Abstract
Acute lung injury (ALI) remains a significant global health issue,
necessitating novel therapeutic interventions. In our latest study, we
pioneered the use of D-mannitol–cerium–quercetin/rutin coordination
polymer nanoparticles (MCQ/R NPs) as a potential treatment for ALI. The
MCQ/R NPs, which integrate rutin and quercetin for their therapeutic
potential and D-mannitol for its pulmonary targeting, displayed
exceptional efficacy. By utilizing cerium ions for optimal nanoparticle
assembly, the MCQ/R NPs demonstrated an average size of less than 160
nm. Impressively, these nanoparticles outperformed conventional
treatments in both antioxidative capabilities and biocompatibility.
Moreover, our in vivo studies on LPS-induced ALI mice showed a
significant reduction in lung tissue inflammation. This groundbreaking
research presents MCQ/R NPs as a promising new approach in ALI
therapeutics.
Keywords: quercetin, rutin, cerium ions, D-mannitol, self-assembly,
acute lung injury
1. Introduction
Damage to the lungs increases permeability, leading to the exudation of
protein-rich fluid that accumulates in the lung interstitium and
alveoli [[36]1]. Recent data indicate that ALI has one of the highest
morbidity and mortality rates among ICU patients [[37]2]. Treatment
strategies for ALI depend on its severity and typically include
mechanical ventilation, pharmacotherapy, and respiratory support. In
the early stages of ALI, a lung-protective ventilation strategy is
commonly recommended, supplemented with medications to reduce
inflammation and prevent further pulmonary damage [[38]3], such as
corticosteroids, surfactants, antioxidants, non-steroidal drugs, and
vasodilators [[39]4,[40]5,[41]6,[42]7,[43]8]. However, the clinical
application of some drugs for ALI treatment, particularly certain
anti-inflammatory agents and antioxidants, is restricted by their side
effects. These side effects include gastrointestinal discomfort,
allergic reactions, and other systemic adverse effects, which can lead
to poor patient compliance and treatment failure [[44]9]. Additionally,
the use of glucocorticoids can lead to complications such as fluid and
sodium retention, hypertension, immunosuppression, and hyperglycemia
[[45]10].
Traditional Chinese medicine (TCM) offers an integrative approach to
treating ALI by enhancing the efficacy of other treatments,
holistically adjusting the body’s condition, enhancing self-healing
capabilities, and reducing unwanted side effects [[46]11]. Rutin and
quercetin, recognized flavonoids of natural origin, are renowned for
their exemplary antioxidative and anti-inflammatory properties
[[47]12,[48]13]. Recent scientific studies have highlighted their
potential pharmacological activity against ALI. For instance, Chen et
al. investigated the mechanism by which quercetin protects against
sepsis-induced ALI, emphasizing its ability to alleviate pulmonary
pathological injuries and counteract oxidative stress through the
SIRT1/AMPK pathway [[49]14]. Similarly, Yeh et al. demonstrated that
rutin could counteract LPS-induced ALI by inhibiting MAPK-NF-κB
activation and increasing antioxidative enzyme activity. Intriguingly,
rutin’s efficacy in ameliorating ALI, attributed to its ability to
chelate extracellular metal ions, was found to be superior to that of
dexamethasone [[50]12]. Furthermore, Basaran et al. suggested that
co-administering rutin with other antioxidants, particularly quercetin,
could enhance therapeutic outcomes, reduce drug resistance, and
attenuate adverse effects [[51]15]. In a study by Yeh and colleagues,
the combined use of quercetin and rutin was observed to enhance
anti-proliferative activity against the MDA-MB-231 cell line in MTT
cell proliferation assays, surpassing the effects of each compound used
separately. Additionally, their combined antioxidative capacity was
found to be superior to their individual effects [[52]16]. However, the
clinical utility of rutin and quercetin is limited by their low oral
bioavailability, poor water solubility, rapid metabolism, and enzymatic
degradation [[53]14,[54]17,[55]18].
The nanomedicine drug delivery systems offer several advantages,
including improved pharmacokinetics and biodistribution, reduced
toxicity, enhanced solubility and stability, controlled drug release,
and targeted delivery of therapeutic agents. Nanocarriers can be
customized by modifying their composition, size, shape, and surface
properties. The primary goal of using nanocarriers in drug delivery
systems is to treat diseases effectively while minimizing the side
effects [[56]19]. One notable advantage of nanomedicine is its ability
to deliver drugs directly to affected areas, thus achieving more
efficient and effective treatment while simultaneously reducing
drug-related side effects [[57]20]. Metal–phenolic networks (MPNs) are
a new class of nanomaterials, formed by the self-assembly of metal ions
and polyphenols, with bioadhesive properties, favorable
biocompatibility, versatile drug loading abilities, and
stimuli-responsive characteristics, thus aiding in the diagnosis and
treatment of diseases [[58]21]. Considered an emerging class of
supramolecular surface modifiers, MPNs show promise in various fields,
including drug delivery. For example, a study described a unique
core–satellite nanosystem integrated with MPNs, where the “core”
consists of a liposome loaded with EDTA, a metal ion chelator [[59]22].
MPN-based drug delivery systems demonstrate excellent stability,
adequate drug loading capacity, good biocompatibility, reduced
premature release, and remarkable therapeutic effectiveness, thus
enabling gradual, multi-stimuli-responsive drug delivery [[60]23]. The
ease of preparation, outstanding characteristics, and promising medical
applications of MPNs have attracted considerable attention. By
coordinating phenolic ligands and metal ions, MPNs demonstrate their
potential as multifunctional theranostic nanoplatforms, possessing
unique attributes like a rapid preparation process, minimal
cytotoxicity, and pH responsiveness [[61]24,[62]25]. The use of
metal–organic frameworks and the formation of metal chelates, such as
those involving cerium ions, can effectively tackle these challenges
[[63]26]. The hydroxyl and carbonyl groups in the structures of
quercetin and rutin serve as strong metal chelators [[64]27]. Studies
have shown that quercetin–metal chelates, due to their geometric
orientation and the presence of metal ion binding sites, demonstrate
superior in vitro pharmacokinetics and bioactivity compared to
quercetin alone [[65]28]. For instance, Hosseinzadeh et al. developed a
delivery system that targets the MDA-MB-231 and A375 cancer cell lines
by chelating curcumin and quercetin with cerium ions [[66]26].
