.
∗∗ Corresponding author at: State Key Laboratory of Supramolecular Structure and
Materials, College of Chemistry, Jilin University, Changchun 13 00 12, PR China.
E-mail addresses: wanll@jlu.edu.cn (L. Wan) , liushuwei@jlu.edu.cn (S. Liu),
hao_zhang@jlu.edu.cn (H. Zhang) .
cation, vascular regulation, wound healing and other physiological
activities, making it the preferred model in innovative tumor ther-
apeutics featured with combination and cascade [5–9] . High con-
centrations of NO ( > 1 μM) can kill tumor cells through a vari-
ety of pathways, including nitrite stress, mitochondrial/DNA dam-
age, apoptosis and et al. [ 10 , 11 ]. Although a variety of NO sources,
for instance, S-nitrosoglutathione, S-nitroso-N-acetyl penicillamine
and nitrate functionalized d - α-tocopheryl polyethylene glycol suc-
cinate, are demonstrated to support NO-mediated tumor treatment
[12–14] , the complicated preparation procedures, high costs and
potential toxicity cause the doubt in clinical utilization.
L-Arginine (L-Arg), an endogenously existing and exogenously
supplemental amino acid with high biocompatibility, can release
https://doi.org/10.1016/j.actbio.2023.07.055
1742-7061/© 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
NO in organisms as its guanidine group is catalyzed by nitric oxide
synthase [15–17] . In addition, l -Arg can react with the excessive
H
2
O
2
in tumor microenvironment (TME) to produce NO via non-
enzymatic pathway, achieving targeting NO release for tumor ther-
apy with high biosafety [18–23] . Since the direct supply of exoge-
nous l -Arg molecules suffers from the shortcomings of short half-
life and low bioavailability, efficient l -Arg delivery to tumor sites
is required [24–27] . However, the conventional l -Arg delivery sys-
tems constructed through noncovalent linkages [28–32] inevitably
lead to premature leakage of the cargos during blood circulation.
Alternative delivery systems with stable l -Arg loading and control-
lable NO release are greatly welcome [ 33 , 34 ].
In this context, besides guanidine group, l -Arg possesses amino
and carboxyl groups, which is covalently linkable with other
segments to avoid premature leakage [ 35 , 36 ]. Mannich reac-
tion, a phenol-aldehyde type reaction, is expandable to multi-
component condensation in the presence of amine, aldehyde and
the molecules with active hydrogens [37] . With respect to l -Arg, its
amino group is available for Mannich reaction to link with a vari-
ety of functional molecules for designing novel therapeutic systems
[38] . (-)-Epigallocatechin gallate (EGCG) is a widely employed nat-
ural polyphenol in therapeutic applications, which exhibits plenty
of pharmacological functions caused by the abundant phenolic hy-
droxyl groups, such as inhibiting tumor cell proliferation and in-
ducing apoptosis [ 39 , 40 ]. Meanwhile, the active hydrogens adja-
cent to the phenolic hydroxyl groups of EGCG can be conducive
to modifying functional molecules [41] . And the conjugated frame-
work of EGCG favors the π- πstacking of neighboring molecules
[42] . According to the structural characteristics of EGCG and l -Arg,
it is reasonable to design self-delivery supramolecular nanodrugs
(SDSNDs) for tumor treatment with the combination of NO gas
therapy and EGCG-contributed apoptosis amplification.
In this work, Mannich reaction is employed to link l -Arg and
EGCG with the assistance of formaldehyde. The condensation prod-
ucts further assemble into SDSNDs through the π- πstacking of
EGCG framework. The covalent linkage between l -Arg and EGCG
avoids premature leakage of l -Arg, significantly improving the half-
life and bioavailability in drug delivery. Because the guanidine
group of l -Arg and the phenolic hydroxyl groups of EGCG are pre-
served in the as-produced SDSNDs, NO gas therapy by provoking
tumor cell apoptosis and the apoptosis amplification from EGCG
are realized. Further investigations reveal that the release of NO
is stimulated by the excessive H
2
O
2
in TME, and the efficacy in
HepG2 tumor model therapy is co-contributed by NO-resulted tu-
mor cell mitochondrial/DNA damage and apoptosis, EGCG-induced
tumor cell proliferation inhibition and apoptosis.
