Abstract
Bioenergetic therapy based on tumor glucose metabolism is emerging as a
promising therapeutic modality. To overcome the poor bioavailability
and toxicity of arenobufagin (ArBu), a MOF-derived intelligent
nanosystem, ZIAMH, was designed to facilitate energy deprivation by
simultaneous interventions of glycolysis, OXPHOS and TCA cycle. Herein,
zeolitic imidazolate framework-8 was loaded with ArBu and indocyanine
green, encapsulated within metal–phenolic networks for chemodynamic
therapy and hyaluronic acid modification for tumor targeting. ZIAMH
nanoparticles can release ArBu in the tumor microenvironment for
chemtherapy, and ICG enables photothermal therapy under near-infrared
laser irradiation. In vitro and in vivo mechanism studies revealed that
the ZIAMH nanoplatform downregulated glucose metabolism related genes,
resulting in the reduction of energy substances and metabolites in
tumors. Additionally, it significantly promoted cell apoptosis by
upregulating pro-apoptotic proteins such as Bax, Bax/Bcl-2, cytochrome
C. Animal studies have shown that the tumor inhibition efficiency of
ZIAMH nanomedicines was three fold higher than that of free drugs.
Therefore, this study provides a new strategy for glucose
metabolism-mediated bioenergetic therapy and PTT/CDT/CT combined
therapy for tumors.
Graphical Abstract
[50]graphic file with name 12951_2024_3084_Figa_HTML.jpg
Supplementary Information
The online version contains supplementary material available at
10.1186/s12951-024-03084-1.
Keywords: Arenobufagin, Nanomedicine, Glycolysis, Oxidative
phosphorylation, Tricarboxylic acid cycle, Bioenergetic therapy,
Integrated photothermal-chemodynamic-chemotherapy
Introduction
Metabolism is the essential characteristic and material basis of cell
life. Unlike normal cells, tumor cells undergo metabolic reprogramming
during differentiation and proliferation, such as changes in glucose
metabolism. In 1920s Otto Warburg reported that tumor cells mainly
produce adenosine triphosphate (ATP) through glycolysis even under
aerobic conditions (Warburg effect) [[51]1]. Tumor cells have higher
glucose uptake compared to normal cells [[52]2]. Metabolic
intermediates produced by glycolysis promote tumor growth, invasion,
and metastasis the produced lactic acid can damage normal tissue
structure and lowers the pH of the tumor microenvironment (TME),
preventing the tumor from being attacked by immune cells and enhancing
the “fighting capacity” of tumor cells [[53]3–[54]5]. At the same time,
glycolysis combines with the tricarboxylic acid (TCA) cycle and
oxidative phosphorylation (OXPHOS) in cancer cells to jointly provide
energy for cancer cell proliferation [[55]6, [56]7].
Over the past few decades, inhibitors have been developed to target
glucose metabolism pathway exhibiting prolonged survival rate of
experimental animals, some of these drugs are in clinical trial stages
[[57]8–[58]10]. However, due to the heterogeneity and complexity of
tumors, these new drugs showed limited efficacy and cannot prevent the
progression of cancer. In addition, traditional drug chemotherapy and
emerging monotherapies such as photothermal and chemodynamic therapy
have certain limitations in the TME [[59]11, [60]12]. For example,
tumor cells treated with PTT abnormally express multiple heat shock
proteins (HSPs), such as HSP70 and HSP90, to resist thermal damage,
resulting in insufficient tumor cell apoptosis. These proteins are
ATP-dependent and protect cancer cells from the cytotoxic effects of
heat generated during PTT [[61]13]. Glycolysis is an important pathway
for cancer cells to produce ATP, which can promote cancer cell
metastasis [[62]14–[63]16]. Inhibition of glycolysis reduces the
expression of HSPs, inhibits tumor growth and metastasis, and enhances
the therapeutic effect of PTT. Therefore, development of
multifunctional nanodrugs that target cancer cell for treatment may be
a promising direction.
In the few decades, more and more traditional Chinese medicine extracts
are showing efficient activity in tumor treatment [[64]17–[65]19].