Furthermore, cerium oxide has demonstrated potential in preventing and
treating ALI by regulating leukocyte recruitment, reducing inflammation
and oxidative stress, decreasing collagen deposition, and thus
improving pulmonary mechanics [[67]29,[68]30,[69]31]. A recent study
showed that administering 0.5 mg/kg of cerium oxide via intraperitoneal
injection before exposure to sevoflurane significantly decreased lung
injuries in rats, highlighting the potential of cerium oxide in
mitigating pulmonary damage [[70]32].
In cases of acute lung injury, there is a notable increase in the
expression of macrophages within the lung [[71]33]. To explore the
feasibility of efficiently delivering anti-tuberculosis drugs to
alveolar macrophages, researchers used mannitol to create
microparticles encapsulating highly water-soluble drugs in a specific
study. Hegde et al. discovered that, compared to the administration of
drugs alone, these mannitol-fabricated microparticles significantly
increased drug accumulation within the alveolar macrophages of rats
[[72]34]. D-Mannitol, with its distinct 3,4-OH structure, can be
recognized by pulmonary surfactant-associated proteins A and D,
achieving a degree of lung targeting [[73]35]. Moreover, it
significantly enhances the dispersion of nanoparticles in aqueous
environments, effectively reducing their propensity to aggregate.
Mannitol, as a reliable protective agent, also maintains the structural
integrity of nanoparticles during critical processes such as
lyophilization and resuspension [[74]36].
In this study, rutin and quercetin synergistically interacted with
cerium oxide under optimal conditions to form a metal–polyphenol
network. This interaction facilitated the formation of ionic bonds
between mannitol and cerium ions. To confirm the formation of the
metal–polyphenol network, ultraviolet-visible spectrophotometry (UV),
Fourier transform infrared spectroscopy (FTIR), inductively coupled
plasma mass spectrometry (ICP-MS), and dynamic light scattering (DLS)
analyses were performed. The morphological behavior of the
metal–polyphenol network was characterized using transmission electron
microscopy (TEM). The antioxidant activities of the coordination
polymers were measured through free-radical scavenging assays.
Furthermore, the in vitro safety profile was assessed via hemolysis
tests. Most importantly, the therapeutic efficacy against acute lung
injury was evaluated through a series of experiments involving animal
trials, hematoxylin and eosin (H&E) staining, inflammation factor
detection, inflammatory cell differential counts, proteomics analysis,
and RT-qPCR analysis. This study utilized the lung-targeting properties
of mannitol and the ability of nanoparticles to prolong drug blood
concentration to investigate the therapeutic advantages of
rutin–quercetin metal–polyphenol networks compared to free drugs.
2. Results
2.1. Size, Zeta Potential, and Encapsulation Efficiency Distribution of MCQ/R
NPs
Based on the single-factor screening results presented in [75]Table 1
showing the composition of NPs, the optimal formulation for the size
and zeta potential of MCQ/R NPs was selected. Repeated experiments were
conducted according to this formulation. The finalized protocol
involved drawing 110 μL of a mixed solution of rutin and quercetin
(22.73 mg/mL) and 250 μL of Tris-HCl 8.8 solution into a reaction
flask, followed by magnetic stirring at room temperature for five
minutes. Subsequently, 31 μL of cerium dioxide solution was added
dropwise to the reaction system and allowed to react for another five
minutes. Under continuous magnetic stirring, 4 mL of mannitol (10
mg/mL) was added dropwise to the reaction flask and stirred for 24 h.
The coordination polymer exhibited satisfactory results in terms of
particle size and zeta potential. As shown in [76]Table 2, the results
of F13 showed that the particle size of MCQ/R NPs was approximately
156.5 ± 2.875 nm, the zeta potential was −23.5 ± 2.28 mV, and the PDI
was 0.147 ± 0.043. Considering these three aspects, the relatively
small PDI indicates that MCQ/R NPs formulated under F13 are more stable
compared to other formulations ([77]Figure 1).
Table 1.
Composition of NPs.
S/N Volume of CeO[2] (μL) Volume of Medicines (μL) Volume of Tris-Hcl
8.8 (μL) Volume of D-Mannitol (mg/mL)
F1 21 110 200 4 (20 mg/mL)
F2 31 110 200 4 (20 mg/mL)
F3 41 110 200 4 (20 mg/mL)
F4 31 65 200 4 (20 mg/mL)
F5 31 110 200 4 (20 mg/mL)
F6 31 175 200 4 (20 mg/mL)
F7 31 110 150 4 (20 mg/mL)
F8 31 110 200 4 (20 mg/mL)
F9 31 110 250 4 (20 mg/mL)
F10 31 110 250 4 (10 mg/mL)
F11 31 110 250 4 (20 mg/mL)
F12 31 110 250 4 (40 mg/mL)
F13 31 110 250 4 (10 mg/mL)
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Table 2.
Diameter, zeta potential, and polydispersity index of the NPs (n = 3).