2. Materials and methods
2.1. Chemicals and reagents
(-)-Epigallocatechin gallate (EGCG) was purchased from Al-
addin Industrial Corporation (Shanghai, China). l -Arginine (L-
Arg), acetonitrile and paraf ormaldehyde were bought from Mack-
lin Biochemical Co., Ltd. (Shanghai, China). Formaldehyde (HCHO)
was purchased from Yongsheng Fine Chemical Co., Ltd (Tianjin,
China). 2,4-Dinitrophenylhydrazine (DNPH) was purchased from
Xiya reagent (Shandong, China). Folin-Ciocalteu’s phenol reagent,
cell counting kit-8 (CCK-8), Annexin V-FITC/PI apoptosis detection
kit, saline and Dulbecco’s modified eagle medium (DMEM) were
obtained from Beijing Solarbio Science & Technology Co., Ltd. (Bei-
jing, China). Trypsin-EDTA solution and phosphate buffer saline
(PBS) were supplied by Biosharp. Fetal bovine serum (FBS) was
provided by Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou,
China). Indocyanine green (ICG) was provided from Shanghai
yuanye Bio-Technology Co., Ltd. Griess reagent, hydrogen peroxide
assay kit, hoechst 33,342 staining solution for live cells, 3-amino,4-
aminomethyl-2
,7
-difluorescein, diacetate (DAF-FM DA), Calcein/PI
cell viability/cytotoxicity assay kit, enhanced mitochondrial mem-
brane potential assay kit with JC-1, immunostaining permeabiliza-
tion buffer with Triton X-100 and crystal violet staining solution
were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai,
China). γ-H2AX (phospho S139, Alexa Fluor 568) antibody was
purchased from Abcam (Cambridge, UK). All reagents are used
without further purification.
2.2. Characterization
The transmission electron microscopy (TEM) images and scan-
ning transmission electron microscopy with energy-dispersive X-
ray spectroscopy (STEM-EDS) elemental mapping images were cap-
tured by using a transmission electron microscopy (JEOL JEM-
2100F). The scanning electron microscopy (SEM) images were ob-
tained by field emission scanning electron microscopy (HITACHI
SU8020). The hydrated diameter and zeta potential were recorded
by using a zetasizer (Malvern NanoZS). The ultraviolet-visible-
near infrared (UV–vis-NIR) absorption spectra were performed on
a spectrophotometer (Shimadzu 2600). The Fourier-transform in-
frared (FTIR) spectra were measured by a Fourier transform in-
frared spectrometer (Brucker VRTEX 80 V). The content of N ele-
ment was detected on a CHNS/O elemental analyser (Agilent). The
HCHO content was measured by using a high performance liquid
chromatograph mass spectrometer (HPLC-MS, Agilent1290 - Bruker
micrOTOF QII). The absorbance tests in vitro were performed on
a microplate reader (BioTek Synergy LX). The quantity and mor-
phology of cells were observed by an inverted microscope. The cell
apoptosis was measured by a flow cytometer (BD FACSCalibur). The
fluorescent images were recorded by using the confocal laser scan-
ning microscope (CLSM, Nikon AXR). The fluorescent images were
performed with a near-infrared fluorescence imager (Raptor pho-
tonics Ninox 640 II).
2.3. Preparation of L-Arg SDSNDs
EGCG (1 mM) and HCHO (2 mM) were first stirred for seconds
in an aqueous solution containing 5% ethanol, then mixed with l -
Arg (2 mM) and stirred for 1 h at room temperature. The l -Arg
SDSNDs were obtained by centrifugation at 15, 0 0 0 rpm for 10 min,
and washed 3 times with diluted water.
2.4. Preparation of ICG-L-Arg SDSNDs
The pre-prepared l -Arg SDSNDs (1 mg/mL) were stirred with
ICG (0.5 mg/mL) at room temperature for 24 h. The ICG- l -Arg SD-
SNDs were obtained by centrifuging at 15, 0 0 0 rpm for 10 min.
2.5. Determination of EGCG content in L -Arg SDSNDs
The Folin-Ciocalteu method was adopted to determine the
EGCG content. Folin-Ciocalteu’s phenol reagent A and B were
freshly prepared. Briefly, l -Arg SDSNDs solution was mixed with
reagent A for 10 min, then mixed with reagent B followed by
30 min incubation at room temperature. And the absorbance at
750 nm was measured by a spectrophotometer. The calibration
curve was also measured by using EGCG at different concentrations
(0 to 150 μg/mL).
2.6. Measurement of HCHO content
The HCHO residue was measured by using HPLC-MS. The l -Arg
SDSNDs solution (4 mg/mL) was incubated with DNPH at 40 °C
for 60 min. Then the supernatant was collected for HPLC-MS test.
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M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
Standard HCHO solutions (0, 1 and 2 μg/mL) were also detected in
the same way.
2.7. Verification of interaction
The l -Arg SDSNDs were incubated with DMSO, urea, NaCl and
Triton X-100 solution, respectively. After 12 h incubation, the pre-
cipitates were centrifugated for TEM tests.
2.8. Exploration of composition release
A dialysis bag containing l -Arg SDSNDs is placed and stirred in
PBS aqueous solution at 37 °C. At the designated time points (0,
0.5, 1, 2, 4, 8, 12 and 24 h), the PBS aqueous solution was tested
using UV–vis absorption spectra.
