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DOI: ./VIW.
RESEARCH ARTICLE
Nanoantidote for repression of acidosis pH promoting
COVID-19 infection
Qidong Liu1,2Huitong Ruan3Zhihao Sheng4Xiaoru Sun1Siguang Li2
Wenguo Cui3Cheng Li1
Department of Anesthesiology and Perioperative Medicine, Shanghai Fourth People’s Hospital, School of Medicine, Tongji University, Shanghai, P. R.
China
Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopedic Department, Tongji Hospital, School
of Medicine, Tongji University, Shanghai, P. R. China
Department of Orthopaedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology
and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China
Department of Anesthesiology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai, P. R. China
Correspondence
Cheng Li, Department of Anesthesiology
and Perioperative Medicine, Shanghai
Fourth People’s Hospital, School of
Medicine, Tongji University, No. ,
Sanmen Rd, Shanghai , P. R. China.
Email: chengli_@tongji.edu.cn
Siguang Li, Key Laboratory of Spine and
Spinal Cord Injury Repair and
Regeneration of Ministry of Education,
Orthopedic Department of Tongji
Hospital, School of Medicine, Tongji
University, No. , Xincun Rd, Shanghai
, P. R. China.
Email: lisiguang@tongji.edu.cn
Wenguo Cui, Department of
Orthopaedics, Shanghai Key Laboratory
for Prevention and Treatment of Bone and
Joint Diseases, Shanghai Institute of
Traumatology and Orthopaedics, Ruijin
Hospital, Shanghai Jiao Tong University
School of Medicine, Ruijin nd Rd,
Shanghai , P. R. China.
Email: wgcui@hotmail.com
Qidong Liu, Huitong Ruan, and Zhihao
Sheng contributed equally to this work.
Funding information
National Key Research and Development
Program of China, Grant/Award Number:
Abstract
Acidosis, such as respiratory acidosis and metabolic acidosis, can be induced by
coronavirus disease (COVID-) infection and is associated with increased
mortality in critically ill COVID- patients. It remains unclear whether acidosis
further promotes SARS-CoV- infection in patients, making virus removal diffi-
cult. For antacid therapy, sodium bicarbonate poses great risks caused by sodium
overload, bicarbonate side effects, and hypocalcemia. Therefore, new antacid
antidote is urgently needed. Our study showed that an acidosis-related pH of .
increases SARS-CoV- receptor angiotensin-converting enzyme (ACE) expres-
sion on the cell membrane by regulating intracellular microfilament polymer-
ization, promoting SARS-CoV- pseudovirus infection. Based on this, we syn-
thesized polyglutamic acid-PEG materials, used complexation of calcium ions
and carboxyl groups to form the core, and adopted biomineralization methods to
form a calcium carbonate nanoparticles (CaCO-NPs) nanoantidote to neutral-
izeexcesshydrogenions(H
+), and restored the pH from . to approximately
. (normal blood pH). CaCO-NPs effectively prevented the heightened SARS-
CoV- infection efficiency due to pH .. Our study reveals that acidosis-related
pH promotes SARS-CoV- infection, which suggests the existence of a positive
feedback loop in which SARS-CoV- infection-induced acidosis enhances SARS-
CoV- infection. Therefore, antacid therapy for acidosis COVID- patients is
necessary. CaCO-NPs may become an effective antacid nanoantidote superior
to sodium bicarbonate.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the
original work is properly cited.
© The Authors. VIEW published by Shanghai Fuji Technology Consulting Co., Ltd, authorized by Professional Community of Experimental Medicine, National Associ-
ation of Health Industry and Enterprise Management (PCEM) and John Wiley & Sons Australia, Ltd.
VIEW. ;:. wileyonlinelibrary.com/journal/view 1of15
https://doi.org/./VIW.
2of15 LIU .
YFA; National Natural
Science Foundation of China,
Grant/Award Numbers: ,
, ; Natural Science
Foundation of Shanghai, Grant/Award
Number: ZR; The Foundation
of Shanghai Fourth People’s Hospital
Affiliated to Tongji University,
Grant/Award Number: sykyqd
KEYWORDS
ACE, acidosis, antacid, COVID-, F-actin, nanoantidote, SARS-CoV-
1 INTRODUCTION
COVID- is caused by SARS-CoV-, a novel coronavirus
that leads to many symptoms, from asymptomatic to crit-
ical illness and even death.COVID- patients who die
are often critically ill.In severe cases, the disease can lead
to acute respiratory distress syndrome, metabolic acidosis,
and multiple organ dysfunction syndromes, among other
complications.Metabolic acidosis, especially diabetic
ketoacidosis (DKA), in COVID- patients has been widely
concerning.SARS-CoV- infection not only induces DKA
in those with diabetes but also directly causes diabetes mel-
litus and DKA.b,, Additionally, COVID- infection also
induces respiratory acidosis in critically ill patients.To
rapidly reverse acute acidemia, the mainstay of therapy
in the past placed great emphasis on intravenous sodium
bicarbonate.Sodium bicarbonate decreases the primary
composite outcome and day mortality in an a priori-
defined stratum of patients with acute kidney injury.