S/N Diameter (nm) Polydispersity Index Zeta Potential (mV)
F1 259.3 ± 3.13 0.273 ± 0.02 −16.5 ± 1.31
F2 187.4 ± 4.93 0.170 ± 0.07 −13.9 ± 0.80
F3 321.2 ± 18.47 0.260 ± 0.03 −30.8 ± 2.65
F4 141.9 ± 1.17 0.175 ± 0.03 −24.7 ± 2.50
F5 163.9 ± 1.66 0.210 ± 0.02 −24.8 ± 1.54
F6 288.6 ± 4.29 0.302 ± 0.05 −25.6 ± 0.85
F7 156.2 ± 1.25 0.198 ± 0.81 −17.9 ± 3.41
F8 170.3 ± 3.68 0.265 ± 0.94 −22.6 ± 2.11
F9 138.5 ± 4.51 0.147 ± 0.65 −23.5 ± 1.60
F10 138.5 ± 4.51 0.147 ± 0.65 −17.9 ± 3.41
F11 170.3 ± 3.68 0.265 ± 0.94 −22.6 ± 2.10
F12 156.2 ± 1.25 0.198 ± 0.81 −23.5 ± 1.61
F13 156.5 ± 2.875 0.147 ± 0.043 −23.5 ± 2.28
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Figure 1.
[80]Figure 1
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Particle sizes and zeta potential of MCQ/R NPs. (A) MCQ/R NPs’ zeta
potential was measured with the F13 formulation. (B) MCQ/R NPs’
particle size was measured with the F13 formulation.
2.2. TEM and ICP-MS Analysis of Coordination Polymers
As depicted in [82]Figure 2A,B, showing the characterization of
coordination polymers, the morphological characteristics of the
coordination polymers were observed using transmission electron
microscopy (TEM). We identified that the coordination polymers
exhibited a square shape with a relatively uniform size. To further
determine the cerium content in the coordination polymers, inductively
coupled plasma mass spectrometry (ICP-MS) was employed. The results
indicated a cerium content of 117.91 mg/kg. The formation process of
the coordination polymer of rutin, quercetin, mannitol, and cerium ions
in a tris-HCL (pH 8.8) environment was visualized through TEM. Rutin,
quercetin, and mannitol acted as ligands, forming coordination bonds
with the vacant orbitals of cerium ions, leading to the generation of
primary coordination complexes. These primary complexes might further
undergo polymerization reactions under specific conditions, resulting
in coordination polymers with a uniform size and regular morphology.
The TEM images revealed the morphological features of these
coordination polymers, showcasing a consistent square structure. This
morphological trait underscores the significant orderliness and
uniformity in the process of the formation of coordination polymers
between rutin, quercetin, mannitol, and cerium ions under specific
reaction conditions. The relatively uniform square structure might also
reflect the lattice structure and coordination of geometric
characteristics within the coordination polymer, suggesting a specific
coordination mode during the complexation of rutin, quercetin,
mannitol, and cerium ions.
Figure 2.
[83]Figure 2
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Characterization of coordination polymers. (A,B) TEM image of MCQ/R
NPs; (C) the UV–Vis spectra of MCQ/R NPs; (D) the FTIR spectra of MCQ/R
NPs. For the FTIR spectra, D-M represents a mannitol solution, Ru(Qu)
represents a quercetin (rutin) free drug solution, and NPs represent
MCQ/R NP nanoformulations. For the UV–Vis spectra, MCQ/R NPs-PM
represents the unreacted physical mixture of the MCQ/R NP formulation,
and MCQ/R NPs-MPM represents the MCQ/R NP nanoformulations.
2.3. UV–Vis Spectra of MCQ/R NPs
The UV–visible absorption spectra of MCQ/R NPs and their physical
mixture solution were recorded between 200 and 800 nm using a
UV–visible spectrophotometer (SHIMADZU, UV-2600, Kyoto, Japan). The
DMSO solution of rutin–quercetin appeared yellow, and upon mixing with
cerium ions, a noticeable color change was observed, turning the MCQ/R
NPs to a brownish hue. For the free drug solution, post-reaction with
cerium ions, the color transitioned from yellow to brownish. This was
hypothesized to be attributed to the successful formation of the
coordination polymer. Thus, a series of characterizations were
undertaken. As illustrated in [85]Figure 2, compared to the solution of
its physical mixture, the spectrum of MCQ/R NPs exhibited a significant
shift in the wavelength of maximum absorbance. The UV spectrum of MCQ/R
NPs revealed absorption peaks at λ = 356, 302, and 256 nm, while the
free drug physical mixture solution displayed peaks at λ = 374, 310,
and 256 nm. CeO[2] showed an absorption peak around λ = 310 nm, and
mannitol exhibited no absorption peaks. Given that the chelation site
of cerium ions is within the cinnamoyl system (B-ring) of the
rutin–quercetin brass structure, if a chelation reaction occurred,
there would be a blue shift in the maximum absorption wavelength of the
B-ring while the A-ring remained almost unchanged. The results
indicated an absorption peak at λ = 374 nm for the free drug physical
mixture solution. Upon the addition of cerium ions, the maximum
absorbance wavelength for MCQ/R NPs shifted to 356 nm. Meanwhile, the
free drug physical mixture solution showed an absorption peak at λ =
256 nm, which remained unchanged for MCQ/R NPs post-cerium ion
addition. The free drug physical mixture solution had an absorption
peak at λ = 310 nm, which, upon cerium ion addition, shifted to 302 nm
for MCQ/R NPs ([86]Figure 2C). These changes in absorption peaks
validate the occurrence of chelation and its impact on the structure of
rutin–quercetin. Specifically, when cerium ions form a coordination
polymer with rutin and quercetin, the maximum absorption wavelength of
the B-ring undergoes a blue shift, resulting in MCQ/R NPs with specific
UV–Vis spectral characteristics.