2.9. NO detection in vitro
The generation of NO was detected by using Griess reagent
method and DAF-F M DA method. Different solutions (H
2
O
2
, l -Arg,
l -Arg SDSNDs, l -Arg + H
2
O
2
and l -Arg SDSNDs + H
2
O
2
) were in-
cubated at 37 °C for 1 h, respectively. For NO quantitative detec-
tion, the l -Arg SDSNDs at different concentrations (0, 250, 500 and
10 0 0 μg/mL) were incubated in the aqueous solution containing
H
2
O
2
(500 μM) at 37 °C for 10 min, 30 min, 1 h, 6 h and 24 h, re-
spectively. The l -Arg SDSNDs (200 μg/mL) were also incubated in
the aqueous solution containing H
2
O
2
(0, 10, 50, 100 and 200 μM)
at 37 °C for 2 and 4 h. Then the Griess reagent was added and
the supernatant was collected for UV–vis absorption detection. The
calibration curve was also obtained in the same procedure. For an-
other method, DAF -FM DA was added and incubated at 37 °C for
30 min in dark. The supernatant was collected for fluorescent de-
tection (Ex 495 nm, Em 515 nm).
2.10. Cell toxicity assay
The l -Arg SDSNDs with different concentrations were incubated
with mouse fibroblast cell lines (L929) and human hepatoma cell
lines (HepG2), respectively. After 24 h incubation, the cell viability
was measured by using the CCK-8 method.
2.11. Cell apoptosis assay
The l -Arg SDSNDs with different concentrations (0 to
200 μg/mL) were incubated with HepG2 cells. After 24 h in-
cubation, the cell apoptosis was measured by using Annexin
V-FITC/PI apoptosis detection kit on a flow cytometer.
2.12. Cell live/dead assay
The l -Arg SDSNDs with different concentrations (0 to
200 μg/mL) were incubated with HepG2 cells. After 24 h
incubation, the cells were stained with Calcein/PI cell viabil-
ity/cytotoxicity assay kit. The fluorescent images were recorded
using CLSM.
2.13. Cellular NO detection
The cellular NO generation was revealed with DAF-FM DA by
CLSM. The HepG2 cells were incubated with l -Arg SDSNDs (0, 50,
10 0 and 20 0 μg/mL) for 6 h, then washed with PBS. The DAF-FM
DA was added and incubated at 37 °C for 30 min in dark. After-
ward, the hoechst solution was employed to stain cell nucleus for
10 min. Then the fluorescent images were captured (Ex = 405 and
488 nm).
2.14. Mitochondrial membrane potential detection
The HepG2 cells were incubated with l -Arg SDSNDs (0, 50, 10 0
and 200 μg/mL) for 24 h. The JC-1 staining working solution was
added and incubated at 37 °C for 20 min in dark. Then the cells
were washed with JC-1 staining buffer solution 2 times. Afterward,
the mitochondrial membrane potential was observed using CLSM.
2.15. DNA damage assay
The HepG2 cells were incubated with l -Arg SDSNDs (0, 50, 10 0
and 200 μg/mL) for 24 h. Then the cells were fixed, permeabilized,
blocked and stained with γ-H2AX antibody. And the hoechst solu-
tion was employed to stain the cell nucleus for 10 min. Then the
fluorescent images were captured.
2.16. Colony forming cell (CFC) assay
The HepG2 cells were incubated with l -Arg SDSNDs (0, 25,
50, 100, 150 and 200 μg/mL) for 24 h. Then 500 cells of each
group were reseeded, respectively. After a 15-day incubation, the
cells were stained with crystal violet staining solution and pho-
tographed.
2.17. Hemolysis assay
The mouse red blood cells (2%) were incubated with Triton X-
100 (positive group), PBS (negative group) and PBS solution con-
taining l -Arg SDSNDs (0, 25, 50, 100, 150 , 200 and 500 μg/mL, ex-
perimental groups) at 37 °C for 1 h. Each group was centrifugated
at 30 0 0 rpm for 10 min and photographed. The supernatant was
collected for 541 nm absorbance (A) measurement. The hemolysis
ratio was calculated according to the following formula: hemolysis
ratio (%) = (A
experimental
- A
negative
) / (A
positive
- A
negative
)
∗100%.
2.18. Fluorescent images in vivo
The mouse was injected with 10 0 μL ICG- l -Arg SDSNDs
(5 mg/mL) through vein tails. The fluorescent images of the mouse
were captured by using a near-infrared fluorescence imager before
injection (control) and after injection.
2.19. Animal experiment
The animal experiments were carried out with the approval
(NO. 0693) of the Animal Laboratory Ethics Committee of the First
Hospital of Jilin University. The Balb/c nude mice (5 weeks, female)
were bought from Beijing Vital River Laboratory Animal Technol-
ogy Co., Ltd. The mice were subcutaneously injected with HepG2
cells on the right buttocks about 12 days in advance ( −12 d). Af-
ter building the tumor models, the mice were randomly divided
into four groups: control, l -Arg, EGCG and l -Arg SDSNDs. From 0
d to 16 d, the mice were injected without or with 127.5 μg l -Arg,
359.5 μg EGCG and 500 μg l -Arg SDSNDs solutions, respectively,
every two days. All solutions were at the equivalent concentration
of 5 mg/mL l -Arg SDSNDs. At the same time, the body weight and
tumor volume also need to be recorded. On 16 d, the blood was
taken for the liver and kidney function test. And all tumors, hearts,
livers, lungs, kidneys and spleens were fetched for H&E, TUNEL and
Ki67 staining.