However, the use of sodium bicarbonate infusion is also
controversial. More data argue against the bicarbonate.
The adverse effect of sodium bicarbonate is considered to
be the administered sodium load, which can induce hyper-
volemia, hyperosmolarity, and hypernatremia.Bicarbon-
ate is also associated with delayed ketone clearance and
worsened hypokalemia.Metabolic alkalosis, hyperna-
tremia, and hypocalcemia are observed more frequently
in bicarbonate-treated patients., Hypocalcemia may also
lead to cardiovascular disease. Previous study proposed a
Mg-based micromotor for regulating the pH in stomach.
However, further research is needed to develop new and
safer antacid antidote drugs to balance the blood pH
of patients with acidosis, especially acidosis induced by
COVID-.
Although it is known to cause serious complica-
tions, it is unclear whether acidosis promotes SARS-
CoV- virus infection, causing a vicious cycle and wors-
ening COVID-. For SARS-CoV- virus infection, the
first step is binding of the viral trimeric spike protein
to the membrane-bound form of the cellular receptor
angiotensin-converting enzyme (ACE). The ACE
recombinant protein shows a great potential for inhibit-
ing SARS-CoV- infection. Our previous study devel-
oped an inhaled ACE-engineered microfluidic micro-
sphere for neutralization of COVID-. ACE is expressed
not only in lung, but also in blood vessels, as well as
in other tissues., Especially, the lung has many vas-
cular networks for pulmonary circulation. There is vas-
culopathy and vascular thrombosis in lungs from both
humans and rhesus macaques infected with SARS-CoV-
. SARS-CoV- can directly infect human blood vessel
cells. SARS-CoV- infection causes vascular endothe-
lial cell dysfunction and vascular endothelial injury.b,
Higher cellular ACE expression on the membrane is
more conducive to virus infection. SARS-CoV- utilizes
some means to enhance infection efficiency. A previous
study indicated that SARS-CoV- exploits species-specific
interferon-driven upregulation of ACE to enhance virus
infection. ACE can be significantly upregulated after
SARS-CoV- infection by stimulation with inflammatory
cytokines. These studies suggested that after infection,
COVID- may induce upregulation of ACE in cells to
promote further infection for SARS-CoV- virus. In arte-
rial vessels, a decrease in arterial blood pH directly indicted
acidosis. However, it remains largely unknown whether
the decrease in blood pH in patients with COVID--
induced acidosis promotes SARS-CoV- infection by regu-
lating expression of ACE on the cellular membrane, caus-
ing deterioration in critically ill patients.
ACE exists in two forms: cell membrane and plasma
(soluble). Membrane ACE levels can be decreased by
angiotensin II, which induces ACE transport to the per-
inuclear space. Rearrangement of the actin cytoskele-
ton has been shown to play important roles in intra-
cellular trafficking. ACE is reported to localize with
actin. The actin bundling protein fascin- regulates the
expression levels and subcellular localization of ACE.
With regard to infection by another kind of human
coronavirus, NL, which also needs to interact with
ACE, actin cortex remodeling is required for virus
endocytosis. Changes in pH can influence actin poly-
merization and depolymerization. Indeed, interaction of
the actin-depolymerizing factor (ADF)/cofilin family and
F-actin is markedly pH dependent. Arterial blood pH
ranges between . and . under normal physiological
conditions, whereas that of acidosis patients is lower than
LIU . 3of15
.. In some cases, it decreases to . and even . in
patients with acidosis induced by COVID-. Whether
this apparent change in arterial pH value regulates the
level of membrane ACE is unknown.
In this study, we first discovered that acidosis-related
pH apparently promotes the infection efficiency of SARS-
CoV- pseudovirus to ACE overexpression HEKT
(HEKT-ACE) cell, or human umbilical vein endothe-
lial cell (HUVEC). According to this finding, we focused
on the CaCOto explore the potential possibility of neu-
tralizing acids. Recent years, materials science has shown
its more and more advantages in the understanding, diag-
nosis, or treatment of the viral diseases. Some carefully
designed nanomaterials have the potential direct function
of fighting viruses. Based on our previous study expe-
rience about biomaterials, we synthesized polyglutamic
acid-polyethylene glycol (PGlu-PEG) materials in which
polyglutamate (Glu) provides carboxyl groups to interact
with Ca+, preventing the mineralization of large CaCO
blocks, and PEG shell acts to prevent agglomeration and
aggregation of particles. Then use complexation of calcium
ions and carboxyl groups to form the core and adopted
biomineralization methods to form a calcium carbonate
nanoparticles CaCO-NPs. We found that this (CaCO-
NPs) nanoantidote apparently prevents acidosis to reduce
the SARS-CoV- infection efficiency enhanced by acidosis.