2.4. Infrared Spectra of MCQ/R NPs
Infrared spectra were recorded for mannitol, the free drug solution of
quercetin (rutin), and MCQ/R NPs. The results revealed characteristic
absorption peaks of quercetin and mannitol in the infrared spectrum of
MCQ/R NPs, albeit with some shifts. The vibrational frequency of the
carbonyl group (C=O) in the free drug solution of quercetin (rutin) was
located at 1750 cm^−1. Upon nanoparticle formation, the C=O group
exhibited a shift towards a lower wavenumber, settling at 1680 cm^−1,
suggesting the involvement of the quercetin carbonyl group in
coordination. Compared to the vibrational frequency of quercetin at
1310 cm^−1 corresponding to C–OH (at the C-3 position), this peak was
absent in the nanoparticles, indicating that this structure also
participated in coordination with cerium ions ([87]Figure 2D). The
changes in the characteristic absorption peaks of quercetin and rutin
after nanoparticle formation indicate their coordination with cerium
ions.
2.5. Stability Assessment of MCQ/R NPs
The stability experiment results revealed that from Day 1 to Day 15,
the particle size of MCQ/R NPs did not exhibit any significant increase
for a continuous span of 10 days, with an average PDI value of 0.121.
Starting from Day 11, there was a noticeable increase in particle size,
and between Day 12 and Day 15, in comparison to Day 1, the particle
size progressively enlarged, with an average PDI value of 0.313
([88]Figure 3A). Upon visual inspection of the solution, a gradual
formation of flocculent precipitates was observed, indicating that the
coordination polymer maintained good stability during the initial ten
days post-production.
Figure 3.
[89]Figure 3
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Evaluation of the stability, in vitro safety, and antioxidant activity
of MCQ/R NPs. (A) Time stability of MCQ/R NPs solution. * p < 0.05; **
p < 0.01;*** p < 0.001. Day 1 was compared with days 11–15. In the bar
chart, the red bars represent significant differences compared to Day
1, while the remaining blue bars represent no significant differences
compared to Day 1. N = 3. (B) The hemolysis rates of MCQ/R NPs at the
concentrations of 100, 200, 300, and 400 μg/mL. *** p < 0.001 compared
with the concentration of 100 μg/mL. n = 6. (C) The scavenging rates of
MCQ/R NPs at the concentrations of 0.01, 0.05, 0.1, and 0.5–1.0 mg/mL.
*** p < 0.001 compared with the concentration of 0.01 mg/mL. n = 6.
2.6. Hemolysis Assay
As illustrated in [91]Figure 3, we observed that the hemolysis rate
increased with the increase in the concentration of the coordination
polymer from 100 to 400 μg/mL. Notably, the hemolysis rate for all
concentrations of the coordination polymer remained below 5%,
suggesting minimal erythrocyte damage by the drug and indicating a low
risk of hemolysis ([92]Figure 3B).
2.7. Antioxidant Activities of MCQ/R NPs
As depicted in [93]Figure 3, the DPPH radical scavenging activity
increased, with the concentration of the MCQ/R NPs solution ranging
from 0.01 mg/mL to 1 mg/mL. Upon reaching a concentration of 0.5 mg/mL,
the scavenging rate began to stabilize, with only minor increments,
indicating the compound’s commendable free-radical scavenging
capability ([94]Figure 3C).
2.8. Changes in Lung Wet-to-Dry Weight Ratio
Compared to the control group, the model group exhibited a significant
increase in the lung wet-to-dry weight ratio. In contrast, all
treatment groups demonstrated a marked reduction in this ratio when
compared to the model group. Among them, the MCQ/R NPs group showed the
most pronounced effect, suggesting that MCQ/R NPs effectively reduced
the wet-to-dry weight ratio in the ALI model, thereby alleviating the
extent of pulmonary edema ([95]Table 3).
Table 3.
Effect of MCQ/R on lung moisture–dry weight ratio in LPS-ALI mice (n =
3) * p < 0.05.
Group W/D
Control 5.647 *
LPS 6.659
Dexamethasone 5.716 *
MCQ/R 5.591 *
Rutin 5.847 *
Quercetin 5.601 *
Rutin–Quercetin mixture 5.767 *
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2.9. Inflammatory Cell Differential Count
Compared to the control group, the model group displayed a significant
increase in the total cell count and inflammatory cells (neutrophils
and macrophages) in the BALF. In contrast, the MCQ/R NPs group showed a
marked reduction in inflammatory cells compared to the model group,
with comparable cell counts to the control group. This suggests that
MCQ/R NPs effectively reduce the number of inflammatory cells in the
ALI model, alleviating inflammatory cell infiltration and modulating
the inflammatory response associated with ALI ([97]Figure 4A).
Figure 4.
[98]Figure 4
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Evaluation of the in vivo safety and therapeutic efficacy of MCQ/R NPs.
(A) Effect of MCQ/R NPs on the number of inflammatory cells in the BALF
of LPS-ALI mice (Giemsa, ×400). (B) Effect of MCQ/R NPs on lung
histopathology in LPS-ALI mice (HE, ×200). (C–E) Expression of TNF-α,
IL-1β, and IL-6 was detected using ELISA. *** p < 0.001 compared with
MCQ/R group. N = 6. (F,G) Expression of TLR4 and NLRP3 mRNA was
detected using RT-qPCR. *** p < 0.001 compared with MCQ/R group. n = 6.
2.10. Histopathological Examination of Mouse Lung Tissue via Hematoxylin and
Eosin (HE) Staining
As depicted in [100]Figure 4B, optical microscopy (×200 magnification)
revealed the histological features of the right lung tissue in mice.