2.20. Statistical analysis
The data were provided as the mean ±standard deviation. Stu-
dent’s t -test was used to calculate the difference for statistical sig-
nificance (
∗p < 0.05,
∗∗ p < 0.01,
∗∗∗ p < 0.001).
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M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
Fig. 1. The TEM image (a), SEM image (b), and Line STEM-EDS elemental mapping images (c) of l -Arg SDSNDs. The scale bar in (a), (b) and (c) is 50 0, 50 0 and 200 nm,
respectively.
3. Results and discussion
3.1. Preparation and characterization of L -Arg SDSNDs
The l -Arg SDSNDs are prepared through room-temperature
Mannich reaction by stirring an aqueous solution of l -Arg, EGCG
and HCHO for 1 h. As revealed by transmission electron mi-
croscopy (TEM) and scanning electron microscopy (SEM), the as-
prepared l -Arg SDSNDs appear as monodispersed spherical parti-
cles ( Fig. 1a and b ). The scanning transmission electron microscopy
with energy-dispersive X-ray spectroscopy (STEM-EDS) elemental
mapping images of l -Arg SDSNDs indicate the existence of C, O
and N elements (Fig. 1c and S1). In the Fourier-transform infrared
(FTIR) spectra, the broad peak from 30 0 0 to 3600 cm
−1
is assigned
to the overlap of O-H and N-H stretching vibration, and the peaks
at 167 5 and 1213 cm
−1 are attributed to C = N and C-N-C stretch-
ing vibration, which imply the guanidine group of l -Arg and the
phenol hydroxyls of EGCG are preserved in SDSNDs ( Fig. 2a ) [43] .
The appearance of C-O-C vibration peak at 1117 cm
−1 proposes
the generation of ether bond, indicating the occurrence of phenol-
formaldehyde type reaction. Moreover, the ultraviolet-visible (UV–
vis) absorption spectra exhibit a slight red-shift of EGCG charac-
teristic peak to 280 nm, which may be attributed to the molecular
π- πstacking and the modification of EGCG in SDSNDs ( Fig. 2b ).
The disassembly of l -Arg SDSNDs is investigated to reveal the non-
covalent bonds in the formation of SDSNDs using dimethyl sul-
foxide (DMSO), urea, NaCl and Triton X-100 to disrupt π- πin-
teraction, hydrogen bond, electrostatic interaction and hydropho-
bic interaction, respectively [44] . As indicated in Fig. S2, the l -
Arg SDSNDs disassemble only after incubating in DMSO solution,
proving that π- πstacking is dominant in the formation of SD-
SNDs. By combining the aforementioned results and the previous
report [45] , a proposed illustration of SDSND formation is shown
in Scheme 1 .
In order to regulate the size of l -Arg SDSNDs, the molar feeding
ratio of l -Arg/HCHO/EGCG is adjusted and the hydrated diameters
of the products are measured by dynamic light scattering (DLS).
As shown in Table S1, if the amount of l -Arg is higher than HCHO,
SDSNDs are failed to prepare. Whereas the hydrated diameters of
SDSNDs increase with the increment of EGCG proportion, reflecting
the increased SDSNDs size. In this context, the π- πstacking and
the formation of SDSNDs are mainly dependent on the conjugated
framework of EGCG. The π- πstacking is favored as more EGCG
participates in the formation of SDSNDs. To achieve better cellu-
lar uptake efficiency, the SDSNDs with different l -Arg/HCHO/EGCG
molar feeding ratio are prepared (Table S1). The SDSNDs are fur-
ther labeled with FITC for flow cytometry analysis. As shown in
Fig. S3, the SDSNDs with high l -Arg proportion are favored for cel-
lular uptake, which is attributed to the homing of Arg-modified
SDSNDs for Arg-starved HepG2 cells [46] . Ethanol is also intro-
duced to regulate the size of l -Arg SDSNDs, because it can adjust
solvent polarity and therewith interfere the π- πstacking (Table
S2). And the solution containing 5% ethanol is most favored for
preparing small SDSNDs. With the consideration of the size and ef-
ficient uptake for therapeutic applications [47] , the l -Arg SDSNDs
prepared with l -Arg/HCHO/EGCG molar feeding ratio of 2/2/1 and
5 v/v% ethanol are selected for further investigations. Under this
condition, the as-prepared SDSNDs present the average diameter
of 114 . 7 ±28.5 nm, average hydrated diameter of 152.4 nm and
zeta potential of −43.9 ±5.7 mV (Figs. S4b, 2c and d).
L-Arg SDSNDs are tried to prepare in the absence of HCHO,
but no monodispersed particles are produced (Fig. S5), showing
that HCHO is indispensable in the preparation of l -Arg SDSNDs.