We further studied the molecular mechanism and found
that acidosis-related pH promotes SARS-CoV- infection
by increasing ACE expression on the membrane via inhi-
bition of actin polymerization. Using the CaCO-NPs anti-
dote suppressed the effects of acidosis-related pH in pro-
moting SARS-CoV- pseudovirus infection. Our research
suggests that a “positive feedback loop,” that is, acidosis
caused by SARS-CoV- infection may promote SARS-CoV-
infection in patients, resulting in a vicious cycle. There-
fore, it is important to perform antacid treatment for crit-
ically ill COVID- patients. Given the existing problems
of sodium bicarbonate, we propose that this CaCO-NPs
is a new and better antidote for treatment of acidosis that
inhibits infection of SARS-CoV-.
2RESULTS
2.1 Acidosis-related acidic pH
conditions induced higher SARS-CoV-2
viral infection efficiency than normal
physiological pH conditions
We cultured human ACE-overexpressing HEKT
(HEKT-ACE) cells with normal culture medium
DMEM containing % fetal bovine serum (FBS).
HEKT-ACE cell line can simulate the human cell
expressing ACE protein for studying the SARS-CoV-
virus infection. SARS-CoV- pseudovirus used its trimeric
spike protein to bind with the ACE protein on HEKT
cell for further infection. HEKT cell line can be con-
tinuously propagated for many passages, which facilitate
the enough supplement of cells for the study. The next day
after the cell seeding, we changed the culture medium to
one at pH ., pH ., or pH . HEPES solution medium.
Then added to a SARS-CoV- pseudovirus that has S
protein and express the green fluorescent protein (GFP).
Six hours later, we changed the culture medium to be
normal fresh culture medium (DMEM, % FBS) for
-hour culture. GFP fluorescence was determined to
indicate SARS-CoV- pseudovirus infection. We found
that compared to the normal human blood-related pH
value of pH ., the COVID- patient acidosis-related pH
value (pH .) apparently increased the SARS-CoV-
infection efficiency, as indicated by more GFP fluores-
cence (Figure A,B). We further found that pH . resulted
in reduced efficiency compared to pH ., which indicated
weakly alkaline inhibition of virus infection (Figure A,B).
To determine whether h of treatment with medium at
different pH values affects HEKT-ACE cell viability
to influence virus infection, we performed a CCK- assay
and found no significant difference in cell viability after
h of treatment in different pH values (Figure C). ACE
is expressed in the cardiovascular system. SARS-CoV-
virus infects human blood vessel cells and causes vascular
endothelial cell dysfunction and vascular endothelial
injury.b, Here, we investigated that whether pH value
influences the SARS-CoV- virus infection of human
blood vessel cells. So, we used the HUVECs, which express
the ACE itself, and cultured in different pH value
culture mediums to simulate the vascular endothelial
cell in different pH value environments in blood vessel.
We infected HUVECs with the SARS-CoV- pseudovirus
and confirmed that acidic conditions promote infection
(Figure D,E). Six hours of treatment with medium at
different pH values also did not affect the viability of
HUVECs (Figure F). These data indicate that acidosis-
associated acidic pH conditions significantly promote
SARS-CoV- infection compared with the normal blood
pH, which suggests a potential vicious cycle in patients
with COVID- acidosis (Figure G).
2.2 Schematic and characterization of
antacid CaCO3-NPs
To construct the antiacid nanodote, the functional poly-
mer mPEG-P(Glu) was synthesized, and the degree of poly-
merization was calculated to be using H-NMR. Charac-
terization of mPEG-P(Glu) copolymers: MHz, in DO,
4of15 LIU .
FIGURE 1 Acidic conditions result in a higher viral infection rate than alkaline conditions. (A) GFP fluorescence indicates SARS-CoV-
pseudovirus infection in HEKT-ACE cells. The infection rate was higher at pH . than at pH . or .. The group with no virus was used
as the negative control, with no significant fluorescence of HEKT-ACE cells. (B) Statistics of the median fluorescence intensity of GFP in
(A). (C) CCK- assays indicated that different pH values (., ., and .) did not influence cell viability during a -h treatment. (D)
SARS-CoV- pseudovirus infection of HUVECs in medium with different pH values. (E) Statistics of the median fluorescence intensity of GFP
in (D). (F) CCK- assays detected the viability of HUVECs at different pH values (., ., and .) during a -h treatment. The data shown are
means ±SDs (n=). (G) Proposed consequences of acidic conditions: pH causes a higher viral infection rate than normal blood pH
conditions. *p<., **p<.. Asterisks (*) indicate statistical significance for the pH . and . groups compared with the pH . group; ns:
no significant difference. Scale bar: μm
LIU . 5of15
PEG: -OCHCH,δ=. ppm, PGlu: -CHCHCOO-:
δ=. ppm, δ=. ppm (Figure A). Then, CaCO-
NPs were prepared using mPEG-P(Glu) block copolymers
to interact with Ca+and CO−in aqueous solution. Glu
provides carboxyl groups to interact with Ca+,prevent-
ing the mineralization of large CaCOblocks, and the PEG
shell acts to prevent agglomeration and aggregation. The
average diameter of the CaCO-NPs was approximately
nm, with a PDI value less than ., indicating that the
particles are uniform (Figure B). The morphology and ele-
ment distribution of CaCO-NPs were analyzed by energy
dispersive spectrometry (TEM-EDS), showing that this
nanoparticle was composed of Ca, C, and O and was homo-
geneous (Figure C,D). The X-ray diffraction (XRD) anal-
ysis was also performed to investigate the crystalline struc-
tures of CaCO-NPs, and the result turned out to be vaterite
and calcite (Figure E). We detected that the zeta poten-
tial (ZP) of CaCO-NP was −. ±. mV. This weak
negative charge of NPs suggested that there may be no sig-
nificant interaction with proteins by interaction of charges
and there may be low cytotoxicity (Figure F). CaCO-NPs
were able to react with hydrogen ions (H+) to exert the
acidic microenvironment (Figure G). We applied differ-
ent amounts of CaCO-NPs (, , , , , μg/ml
final concentration) to regulate the pH value of the pH .