Compared to the model group, the control group exhibited a normal
cellular morphology and overall lung structure, with no evident signs
of fibrotic changes, hemorrhage in the alveolar septa, or inflammatory
infiltration. In contrast, the LPS-induced model group displayed marked
heterogeneity in alveolar size and shape, with some areas even showing
a loss of structural integrity. The orderly arrangement was disrupted,
and the thickness of the alveolar septa increased, indicating fibrotic
alterations. Additionally, hemorrhages in the alveolar septa and signs
of inflammatory infiltration were evident, aligning with the typical
pathological features of ALI. Upon comparison across experimental
groups, improvements or reductions in alveolar size, the thickness of
the alveolar septa (indicative of fibrosis degree), the degree of
inflammatory infiltration, and pulmonary interstitial edema were
observed in the dexamethasone group, rutin group, quercetin group,
rutin–quercetin mixed-solution group, and the MCQ/R NPs group relative
to the model group. Specifically, these groups exhibited a trend
towards normalization of lung tissue structure. When ranking based on
significance, the dexamethasone and MCQ/R NPs groups emerged as the
most effective in ameliorating the pathological changes. The
rutin–quercetin mixed-solution group followed, with the quercetin group
trailing and the rutin group showing the least efficacy. In summary,
all treatment groups demonstrated significant improvements in alveolar
size, the thickness of the alveolar septa, the degree of inflammatory
infiltration, and interstitial edema compared to the model group.
Notably, the dexamethasone and MCQ/R NPs groups showcased the most
pronounced effects, highlighting their potential benefits in
alleviating pulmonary fibrosis and inflammation.
2.11. Serum Inflammatory Cytokine Levels of IL-6, IL-1β, and TNF-α
Compared to the control group, the model group exhibited a significant
elevation in the expression of the serum cytokines IL-6, IL-1β, and
TNF-α. The other treatment groups managed to reduce their expression,
with the differences being statistically significant. Notably, the
MCQ/R NPs group was the most effective in significantly reducing the
expression levels of IL-6, IL-1β, and TNF-α ([101]Figure 4C–E). We
found that, in the MCQ/R NPs group, the expression levels of IL-6,
IL-1β, and TNF-α were lower than those in the control group. We
speculate that this could be due to several reasons. Firstly, for the
high-dose rutin–quercetin mixture, these combinations have potent
anti-inflammatory properties that can significantly inhibit the
production of inflammatory cytokines, even beyond normal physiological
levels, resulting in lower expression levels than the control group.
Secondly, the antioxidant mechanism may further reduce the inflammatory
response by decreasing the production of free radicals and the
activation of inflammatory signaling pathways, thereby delaying the
onset of the LPS-induced cytokine storm. Thirdly, the synergistic
effect of rutin and quercetin with nanoparticles enhances the drug’s
efficacy, making it more effective than when used alone. Finally, the
use of nanoparticles improves drug delivery efficiency, allowing the
drug to more effectively reach and act on target cells, significantly
suppressing the production of inflammatory cytokines (see
[102]Supplementary Materials).
2.12. Expression of TLR4 and NLRP3 mRNA in Lung Tissue
As illustrated in [103]Figure 4, when compared to the control group,
the model group displayed a significant upregulation in the expression
of TLR4 and NLRP3 mRNA (p < 0.01). In contrast to the model group, all
treatment groups exhibited a decrease in TLR4 and NLRP3 mRNA
expression. Notably, the MCQ/R NPs group demonstrated a significant
reduction in TLR4 and NLRP3 mRNA expression when compared to both the
model group and the free drug group ([104]Figure 4F,G).
2.13. Proteomics Analysis
Mass spectrometry and proteomics data retrieval identified 2828
proteins. A statistical analysis was performed of the model group
versus the control group, the model group versus the rutin–quercetin
mixture group, and the model group versus the MCQ/R group, with a
criterion of fold changes > 1.5 (p < 0.05). The comparative proteomic
analysis revealed a set of differentially expressed proteins, with
significant implications. Specifically, between the model and control
groups, 196 proteins exhibited pronounced differences; when the model
group was compared with the rutin–quercetin mixture group, this number
escalated to 290 proteins; and the comparison between the model and
MCQ/R group identified 224 proteins with notable variances. The
differential proteins from each group were subjected to KEGG pathway
enrichment analysis. Our findings revealed that all three groups showed
significant enrichment in the coronavirus disease–COVID-19 pathway,
which aligns with our construction and treatment of the ALI model.
Further analysis of the Coronavirus disease–COVID-19 pathway indicated
that the MCQ/R group showed the enrichment of more targets compared to
the rutin–quercetin mixture group. Moreover, the total number of
enriched pathways in the MCQ/R group was less than that in the
rutin–quercetin mixture group. This suggests that MCQ/R, in comparison
with the rutin–quercetin mixture, can modulate the treatment of ALI
more effectively by targeting a greater number of key regulatory points
in a more focused manner ([105]Figure 5A,B).
Figure 5.
[106]Figure 5
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Proteomics analysis. (A) In terms of the KEGG enrichment outcomes, a
Venn diagram depicting the proteins enriched in the Coronavirus
disease–COVID-19 pathway was constructed to compare the differences
between the model group and the control group, the model group and the
rutin–quercetin mixture group, as well as the model group and the MCQ/R
group, along with the corresponding enriched genes in the Coronavirus
disease–COVID-19 pathway. (B) In the model group, 196 differentially
expressed proteins were subjected to KEGG pathway analysis compared to
the control group. Additionally, 290 differentially expressed proteins
were analyzed in comparison to the rutin–quercetin mixture group, and
224 differentially expressed proteins were compared to the MCQ/R group.