To explore the biosafety, the HCHO residue is detected using 2,4-
520
M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
Fig. 2. The FTIR spectra (a) and UV–vis absorption spectra (b) of EGCG, l -Arg and l -Arg SDSNDs. The hydrated diameter (c) and zeta potential (d) of l -Arg SDSNDs.
dinitrophenylhydrazine (DNPH), because DNPH can be transformed
to DNPH derivative by HCHO [48] . In comparison with standard
HCHO solutions, the high performance liquid chromatograph mass
spectrometer (HPLC-MS) of 4 mg/mL l -Arg SDSNDs is far lower
than that of 1 μg/mL HCHO solution (Fig. S6). It means that the
l -Arg SDSNDs are completely safe by avoiding the potential health
risk of HCHO for further in vivo utilization [46] .
The quantitative analysis of EGCG and l -Arg contents is also in-
vestigated. EGCG content is measured according to Folin-Ciocalteu
method [46] . According to the absorbance at 750 nm, the calibra-
tion curve of EGCG is fitted ( Fig. 3a and b ). The absorbance of
200 μg/mL l -Arg SDSNDs is 0.1189. Thus, the EGCG content is cal-
culated as 143.8 μg/mL, approximately 71.9 wt% in the l -Arg SD-
SNDs. The content of N element in l -Arg SDSNDs is determined by
a CHNS/O elemental analyser, which is measured as 8.2 wt%, cor-
responding to the l -Arg content of 25.5 wt%.
3.2. Exploration of L -Arg and NO release
The l -Arg SDSNDs are incubated in phosphate buffer saline
solution (PBS, pH 7.4 ) at 37 °C to explore the possible release
of l -Arg and EGCG. All the spectra are basically same with PBS,
and no characteristic peaks of l -Arg and EGCG are observed
( Fig. 3c ). This result illustrates that l -Arg SDSNDs avoid the un-
necessary cargo release in normal physiological environment. Nev-
ertheless, l -Arg SDSNDs can produce NO under simulated TME,
which is proved both by Griess reagent method and 3-amino,4-
aminomethyl-2
,7
-difluorescein, diacetate (DAF-FM DA) method
[49] . In Griess reagent method, the absorption peak at 540 nm rep-
resents the generation of NO as l -Arg and l -Arg SDSNDs are trig-
gered with H
2
O
2
( Fig. 3d ). For DAF-FM DA method, DAF-FM DA can
react with NO and generate strong fluorescence at 515 nm. The en-
hanced fluorescence intensity with the increased l -Arg SDSND con-
centration demonstrates the effective generation of NO ( Fig. 3e ).
The quantitative analysis of NO generation is further studied by
Griess reagent method. According to the calibration curve ( Fig. 3f ),
the dependence of NO content on the concentration of l -Arg SD-
SNDs and the duration of H
2
O
2
trigger is summarized in Fig. 3 g .
The NO content indicates a positive correlation with the concen-
tration of l -Arg SDSNDs, which consists with the results in Fig. 3e .
In addition, the maximum absorbance appears after 1 h H
2
O
2
trig-
ger, probably attributing to the NO effuse along with the duration.
Besides, more NO is generated with more H
2
O
2
stimulation (Fig.
S7). These results demonstrate that the l -Arg SDSNDs can generate
NO with H
2
O
2
trigger ( Fig. 3h ) [18] . The no leakage of l -Arg and
H
2
O
2
-stimulated NO release are capable to facilitate antitumor gas
therapy.
3.3. Cellular NO generation
The NO gas antitumor of l -Arg SDSNDs is studied in vitro . First,
the physical stability of l -Arg SDSNDs is monitored. After incubat-
ing at room temperature for 1 month, the morphology, size and
hydrated diameter of SDSNDs are basically unchanged in compar-
521
M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
Scheme 1. Schematic illustration of the preparation of l -Arg SDSNDs and their application in NO gas tumor therapy.
ison with the newly prepared ones, reflecting the good physical
stability (Figs. S8 and S9). Then the physiological stability of l -Arg
SDSNDs is conducted by incubating in H
2
O, PBS, Dulbecco’s mod-
ified eagle medium (DMEM) and DMEM with fetal bovine serum
(FBS) at 37 °C. After incubating for 16 days, l -Arg SDSNDs dis-
perse well in all solutions (Fig. S10), exhibiting the good stabil-
ity in physiological environment. To explore the dispersion stabil-
ity in biological media, the TEM image of SDSNDs is measured af-
ter incubation in culture medium for 16 days. The morphology and
size of SDSNDs are basically unchanged, indicating the good dis-
persion stability in biological media (Fig. S11). The aforementioned
good stability guarantees the further utilization in the biological
applications. The toxicity of l -Arg SDSNDs to mouse fibroblast cell
lines (L929) and human hepatoma cell lines (HepG2) is evaluated
through the CCK-8 method. With the increment of SDSND concen-
tration, the viability of L929 cells still keeps at a high level (Fig.