and pH . solutions. With increasing amounts of CaCO-
NPs, the pH value of the pH . solution increased grad-
ually and reached approximately pH . at μg/ml.
While for the pH . solution, the CaCO-NPs caused no
significant changes in pH value (Figure H). Precise pH
test paper was also used to assess how the CaCO-NPs
( μg/ml) altered the pH value of the pH . or . solu-
tion (Figure I). Further, we detected the CaCO-NPs func-
tion in different pH values FBS to simulate the CaCO-NPs
working in the blood, and determined whether the CaCO-
NPs can neutralize the hydrogen ions to restore normal
serum pH. First, we determined that the pH value of FBS
is about ., which was also similar with the pH of normal
blood in human beings. μg/ml CaCO-NPs in serum
had no apparent influence on the serum pH value. In
order to simulate the acidosis blood, the pH of serum was
downregulated to . by adding concentrated hydrochlo-
ric acid slowly. Then the CaCO-NPs was added at the
concentration of μg/ml. It is found that the pH was
apparently restored by supplementation with CaCO-NPs
(Figure J).
2.3 CaCO𝟑-NPs neutralize acid to inhibit
viral infection of SARS-CoV-2
We added CaCO3-NPs (final concentration μg/ml) to
pH . medium and observed an apparent attenuation of
the promotion of virus infection caused by pH .. The
infection efficiency was restored to approximately the level
of that of the pH . medium. Similar in the HEKT-
ACE cell line, CaCO3-NPs ( μg/ml) inhibited virus
infection efficiency in HUVECs at pH . (Figure A–
C). These results not only confirm that the acidic condi-
tion promotes virus infection but also indicate the effec-
tiveness of the CaCO3-NPs in attenuating virus infection
(Figure D).
2.4 Acidic pH conditions induced more
ACE2 expression on the cell membrane
than in the cytoplasm
To determine whether the different infection efficiencies
of SARS-CoV- under different pH conditions are caused
by pH-dependent ACE expression regulation in cells, we
treated HEKT-ACE cells (Figure A,B)orHUVECs
(Figure C,D) and found no apparent difference in total
ACE protein expression under the different pH condi-
tions. The mechanism for SARS-CoV- infection involves
requisite binding of the virus to the membrane-bound form
of ACE. Therefore, we further performed immunoflu-
orescence staining to detect ACE and determined that
the level of ACE on the cell membrane of HEKT-
ACE cells (Figure E) and HUVECs (Figure F)wassig-
nificantly increased under pH . conditions. In contrast,
supplementation with CaCO3-NPs attenuated results with
the pH . condition to a level similar to that of the pH
. group (Figure E,F). These results show that ACE
expression was induced by acidic-related pH conditions
andattenuatedbyCaCO
3-NPs, which not only uncovers
the mechanism by which acidic pH promotes SARS-CoV-
infection but also suggests that CaCO3-NPs are a poten-
tial antidote to prevent SARS-CoV- infection in acidosis
patients (Figure G).
2.5 Acidic pH conditions are not
conducive to F-actin polymerization
ACE is reported to colocalize with actin. The actin
bundle protein fascin- regulates the expression level and
subcellular localization of ACE. Thus, we detected
the morphology of F-actin by phalloidin labeling green
fluorescence staining and found obvious actin polymer-
ization and stable actin bundles in HEKT-ACE cells
(Figure A) and HUVECs (Figure B)atpH.,thatis,
normal blood pH conditions. Additionally, polymerization
of F-actin was stable in pH . medium. However, pH
. induced a fuzzy dispersion morphology of F-actin in
cells (Figure A,B). Addition of CaCO3-NPs neutralized
6of15 LIU .