3. Discussion
To effectively deliver rutin and quercetin polyphenols, coordination
polymers were constructed using a metal–phenolic network approach
within the rutin and quercetin molecules. Cerium dioxide was used as
the metal source. Mannitol was added to prevent the aggregation of the
coordination polymers, control particle size, and possibly facilitate
targeted delivery. The coordination bonds between rutin and quercetin
with cerium ions were characterized by FTIR and UV spectroscopy,
suggesting interactions between hydroxyl and carbonyl groups and cerium
ions, likely resulting in the formation of coordination polymer
nanoparticles. Screening results revealed a favorable nanoparticle size
and zeta potential. Their stability was highlighted by insignificant
variations in size and potential, likely due to the presence of
mannitol in the suspension. Since safety is essential for clinical
application, the biocompatibility of the coordination polymers was
assessed using hemolysis tests. Although hemolysis increased with
higher concentrations of the coordination polymer, all samples showed
hemolysis rates below 5%, making them suitable for clinical
applications. Given the pivotal role of oxidative stress in ALI
progression, quenching excessive ROS is a promising therapeutic
strategy. Due to their catechol moieties, polyphenols are generally
recognized for their antioxidative potential. This hypothesis was
validated by assessing the metal–phenolic network’s scavenging activity
against DPPH radicals. As the concentration of MCQ/R NPs solution
increased, DPPH radical scavenging stabilized, and at 0.5 mg/mL, the
scavenging rate exceeded 70%. Animal model studies revealed that MCQ/R
NPs attenuated the morphological changes in rat lung tissues induced by
lipopolysaccharide (LPS). Optical microscopy revealed significant
differences in the lung tissue sections of model mice, characterized by
uneven alveolar dimensions, compromised structural integrity, thickened
alveolar septa, fibrotic changes, hemorrhage, and inflammatory
infiltration. In stark contrast, mice treated with dexamethasone,
rutin, quercetin, a mixture of rutin and quercetin, and MCQ/R NPs
showed varying degrees of damage alleviation, with lung tissue
structures approaching normalcy. Notably, compared to free drugs and
the positive control, MCQ/R NPs showed superior efficacy in
downregulating TLR4 and NLRP3 mRNA expressions, highlighting
significant potential in improving the prognosis of acute lung injury.
Additionally, differentially expressed proteins from each group were
analyzed using KEGG pathway enrichment. The study found significant
enrichment in the coronavirus disease–COVID-19 pathway in all three
groups, aligning with our ALI model construction and treatment. Further
analysis of the coronavirus disease–COVID-19 pathway indicated that the
MCQ/R group had more target enrichment compared to the rutin–quercetin
mixture group. Moreover, the total number of enriched pathways in the
MCQ/R group was lower than in the rutin–quercetin mixture group. This
suggests that MCQ/R, compared to the rutin–quercetin mixture, can
modulate ALI treatment more effectively by targeting more key
regulatory points in a focused manner.
4. Methods, Materials, and Animals
4.1. Materials and Animals
Rutin (purity ≥ 98%) was purchased from Chengdu Efa Biotech Co., Ltd.
(Chengdu, China). Quercetin (purity ≥ 98%) was sourced from Chengdu Efa
Biotech Co., Ltd. (Chengdu, China). D-Mannitol was obtained from
Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).
Tris-HCl (pH 8.8) was acquired from Shanghai Biyuntian Biotech Co.,
Ltd. (Shanghai, China). Dialysis bags (3.5 KDa) and ultrafiltration
tubes (3 KDa) were provided by Guangzhou Yuecan Laboratory Instrument
Co., Ltd. (Guangzhou, China). Reactive oxygen species (ROS) assay kit
was procured from Shanghai Biyuntian Co., Ltd. (Shanghai, China).
Wright-Giemsa stain was bought from Sigma-Aldrich (St. Louis, MI, USA).
Cerium dioxide solution (CeO[2]) was obtained from Shanghai Aladdin
Biochemical Technology Co., Ltd. (Shanghai, China).
BALB/c mice, SPF grade, aged 6–8 weeks and weighing 20–25 g, were
supplied by the Animal Experimental Center of Southern Medical
University (Guangzhou, China). The animals were housed at a temperature
of (24 ± 2) °C with a relative humidity of 50–60%. They had ad libitum
access to food and water.
4.2. Synthesis and Characterization of MCQ/R NPs
The synthesis of MCQ/R NPs nanoparticles proceeded as follows: An
appropriate volume of a mixed solution of rutin and quercetin was
combined with an adequate amount of Tris-HCl (pH 8.8) in a reaction
flask and stirred magnetically at room temperature for five minutes.
Subsequently, an optimal volume of cerium dioxide solution was
introduced into the system and allowed to react for an additional five
minutes. Finally, under continuous magnetic stirring, D-mannitol was
incrementally added dropwise to the reaction mixture and stirred for 24
h ([108]Figure 6). The resulting solution was then dialyzed for 24 h
and stored in ambient conditions. UV–Vis, FTIR, ICP-MS, and TEM were
used for a comprehensive characterization. Malvern Zetasizer Nano ZS
facilitated precise measurements of particle diameter, zeta potential,
and PDI, offering intricate insights into nanoparticle morphology,
structure, and cerium ion content.
Figure 6.
[109]Figure 6
[110]Open in a new tab
Preparation procedure of MCQ/R NPs. An appropriate volume of a mixed
solution of rutin and quercetin is added to a reaction flask containing
Tris-HCl (pH 8.8). The mixture is stirred magnetically at room
temperature for five minutes. Subsequently, an optimal volume of CeO[2]
is reacted with for an additional five minutes. Finally, under
continuous magnetic stirring, D-mannitol is titrated into the reaction
mixture and stirred for 24 h.