S12). While the viability of HepG2 cells reduces to approximately
50% at 200 μg/mL (Fig. S13). The selective toxicity of l -Arg SDSNDs
to cancerous HepG2 cells is attributed to two reasons. On the one
aspect, the great demand of nutrition in cancerous cell survival and
proliferation facilitates the uptake of SDSNDs via micropinocytosis
[50] . On the other aspect, the excessive H
2
O
2
in tumor cells accel-
erates the release of NO from l -Arg SDSNDs (Figs. S14 and S15).
Both of the two effects favor the selective NO damage towards tu-
mor cells.
To certify this consideration, the NO content of l -Arg SDSNDs-
treated HepG2 cells is conducted by virtue of DAF-FM DA-based
fluorescent detection. As presented in Fig. 4a , the intensity of flu-
orescent images is positively related to the concentrations of l -
Arg SDSNDs, illustrating more NO is produced. Accordingly, the
three-dimensional (3D) surface plots and relative intensity intu-
itively manifest the effective generation of NO from l -Arg SDSNDs
in HepG2 cells (Figs. 4b and S16). For further quantitative analysis,
the intracellular NO concentration of HepG2 cells after incubation
with different concentration of l -Arg SDSNDs is studied by using
Griess reagent. There is far beyond 1 μM NO generated with the in-
creasing SDSNDs concentration, which is enough to induce apopto-
sis [10] , indicating the good capability of l -Arg SDSNDs for NO de-
livery (Fig. S17). The mitochondrial membrane potential is further
assessed to evaluate NO-induced mitochondrial dysfunction using
JC-1 fluorescent probe. When mitochondrial membrane potential is
high, JC-1 aggregates in the mitochondrial matrix and produces red
fluorescence. When mitochondrial membrane potential is low, JC-1
exists as monomer and exhibits green fluorescence. In the experi-
ment, stronger green fluorescence is observed with the increment
of l -Arg SDSNDs concentration, representing the lower mitochon-
drial membrane potential (Figs. 4c and S18). This means that the
generated NO from l -Arg SDSNDs reduces the mitochondrial mem-
brane potential of HepG2 cells. The increase of intracellular NO
levels also leads to DNA damage of tumor cells. In the γ-H2AX im-
munofluorescence study, the l -Arg SDSNDs-treated HepG2 cells ex-
hibit stronger fluorescence, revealing the occurrence of DNA dam-
age ( Fig. 4d ). The relative intensity of the γ-H2AX fluorescent im-
ages is also measured by imageJ, showing the enhanced intensity
with SDSND concentrations (Fig. S19). Both the DNA damage and
the decrease of mitochondrial membrane potential are the hall-
marks of apoptosis. The control experiments are also conducted in
L929 cells. There is barely NO generation (Figs. S20 and S21), be-
cause of the low H
2
O
2
concentration in L929 cells (Fig. S15). After-
ward, there is basically no mitochondrial membrane potential dys-
function (Figs. S22 and S23) and DNA damage (Figs. S24 and S25)
after SDSNDs treatment, because of few NO generation.
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M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
Fig. 3. (a) The vis-NIR absorption spectra of EGCG at different concentrations (0 to 15 0 μg/mL) mixed with Folin-Ciocalteu’s phenol reagent. (b) The fitting calibration curve
according to the absorbance at 750 nm in (a). (c) The UV–vis absorption spectra of the supernatants of l -Arg SDSNDs incubated in PBS for different duration. (d) The UV–vis
absorption spectra of the supernatants of different solutions (H
2
O
2
, l -Arg, l -Arg SDSNDs, l -Arg + H
2
O
2 and l -Arg SDSNDs + H
2
O
2
) mixed with Griess reagent. (e) The
fluorescent spectra of the supernatants of l -Arg SDSNDs incubated with DAF-F M DA (Ex 495 nm, Em 515 nm). (f) The calibration curve of NO detection by Griess reagent.
(g) The generate d NO quantitative detection of the l -Arg SDSNDs at different concentrations
(0, 250, 500 and 1000 μg/mL) by Griess reagent. (h) Schematic illustration of
NO ge neration from l -Arg SDSNDs.