FIGURE 2 Schematic and characterization of antacid CaCO-NPs. (A) Characterization of mPEG-P(Glu) copolymers by H-NMR
( MHz, in DO). PEG (-OCHCH,δ=. ppm), PGlu (-CHCHCOO-: δ=. ppm, δ=. ppm). (B) Size distribution of CaCO-NPs
measured by dynamic light scattering (DLS). (C) Morphology observation by TEM-EDS of the CaCO-NPs. The scale bar represents nm.
(D) Elemental analysis of CaCO-NPs by TEM-EDS in selected Area # (yellow box on the merged picture). (E) XRD curves of CaCO-NPs.
Circle icon: vaterite, No. -; pentagram icon: calcite, No. -. (F) Detection of zeta potential of CaCO-NPs. (G) Schematics indicating
the chemical reaction between CaCO-NPs and hydrogen ions showing how the nanoantidote plays a role in alleviating acidosis. (H) Different
amounts of CaCO-NPs regulate the pH value of pH . (left panel) and pH . (right panel) solutions. (I) Precise pH test paper was used to
assess CaCO-NPs ( μg/ml final concentration) regulation of the pH value of pH . and pH . solutions. The upper image is a color chart
of the pH value, and the bottom image is the color of the different experimental groups. (J) pH value of FBS was regulated by CaCO-NPs
( μg/ml final concentration). Left image indicated the pH value of normal serum and normal serum with CaCO-NPs. Right image indicated
the pH value of pH . serum and pH . serum with CaCO-NPs. The data shown are means ±SD (n=). *p<., **p <.,***p<.
LIU . 7of15
FIGURE 3 CaCO3-NPs-mediated pH influences virus infection. (A) GFP fluorescence indicated that CaCO-NPs ( μg/ml) attenuated
the enhanced SARS-CoV- pseudovirus infection of HEKT-ACE cells and HUVECs caused by pH ., as shown in (B). (C) Statistics of the
median fluorescence intensity of GFP in HEKT-ACE cells (left panel) and HUVECs (right panel). (D) Proposed consequences of reduced
viral infection rate due to CaCO-NPs neutralization of hydrogen ions under acidic conditions at the approximate blood pH (pH .). The data
shown are means ±SD (n=). * p<., *** or ### indicates p<.. Asterisks (*) indicate statistical significance for the pH . and .
groups compared with the pH . group. Hashes (#) indicate statistical significance for the pH .+CaCO3-NPs group compared with the pH
. group. Scale bar: μm
the hydrogen ions in the pH . medium and restored
the apparent actin bundles in HEKT-ACE cells
(Figure A) and HUVECs (Figure B), which was similar
to the effects of pH . medium. This result reveals the
effect of pH on regulating actin polymerization and
stabilization (Figure C).
2.6 Promotion of actin polymerization
inhibits ACE2 expression on the cell
membrane and SARS-CoV-2 infection
To assess whether induction of actin polymerization
influences ACE expression on the cell membrane and
8of15 LIU .
FIGURE 4 Acidic pH conditions induce a higher expression level of ACE on the cell membrane than in the cytoplasm. (A)
Representative Western blots indicate that neither pH . nor pH . conditions changed the whole level of ACE expression in
HEKT-ACE cells. (B) The statistics of the relative gray value ratio of ACE expression in (A). (C) Representative Western blots indicate
that neither pH . nor pH . conditions changed the whole level of ACE expression in HUVECs. (D) The statistics of the relative gray value
ratio of ACE expression in (C). (E) Immunofluorescence staining of ACE protein (green) indicated that compared with pH . or pH ., pH
. increased levels of ACE on the membrane of HEKT-ACE cells and (F) HUVECs, which was attenuated by CaCO-NPs ( μg/ml).
Dil staining showed the cell membrane (red), and DAPI staining showed the nucleus (blue). (G) Proposed consequences of CaCO-NPs
neutralizing hydrogen ions under acidic conditions to counteract the effects of pH . increasing levels of ACE on the membrane. Scale bar:
μm (upper) or μm (lower). The data shown are means ±SD (n=). ns: no significant difference of statistics
LIU . 9of15
FIGURE 5 Acidic pH represses F-actin polymerization, which is restored by CaCO3-NPs to normal blood pH conditions. (A)
Immunofluorescence of F-actin (phalloidin labeling) showed that there was more actin polymerization to form the large F-actin bundle in
HEKT-ACE cells and (B) HUVECs at pH ., the normal blood pH condition, than in pH . medium. CaCO3-NPs restored the F-actin
bundle in pH . medium. (C) Proposed consequences of neutralization of hydrogen ions by CaCO-NPs to upregulate the pH value to restore
the F-actin polymerization repressed by pH .. Scale bar: μm (upper) or μm(lower)
10 of 15 LIU .
even regulates SARS-CoV- infection, we added the
jasplakinolide ( μM), the widely used inducer of actin
polymerization and F-actin stabilization, to HEKT-
ACE cells and HUVECs (Figure A). We observed an
apparent reduction in ACE expression on the mem-
brane in HEKT-ACE cells after jasplakinolide sup-
plementation in pH . medium compared with cells
cultured in pH . medium (Figure B). GFP fluores-
cence of pseudovirus infection indicated that jasplakino-
lide apparent attenuated SARS-CoV- pseudovirus infec-
tion efficiency enhanced by the pH . condition in
HEKT-ACE cells (Figure C,D). In addition, jasplaki-
nolide inhibited the increase in ACE expression on
the HUVECs membrane (Figure E) and virus infection
(Figure F,G) in pH. medium. These results indicate that
SARS-CoV- infection is promoted by acidic conditions
through decreased F-actin polymerization, which is asso-
ciated with the regulation of ACE expression on the cell
membrane.