4.3. Characterization of Coordination Polymers
4.3.1. TEM and ICP-MS Analysis
For TEM analysis, suspensions of each coordination polymer were
deposited onto copper grids coated with carbon film, stained with a 2%
(w/v) tungstophosphoric acid solution, and air-dried at room
temperature. The morphological behaviors of each sample were visualized
using a transmission electron microscope operating at 120 kV (Hitachi,
Tokyo, Japan). For ICP-MS examination, coordination polymer samples
were analyzed using an Agilent 7700 ICP-MS instrument with suitably
pure and concentrated sample solutions. The ICP-MS instrument was
configured with specific parameters: RF power at 1.55 kW, RF matching
at 1.80 V, auxiliary gas flow at 1.50 L/min, carrier gas flow at 0.71
L/min, and makeup gas flow at 0.48 L/min. Sample solutions were
introduced to the instrument over a 45 s uptake period, allowing the
instrument to initiate after a 30 s stabilization interval. Data
generated by the ICP-MS instrument were collected and analyzed with an
integration time set at 0.90 s per mass, providing insights into the
mass and composition of the coordination polymer samples.
4.3.2. UV–vis Spectrophotometry
The structural composition of MCQ/R NPs was determined using
ultraviolet (UV) spectroscopy. The procedure involved the sequential
sampling and testing of appropriate volumes of the MCQ/R NP solution
and a physical mixture solution of the coordinating polymer [[111]37].
4.3.3. FTIR Spectroscopy
Fourier-transform infrared (FTIR) spectroscopy was employed to
determine the structural composition of MCQ/R NPs. The method involved
evaporating and drying an adequate volume of the MCQ/R NP solution to
produce a powder. An appropriate amount of physically mixed
coordinating polymer powder was then sampled, followed by sequential
testing of the aforementioned two powders and D-mannitol powder
[[112]37].
4.4. Stability Test of MCQ/R NPs
The prepared MCQ/R NPs solution was stored at room temperature. Its
stability was assessed through continuous visual observations over
several days and a particle size analysis was conducted using a laser
particle size analyzer.
4.5. Radical Scavenging Ability Experiment
We accurately weighed 5.9148 mg of DPPH using an analytical balance and
transferred it into a brown glass bottle. We then dissolved it in
anhydrous ethanol and diluted it to a volume of 100 mL, resulting in a
DPPH working solution of 150 μmol/L. A volume of 180 μL of this working
solution was mixed with 20 μL of sample solutions at concentrations of
0.01, 0.05, 0.1, 0.5, and 1 mg/mL. The mixtures were gently agitated to
ensure homogeneity. For the positive control, distilled water (dH[2]O)
was used instead of the sample solution. Meanwhile, for the negative
control, anhydrous ethanol was used instead of DPPH. After incubation
in the dark for 30 min, absorbance readings were taken at 517 nm for
all groups using an enzyme-linked immunosorbent assay (ELISA) plate
reader [[113]37]. The DPPH radical scavenging rate was calculated using
the following equation:
[MATH:
DPPH scavenging <
mi>rate (%)=(1−Asamp
le−Anega
tive con
trolAposi
tive con
trol) × 100%
:MATH]
4.6. Hemolysis Test
Cells were repeatedly washed with PBS (pH 7.4) and then centrifuged at
2500 rpm for 10 min. The pellet was resuspended in PBS to achieve a 2%
(v/v) red blood cell suspension. The sedimented red blood cells were
washed 3–4 times with PBS (pH 7.4) until the supernatant was no longer
red. Different concentrations of the MCQ/R NPs solution (100, 200, 300,
and 400 µg/mL) were mixed with an equal volume of the 2% red blood cell
suspension and incubated at 37 °C on a shaker at 65 rpm for 3 h. After
incubation, the mixtures were centrifuged at 3000 rpm for 10 min, and
200 μL of the supernatant was collected into a 96-well plate. The
absorbance of the released hemoglobin in the supernatant was measured
at 540 nm. Red blood cell suspensions incubated with either deionized
water or physiological saline served as positive and negative controls,
respectively [[114]37]. The hemolysis rate was calculated using the
following formula, where the original sample was replaced by an
equivalent volume of PBS (pH 7.4) for incubation with the red blood
cell suspension.
[MATH:
Hemolysis rate (%)=(1−Asamp
le−Aoriginal sampleAposi
tive con
trol−Anega
tive con
trol) × 100%
:MATH]
4.7. Animal Grouping, Administration, Modeling, and Sample Collection
Following 5 days of acclimatization, mice were weighed, ranked by
weight, and stratified. Grouping was randomized using a random number
table. Except for the blank group, acute lung injury models were
induced via the intraperitoneal injection of a 5 mg/kg LPS saline
solution. Twelve hours post-modeling, and based on the literature
conversion, mice were administered equivalent doses. The groups
consisted of blank, LPS (5 mg/kg), dexamethasone (5 mg/kg), rutin (100
mg/kg), quercetin (100 mg/kg), a mixed solution of rutin and quercetin
(100 mg/kg each), and MCQ/R NPs (50 mg/kg). Each group contained 10
mice, with intraperitoneal injections administered every 12 h for 5
consecutive days. The blank and model groups received equivalent
volumes of saline. Blood was drawn from the orbit 12 h after the final
dose, and serum was separated for storage. From each group, three mice
underwent bronchoalveolar lavage by exposing the trachea, inserting a
soft tube, and washing with pre-cooled PBS (pH 7.4), ensuring an 85%
retrieval rate. The BALF was centrifuged at 3000 rpm (centrifuge
radius: 10 cm) for 10 min at 4 °C. A total of 0.8 mL of the supernatant
was aliquoted for total and inflammatory cell counts. After BALF
collection, lung tissues were washed with PBS (pH 7.4). The right lung
was fixed in 4% neutral formalin, the left lung was stored at −80 °C,
and other mice were similarly processed to compute W/D values. Heart,
liver, spleen, and kidney tissues were also collected and stored at −80
°C.