3.4. Apoptosis amplification of EGCG in L-Arg SDSNDs
To evaluate the apoptosis amplification of EGCG in l -Arg SD-
SNDs, Annexin V-FITC/PI apoptosis detection kit is employed and
detected by flow cytometer. As mentioned in the previous reports
[ 39 , 40 ], EGCG can induce apoptosis and inhibit tumor cell pro-
liferation. To clarify the function of EGCG in l -Arg SDSNDs, the
apoptosis of HepG2 cells is estimated after treating by l -Arg, EGCG
and l -Arg SDSNDs with the equivalent concentration of 0, 25, 50,
100, 150 and 200 μg/mL l -Arg SDSNDs. Compared with the control
group, there is obvious cell apoptosis in l -Arg, EGCG and l -Arg SD-
SNDs groups (Figs. S26, S27 and 5 a). Moreover, the cell apoptosis
ratio of 30.0 ±1. 0% in l -Arg SDSNDs group is much higher than
that treated with l -Arg (10.9 ±3.9%) at the equivalent concentra-
tion of 200 μg/mL l -Arg SDSNDs, which results from the apoptosis
amplification of EGCG in l -Arg SDSNDs. On the one hand, it means
that l -Arg SDSNDs remain the activity of EGCG for inducing can-
cerous cell apoptosis and the combination of NO-induced HepG2
cell apoptosis with EGCG-induced one leads to an apoptosis am-
plification. On the other hand, the l -Arg SDSNDs avoid the high
toxicity of EGCG to normal cells (Figs. S12, S27 and S28). In addi-
tion, the proliferation of HepG2 cells is estimated by treating with
l -Arg SDSNDs at different concentrations (
Fig. 5b ). The live/dead
assay demonstrates that more dead cells are observed at higher
SDSND concentrations, though many cells are still alive. To figure
out the proliferation of these alive cells, colony forming cell assay
is conducted. After reseeded and incubated for 15 days, the cell
proliferation exhibits a declining trend ( Fig. 5c ). The relative clone
formation rate in the group of 200 μg/mL is only 26.0%, which re-
veals that the proliferation of l -Arg SDSNDs-treated HepG2 cells is
greatly inhibited ( Fig. 5d ).
3.5. In vivo tumor therapy of L -Arg SDSNDs
Because of the efficacy of l -Arg SDSNDs on HepG2 cells in vitro,
in vivo investigation is further performed on the basis of HepG2
tumor model. The hemolysis assay is first carried out to evalu-
ate the biocompatibility of l -Arg SDSNDs. As shown in Fig. S29,
l -Arg SDSNDs do not destroy red blood cells even the concentra-
tion is up to 500 μg/mL, exhibiting the potential for in vivo utiliza-
tion. To analyze the pharmacokinetics, indocyanine green (ICG) is
labeled on l -Arg SDSNDs via the π- πstacking with EGCG frame-
work to produce ICG- l -Arg SDSNDs according to the red-shift of
the ICG absorption peak at 217 nm in Fig. S30 and the previous re-
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M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
Fig. 4. The CLSM images (a) and 3D surface plots (b) of l -Arg SDSNDs (0, 50, 100 and 200 μg/mL) treated HepG2 cells after incubating with DAF-FM DA . The CLSM images
of l -Arg SDSNDs (0, 50, 10 0 and 200 μg/mL) treated HepG2 cells after incubating with JC-1 (c) and γ-H2AX antibody (d). The scale bar in (a), (c) and (d) is 100 , 50 and
100 μm, respectively.
port
[51] . The appearance of the typical absorption at 795 nm (Fig.
S30) and the photoluminescent emission at 805 nm (Fig. S31) con-
firms the successful labeling of ICG on l -Arg SDSNDs. As indicated
in Figs. S32 and 6 a, the ICG- l -Arg SDSNDs exhibit good fluores-
cence stability, which guarantees the pharmacokinetics studies in
vivo . The half-life of SDSNDs in blood circulation is calculated to
be 8.3 ±1.0 h ( Fig. 6b ) according to the blood fluorescence inten-
sity of the mice after injecting with ICG- l -Arg SDSNDs (Fig. S33),
which is much higher than that of l -Arg (80 ±9 min) and EGCG
(62 ±11 min), showing the improved bioavailability [ 52 , 53 ]. The
distribution and metabolism of ICG- l -Arg SDSNDs in various or-
gans are also evaluated through the quantitative analysis of fluo-
rescence intensity of various organs over time. In the first 12 h,
the SDSNDs mainly exist in liver, lungs, kidneys, spleen and tu-
mor. Subsequently, the SDSNDs content in major organs decreases
over time, while the retention in the tumors can keep for 5 days,
which implies the good metabolism and potential long-time treat-
ment of the tumors ( Fig. 6c -e). To explore the excretion, after be-
ing administrated with ICG- l -Arg SDSNDs, the feces and urine of
mice are collected for fluorescence imaging. Both of them show
strong fluorescent signals at 4, 8 and 12 h ( Fig. 6f ), which indi-
cates that the SDSNDs can excrete via renal excretion and biliary
excretion.
The good metabolization and biodistribution of l -Arg SDSNDs in
tumor tissues intimate the viability for in vivo antitumor. The ani-
mal experiment is conducted by pre-building subcutaneous HepG2
tumor models in advance of 12 days ( Fig. 7a ). The mice bearing
tumor are randomly divided into four groups, namely Control, l -
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M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
Fig. 5. (a) The flow cytometry of l -Arg SDSNDs treated HepG2 cells by using Annexin V-FITC/PI apoptosis detection kit. (b) The CLSM images of l -Arg SDSNDs (0, 50, 100
and 20 0 μg/mL) treated HepG2 cells after incubating with Calcein/PI cell viability/cytotoxicity assay kit. The scale bar is 200 μm. (c) The images of CFC assay. (d) The relative
clone formation rate according to (c).