The above results indicate that acidosis-related pH can
increase SARS-CoV- receptor ACE expression on the cell
membrane, resulting in a significant increase in infection
efficiency. Therefore, CaCO3-NPscanbeusedasan“anti-
dote” for acidosis to neutralize hydrogen ions, effectively
reversing and maintaining the acidic microenvironment to
attenuate SARS-CoV- infection promoted by acidic condi-
tions (Figure ).
3CONCLUSION
In this study, we found that acidosis-related acidic pH
benefits SARS-CoV- pseudovirus infection by increasing
actin polymerization-associated ACE expression on
the membrane. Our results suggest the existence of a
positive feedback loop in which SARS-CoV- infection-
induced acidosis enhances SARS-CoV- infection.
We further report that CaCO-NP is a new poten-
tial antacid nanoantidote for acidosis in COVID-
patients.
SARS-CoV- infection induces metabolic acidosis and
respiratory acidosis caused by acute respiratory distress
syndrome., Patients with DKA have an overall mortal-
ity rate of %, higher than that for all patients without
DKA and patients with diabetes but without DKA. How-
ever, whether acidosis in critically ill COVID- patients
promotes further infection and difficult elimination of
SARS-CoV- remains largely unknown. In this study, we
mimicked the decrease in arterial blood pH in patients
with acidosis to approximately pH . and found that
compared with the normal physiological pH of ., the
virus infection rate approximately doubled under pH .
conditions. These results confirm a positive feedback
loop through which SARS-CoV- induces acidosis, fur-
ther enhancing infection. Our results also suggest the
necessity of antacid treatment for COVID- patients with
acidosis.
Increasing expression of the ACE protein promotes
COVID- infection. Downregulation of ACE decreases
SARS-CoV- infection in vitro. ACE expression level on
cell membrane is dynamically regulated. We found that a
pH change (pH . or .) did not significantly affect ACE
total protein expression but that acidic pH increased ACE
levels on the cell membrane. This not only explains why
an increase in virus infection occurs under acidic condi-
tions but also provides a simple and safe way to inhibit
virus infection. Our study suggests that antacid therapy
can reduce the increase in membrane ACE caused by
acidosis-related pH without changing expression of total
ACE to reduce the efficiency of virus infection. In our
study, regulation of pH with CaCO-NPs did not influence
the total ACE protein level but only altered that on the
cell membrane and in the cytoplasm. This maintains total
ACE in cells to prevent side effects such as cardiovascular
diseasecausedbyACEdownregulation.
The actin bundling protein fascin- regulates the expres-
sion levels and subcellular localization of ACE. NL,
another kind of human coronavirus, infection also requires
interaction with ACE, and actin cortex remodeling is
required for virus endocytosis. We found that a change
in pH had a great influence on actin polymerization. At
pH ., thick and long F-actin was observed in cells; at
pH ., actin was depolymerized, and a clear F-actin struc-
ture could not be seen. Furthermore, the use of jasplaki-
nolide, a classical molecule that promotes actin polymer-
ization, significantly inhibited the increase in cell mem-
brane ACE expression caused by pH . and the increase
in SARS-CoV- pseudovirus infection efficiency. Our study
suggests that depolymerization of actin under acidic con-
ditions leads to an increase in ACE content in the cell
membrane and promotes SARS-CoV- infection. The find-
ings suggest the necessity of antacid therapy in COVID-
patients.
As an antacid, sodium bicarbonate has adverse effects,
such as sodium load, which induces hypervolemia, hyper-
osmolarity, and hypernatremia,and bicarbonate is also
associated with delayed ketone clearance and worsened
hypokalemia.Hence, we used CaCOto neutralize hydro-
gen ions, which does not produce excessive sodium ions
and bicarbonate. Additionally, hypocalcemia frequently
occurs in COVID- patients who have a worse outcome.
Sodium overload caused by sodium bicarbonate aggravates
hypocalcemia, which may lead to cardiovascular disease.