4.8. Pulmonary Tissue Hematoxylin and Eosin (H&E) Staining
Mouse left lung tissues were thoroughly rinsed with water for several
hours. The tissues were then dehydrated through a graded ethanol series
of 70%, 80%, and 90%. This was followed by a 15 min incubation in an
equal mixture of pure ethanol and xylene, and subsequent xylene
treatments for 15 min each until transparency was achieved. The tissues
were then immersed in a 50:50 mixture of xylene and paraffin for 15
min, followed by sequential immersion in paraffin I and paraffin II,
each for 50–60 min. After embedding in paraffin, sections were cut. The
paraffin sections were baked, dewaxed, and rehydrated. The rehydrated
sections were stained with hematoxylin solution for 3 min,
differentiated with hydrochloric acid ethanol for 15 s, briefly rinsed
with water, blued for 15 s, and then rinsed again. Eosin staining was
applied for 3 min, followed by a thorough rinsing. The sections were
then dehydrated, cleared, mounted, and examined under a microscope.
4.9. Serum Levels of TNF-α, IL-1β, and IL-6 Were Measured in Mice
Blood was drawn from the orbital plexus and allowed to clot at room
temperature for 2 h. The samples were then centrifuged at 4 °C at 3000
rpm for 10 min. The supernatant, representing the serum, was collected.
Assays were performed using ELISA according to the manufacturer’s
protocol.
4.10. Quantitative Real-Time PCR (qRT-PCR) Was Employed to Assess mRNA
Expression Levels
Total RNA was extracted from lung tissues utilizing an RNA extraction
kit, followed by the determination of RNA concentration. For the
qRT-PCR analysis, we used the SYBR Premix Ex Taq™ II (Tli RNase H Plus)
with the Archimed-X6 real-time PCR system. The forward (F) and reverse
(R) primers used for the qRT-PCR are listed in [115]Table 4.
Table 4.
Sequences of the primers.
Gene Forward Primer (5′–3′) Reverse Primer (5′–3′)
GAPDH CATCACTGCCACCCAGAAGACTG ATGCCAGTGAGCTTCCCGTTCAG
TLR4 AGCTTCTCCAATTTTTCAGAACTTC TGAGAGGTGGTGTAAGCCATGC
NLRP3 ACCTCAACAGTCGCTACACG ATGGTTTTCCCGATGCC
[116]Open in a new tab
4.11. Proteomics Analysis
Protein concentration was measured at 280 nm using a NanoDrop
spectrophotometer (Thermo, Waltham, MA, USA) with an extinction
coefficient of 1.1 AU. The Filter-Aided Sample Preparation (FASP)
protocol was used for proteolytic digestion of proteins to remove
detergents from the lysate, employing centrifugal units with a
molecular weight cutoff of 30,000. This involved adding 200 μL of 8 M
urea in 0.1 M Tris/HCl, pH 8.5 (UA buffer), to YM-30 Microcon filter
units (Millipore, Burlington, MA, USA) containing CM protein
concentrates and centrifuging at 14,000× g for 15 min at 20 °C,
repeated twice. A total of 50 μL of 0.05 M iodoacetamide in 8 M urea
was added to the filters, incubated for 20 min in the dark, and washed
twice with 100 μL of 8 M UA buffer and three times with 100 μL of 50 mM
NH[4]HCO[3]. A total of 100 μL of 50 mM NH[4]HCO[3] containing trypsin
(Promega, Madison, WI, USA) was added to each filter at a
protein-to-enzyme ratio of 100:1. Samples were incubated overnight at
37 °C, and the digested peptides were collected by centrifuging at
14,000× g for 15 min at 20 °C. The peptides were then analyzed online
using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA) coupled with a nano-flow HPLC (EASY-nLC
1000 system, Thermo Fisher Scientific, USA) and a self-packed
chromatography column with Ultimate XB-C18 3 μm resin (Welch Materials,
West Haven, CT, USA). The peptide mixtures were separated on a C18
reversed-phase column (10 cm length, 75 μm inner diameter) using a 75
min linear gradient of 3–100% buffer B (99.5% acetonitrile, 0.5% formic
acid) in buffer A (99.5% water, 0.5% formic acid) at a flow rate of 350
nL/min. The total LC-MS/MS run time, including loading and cleaning
steps, was approximately 90 min. The electrospray voltage was set to
2.0 kV. During data-dependent MS/MS acquisition, the dynamic exclusion
time was set to 18 s. In the MS1 stage, the resolution was set to
70,000 and the AGC target to 3 × 10^6, with a maximum injection time of
20 ms. In the MS2 stage, the resolution was 17,500, the AGC target was
1 × 10^6, and the maximum injection time was 60 ms. The scan range was
set to 300–1400 m/z, with the top 20 strongest precursor ions selected
for MS/MS analysis. The raw data were subjected to database searching,
and significant proteins were selected using R Studio software, with
p-values of less than 0.05 set as the threshold for the statistical
analysis of differentially expressed proteins between the control and
model groups and the control and high-dose groups. The PPI networks of
the differential proteins of the two groups were integrated and
analyzed.
4.12. Statistical Analysis
Image J (Bio-Rad, Hercules, CA, USA) was used for the quantitative
analysis of target bands on Western blots. Histograms and statistical
analyses were performed using GraphPad Prism (GraphPad Prism 9.0, San
Diego, CA, USA). The results are presented as the mean ± standard
deviation (SD). Analysis of variance (ANOVA) followed by Tukey’s
post-hoc test was used to compare means among multiple groups.
Significance levels were set at p < 0.05, p < 0.01, and p < 0.001.
5. Conclusions
In this study, rutin and quercetin acted both as therapeutic agents and
as self-carrier materials, interacting with cerium ions as the
connecting points and mannitol as a potential targeting delivery
carrier, ultimately self-assembling into MCQ/R nanoparticles. These
nanoparticles demonstrated a clear therapeutic effect in the treatment
of acute lung injury while maintaining good biocompatibility.
Acknowledgments