525
M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
Fig. 6. (a) The quantitative fluorescent intensity analysis of ICG- l -Arg SDSNDs incubated in culture medium for 5 days in Fig. S32. (b) The half-life of l -Arg SDSNDs. (c) The
fluorescent images of mice after treated with ICG- l -Arg SDSNDs before (Control) and after 4, 8, 12, 24, 48, 72 and 120 h. (d) The fluorescent images of major organs (heart,
liver, spleen, lungs and kidneys) and tumor of the mice after treated with ICG- l -Arg SDSNDs. (e) The biodistribution of l -Arg SDSNDs in major organs and tumors according
to Fig. 6d. (f) The fluorescent images
of the feces and urine of the mice after treated with ICG- l -Arg SDSNDs. The scale bar is used for 6c, 6d and 6f.
Arg, EGCG and l - Arg SDSNDs. From 0 to 16 days, the mice in each
group are administrated with corresponding drug solutions, re-
spectively, every two days. At the same time, the body weight and
tumor volume of mice in all groups are measured and recorded. On
the 16t h day, the mice are captured and euthanatized. As shown
in Fig. 7b , the relative tumor volume of l -Arg SDSNDs group is
far smaller than other groups, directly manifesting the good an-
titumor effect of l -Arg SDSNDs. The average tumor weight shows
the same trend, according to which the tumor growth inhibition
(TGI) is calculated ( Fig. 7c and d ). The TGI of l -Arg SDSNDs is up
to 91.4 ±6.5%, much higher than those of other groups, which ex-
hibits the superiority of SDSNDs by combining the antitumor ef-
fect of l -Arg and EGCG. The images of mice bearing tumors and
dissected tumors are shown in Fig. 7e . Furthermore, the slices of
the tumors are stained by H&E, TUNEL and Ki67 to estimate the
state of the tumor cells ( Figs. 7 f, S34 and S35). The l -Arg SDSNDs
therapy group induces highest apoptosis and lowest proliferation
in tumor tissues, suggesting l -Arg SDSNDs possess good antitumor
effect. Note that although l -Arg is the source of NO and EGCG can
induce apoptosis, the therapeutic effect of l -Arg group and EGCG
group is very limited in animal experiments owing to two reasons.
First, the half-lives of free l -Arg and EGCG in blood circulation are
much lower than that of SDSNDs ( Fig. 6b ), and the quick renal
clearance results in the lower bioavailability. Second, the combi-
nation of NO-induced cell apoptosis with EGCG-induced one by l -
Arg SDSNDs leads to the apoptosis amplification ( Fig. 5a ). With re-
spect to biosafety, the relative body weight of mice in all groups is
not significantly different and shows steadily increase from 0 to 16
days (Fig. S36). On the 16th day, the blood of mice is obtained for
major liver and renal function tests. All indexes of each group are
normal (Fig. S37). The state of major organs, including heart, liver,
lungs, kidneys and spleen, are also investigated by H&E staining,
showing no obvious side effect in all groups (Fig. S38). All these
results confirm the good biosafety of l -Arg SDSNDs.
526
M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
Fig. 7. (a) The Schematic illustration of animal experiment. The records of relative tumor volume (b) and average tumor weig ht (c) of the mice in each group (Control, l -Arg,
EGCG and l -Arg SDSNDs). (d) The TGI rate according to (c). (e) The images of mice bearing tumor and tumors dissected in each group on 16 d (f) The images of H&E stained
tumor tissue slices of the mice in each group. The scale bar in (f) is 20 μm.
4. Conclusion
In summary, carrier- and surfactant-free SDSNDs are prepared
by combining the Mannich reaction of l -Arg, EGCG and HCHO and
the assembly of the products via molecular π- πstacking. The for-
mation of l -Arg SDSNDs not only improves the half-life of l -Arg,
but also avoids the leakage of l -Arg in physiological environment.
With the stimulation of excessive H
2
O
2
in TME, the retained guani-
dine group of l -Arg can release NO for gas antitumor by provok-
ing tumor cell mitochondrial/DNA damage and apoptosis. Mean-
while, the combination of the preserved phenolic hydroxyl groups
of EGCG with NO leads to tumor cell apoptosis amplification and
proliferation inhibition, exhibiting good efficacy in HepG2 tumor
models. This work provides “no loss and NO release” SDSNDs,
which is an alternative for further designing innovative tumor ther-
apeutic systems with NO-associated synergism and cascade.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the Science and Technology Devel-
opment Program of Jilin Province (YDZJ202201ZYTS055), the Sci-
527
M. Zhang, H. Jin, Y. Liu et al. Acta Biomaterialia 169 (2023) 517–529
ence and Technology Project of Jilin Provincial Department of Ed-
ucation (JJKH20221079KJ), the Project funded by China Postdoc-
toral Science Foundation (2021TQ0125, 2022M711303), the Inter-
disciplinary Integration and Innovation Project of Jilin University
(JLUXKJC2021QZ10), and Special Project from MOST of China.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.actbio.2023.07.055 .
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