Calcium supplement by oral or administration of CaClis
commonly used to treat hypocalcemia. Previous study
indicated that the metal organic frameworks (MOFs)
LIU . 11 of 15
FIGURE 6 Actin polymerization reduces ACE levels on the cell membrane and SARS-CoV- pseudovirus infection. (A) Schematic
illustration of the strategy by which SARS-CoV- infection is regulated by inducing F-actin polymerization and stabilization with
jasplakinolide in pH . medium. (B) Jasplakinolide ( μM) reduces membrane ACE increased by pH . in HEKT-ACE cells. (C) GFP
fluorescence indicates that jasplakinolide ( μM) reverses the effects of the pH . condition that promotes pseudovirus infection in
HEKT-ACE cells. (D) The statistics of the median fluorescence intensity of GFP in (C). (E) Jasplakinolide ( μM) reduces HUVECs
membrane ACE levels that are increased under pH . conditions. (F) GFP fluorescence indicates that jasplakinolide reverses the effects of
the pH . condition that promotes pseudovirus infection in HUVECs. (G) The statistics of the median fluorescence intensity of GFP in (F).
Scale bar: μm (upper) or μm (lower). The data shown are means ±SD (n=). **p<., ***p<.
12 of 15 LIU .
FIGURE 7 The proposed consequence of the promotion of SARS-CoV- infection by acidic pH, which is inhibited by
CaCO3-NPs-mediated pH increase or jasplakinolide. Acidosis-related pH increases SARS-CoV- receptor ACE levels on the cell membrane,
resulting in a significant increase in virus infection efficiency. Therefore, CaCO3-NPs can be used as an “antidote” of acidosis to neutralize
hydrogen ions, effectively reversing and maintaining the acidic microenvironment to attenuate SARS-CoV- infection promoted by acidic
conditions
biomaterials release Ca+on demand to promote extra-
cellular matrix mineralization and upregulate osteogenic
gene expression. Therefore, in our study, a CaCO-NPs
was used to neutralize hydrogen ions and as a calcium
supplement to help alleviate hypocalcemia. Sodium bicar-
bonate is a weakly alkaline salt that is soluble in water and
results in a weakly alkaline pH. In excess, the blood pH
will rise, with possible alkalosis. However, CaCOdoes
not significantly changes the blood acid–base balance
under normal physiological conditions. So, compared
with sodium bicarbonate, CaCOis relatively safe. Novel
biomaterials treatment for COVID- has the potential
new strategies and opportunities. CaCO-NPs are rapidly
degraded under a slightly acidic environment and have
potential as drug delivery systems and may be admin-
istered through inhalation, intravenous injection, and
topical application and so on, which suggests the CaCO-
NPs being an efficient treatment strategy in the future. We
utilized CaCO-NPs to neutralize the acidic environment
under acidosis-associated acidic pH conditions to repress
SARS-CoV- virus infection. Compared with sodium
bicarbonate, CaCO-NPs may be a better alternative for
the treatment of acidosis in COVID- patients.
Our work reveals that acidosis-related acidic pH con-
ditions promote SARS-CoV- infection. Mechanistically, a
lower pH value increases ACE expression on the mem-
brane by inhibiting actin polymerization. SARS-CoV-
infection induces a positive feedback loop involving acido-
sis in COVID- patients and increases ACE levels on the
membrane to facilitate viral entry, which induces a vicious
cycle in severely ill patients. Our study also suggests that it
may be necessary to perform antacid therapy for acidosis in
severely ill COVID- patients. In view of the controversy
over the use of sodium bicarbonate, we propose the poten-
tially safer CaCO-NPs as treatment.
4EXPERIMENTAL SECTION/
METHODS
4.1 Cell culture
The HEKT cell line with ACE overexpression
(HEKT-ACE cells) (Genomeditech, GM-C,
China) was cultured in DMEM (Gibco) with % FBS
(Lonsera, S-S, Uruguay). HUVECs were cultured
in RPMI- medium (Gibco) with % FBS. Cells were
cultured at ◦C, % COatmosphere.
4.2 Different pH value culture medium
The basic ingredients of the extracellular solution medium
were as follows: mM NaCl, mM KCl, mM HEPES,
mM glucose, mM CaCl, and mM MgCl.ANaOH
solution was used to adjust the pH value of solutions, as
monitored by using a pH meter (Mettler Toledo, FiveEasy
Plus, Switzerland).
4.3 Infection of SARS-CoV-2
pseudovirus
SARS-CoV- spike pseudovirus (Genomeditech, batch
number VF) based on the HIV lentivirus packag-
ing system is a replication-defective pseudovirus that con-
tains the spike protein and expresses the GFP. Green
fluorescence can be used to determine infection by the
pseudovirus. The day before the experiment, HEKT-
ACE cells were seeded into a -well culture plate ( ×
cells/well) for virus infection the next day. The pseu-
dovirus was completely thawed at ◦C, and a :-fold
LIU . 13 of 15
dilution of the pseudovirus infection solution was used
with different pH solutions. The diluted virus infection
solution was added to the culture plate. After h, the
medium was replaced with normal DMEM (with % FBS)
medium for culture for h. Confocal microscopy (Olym-
pus, FV, Japan) was used to observe GFP fluores-
cence.
4.4 Western blotting
Protein lysis buffer (M-PER Mammalian Protein Extrac-
tion Reagent, Thermo, USA) was used to extract total cell
protein. A protease inhibitor cocktail (Bimake, Shanghai,
China) was added to the lysis buffer to prevent protein
degradation. After the SDS-PAGE electrophoresis of
protein, the proteins were transferred onto polyvinylidene
fluoride (PVDF) membranes (BioRad, USA) to detect the
level of ACE expression. Anti-ACE (--AP, :
dilution, Proteintech, USA) was used as the primary anti-
body, and anti-GAPDH (ab, : dilution, Abcam,
USA) was used as a loading control. The secondary anti-
bodies used were anti-mouse IgG HRP-linked antibody
( V, : dilution; Cell Signaling Technology, USA)
and anti-rabbit IgG horseradish peroxidase (HRP)-linked
antibody ( V, : dilution; Cell Signaling Tech-
nology). The ChemiDoc XRS+system (Bio-Rad) was
applied to determine the protein level with enhanced
chemiluminescence (Clarity Western ECL substrate,
Bio-Rad).
4.5 Immunofluorescence staining
HEKT-ACE cells or HUVECs were seeded onto polyly-
sine (Beyotime, China)-coated cover glass and fixed in
% paraformaldehyde (PFA) solution for min. Then,
.% Triton X- solution was added for min. The
slides were blocked for h in % FBS PBS solution.
For detection of ACE expression, the cells were incu-
bated with the anti-ACE primary antibody (: dilu-
tion, Proteintech), with rabbit IgG-H&L (Alexa Fluor
) (ab, : dilution, Abcam) as the sec-
ondary antibody. Phalloidin Alexa Fluor (A,
Thermo, USA) was used to detect F-actin. Dil (DIIC(),
,’-dioctadecyl-,,’,’-tetramethylindocarbocyanine per-
chlorate) (Yeasen, China) was used to stain the cell mem-
brane (red), and the nucleus was stained (blue) with DAPI
(Sigma, USA). Images were captured using a confocal
microscope (Olympus).
4.6 Synthesis of methoxypoly(ethylene
glycol)-block-poly(sodium glutamate)
(mPEG-P(Glu)) polymers
mPEG-P(Glu) polymers were synthesized as previously
described. Briefly, l-glutamic acid γ-benzyl ester (cat.
Merck, USA) was reacted with triphosgene (cat.
, Merck, USA) to obtain the N-carboxyanhydride of
γ-benzyl l-glutamate (NCA-BLG) via the Fuchs–Farthing
method. Then, NCA-BLG was polymerized in N,N-
dimethylformamide, as initiated by the amino group
of CHO-PEG-NH(Mw K, Xiamen Sinopeg Biotech
Co., Ltd., China), to synthesize mPEG-poly(γ-benzyl l-
glutamate) (mPEG-PBLG). Finally, the benzyl groups
of mPEG-PBLG were removed by mixing with . N
NaOH at room temperature to obtain mPEG-P(Glu) poly-
mers, which were determined by H-NMR spectroscopy
( MHz; solvent: DO).
4.7 Construction and characterization
of CaCO3-NPs
Nanoparticles were prepared via the biomineralization
method. First, . ml mPEG-P(Glu) ( mg/ml) and
. ml CaClaqueous solution ( mg/ml) were mixed.
Then, . M Tris-HCl buffer (pH .) was slowly added
to adjust the pH value to pH . to form Ca+chelate com-
pounds, and . ml NaCOsolution ( mg/ml) was added
dropwise to the mixture until opalescence was observed,
indicating the formation of CaCO-NPs. The mixture was
stirred at ◦C overnight and centrifuged at , rpm for
min to remove excess ions and copolymers. The size
distribution was characterized by DLS (Zetasizer Nano S,
Malvern, UK), and the morphology and element composi-
tions were observed by transmission electron microscopy
(TEM, TALOS FX), the XRD analysis was carried out
by Aeris XRD instrument (Malvern Panalytical) and ana-
lyzed by MDI jade . software.
4.8 Statistical analysis
Student’s t-test was used when two independent groups
were compared; one-way ANOVA extended more than two
groups. Detailed instructions are provided in figure leg-
ends, and p-values <. were considered statistically sig-
nificant. GraphPad Prism software (version .; Graph-
Pad Software, La Jolla, CA, USA) was used to calculate
statistics.
14 of 15 LIU .
ACKNOWLEDGMENTS
This study was supported by the National Key Research
and Development Program of China (YFA),
National Natural Science Foundation of China (No.
, , ), the Natural Science Founda-
tion of Shanghai (No. ZR), and the Foundation
of Shanghai Fourth People’s Hospital Affiliated to Tongji
University (No. sykyqd). During these difficult times
of fighting the COVID- epidemic, we thank Dr. Ge Gao,
Dr. Xinglei Song, Dr. Qi Shao, and Dr. Man Zhang for giv-
ing the helpful support. All the schematics were created
with BioRender.com.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
ORCID
Zhihao Sheng https://orcid.org/---
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How to cite this article: Q. Liu, H. Ruan, Z.
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