Arenobufagin (ArBu) is a monomer extracted from secretions of Bufonis
Venenum, with a content up to 2%. It is the main active components of
Chinese medical anti-tumor preparation Huachansu injection [[66]20,
[67]21]. The potent anti-tumor effect of ArBu and the mode of action
behind the cytotoxic characteristics have been reported in previous
studies [[68]22–[69]24]. Zhang et al.[[70]25] found that ArBu can
induce apoptosis in HepG2 and its drug-resistant strain HepG2/ADM via
the mitochondrial pathway, namely, the down-regulation of some key
proteins in the PI3K/Akt/mTOR signaling pathway. In addition to
apoptosis induction, ArBu may also play an antagonistic role in
metabolic reprogramming mediated by mTOR, which means that ArBu can not
only directly kill tumor cells, but also can inhibit their growth by
regulating the metabolic patterns of tumor cells [[71]25]. However, the
potent Na/K-ATPase binding activities, low solubility and poor
bioavailability of ArBu limited its anticancer function [[72]26,
[73]27], which requires the development of appropriate cargo
nanomaterials to explore its clinical applications.
Zeolitic imidazolate framework-8 (ZIF-8) is the most typical
metal–organic framework materials (MOFs) in the ZIF family, which is
coordinated by Zn^2+ cations and 2-methylimidazole anions [[74]28].
Since zinc is an ample transition metal in human body and the side
chain of histidine incorporates imidazole, ZIF-8 exhibited thermal
stability and safety [[75]29]. In addition, due to its pH
responsiveness, ZIF-8 is easy to decompose under the acidic conditions
of the TME, leading to the release of incorporated drugs [[76]30]. To
date, multiple nano-drug delivery systems containing ZIF-8 have been
developed [[77]31–[78]33]. These systems can combine with existing
tumor treatment methods such as photothermal and photodynamic therapies
[[79]34]. PTT is an effective cancer treatment method, which can
directly and precisely treat local areas in a non-invasive manner
[[80]35]. Since tumor cells exhibit high thermal sensitivity, high
temperature can destroy tumor cell proteins or increase natural drug
release from nanocarriers, thereby achieving chemosensitization
[[81]36, [82]37]. These combinations are expected to improve treatment
outcomes and reduce side effects.
Metal–phenolic networks (MPNs) are self-assembled from metal ions and
polyphenol ligands. Polyphenols are widely found in natural plants,
which have antioxidant and anti-tumor effects [[83]38, [84]39]. Due to
their good biocompatibility and excellent adhesive ability, MPNs can
effectively encapsulate nanoparticles with various structure [[85]40].
In tumor microenvironment, MPNs initiate Fenton or Fenton-like
reactions to convert weak oxidizing H[2]O[2] into strong oxidizing
hydroxyl radical (·OH) [[86]41, [87]42]. This conversion leads to
increased intracellular oxidation levels, triggering a series of
reactions such as DNA necrosis, protein inactivation and lipid
oxidation, ultimately inducing apoptosis of cancer cells.
Herein, based on our group’s previous review of nanomedicines
modulating cancer metabolism [[88]43], we constructed a versatile
ZIF-8@ArBu&ICG@MPN@HA (ZIAMH) nanosystem to regulate glucose metabolism
in the TME, i.e., simultaneously suppress glycolysis, OXPHOS and
tricarboxylic acid (TCA) cycle, thereby inhibiting tumor growth and
metastasis. As shown in scheme [89]1, MOFs-derived ZIF-8 loaded with
ArBu and indocyanine green (ICG) were first prepared. Subsequently, a
metal-polyphenol network self-assembled from ferric iron and gossypol
encapsulated the above material for chemodynamic therapy (CDT), and
followed by coating with a hyaluronic acid (HA) film to confer the
targeting abilities of ZIAMH. During cancer therapy, ZIAMH
nanoparticles could target to the tumor sites under the action of HA to
the overexpressed CD44 receptors of cancer cells. The ArBu encapsulated
in ZIF-8 was able to exert chemotherapy (CT) while enhancing its
solubility and reducing its toxicity. Besides, the Fe^3+ in the ZIAMH
nanomaterials was released and reduced to Fe^2+ by gossypol, which
exhibited enhanced CDT effects based on the Fenton reaction to generate
·OH in the tumor tissue. Under 808 nm near-infrared (NIR) laser
irradiation, ICG-loaded ZIAMH nanometarials exhibited photothermal
conversion capabilities and can kill cancer cells for photothermal
therapy (PTT). Combining the above multiple advantages, ZIAMH could
synchronously suppress tumor cell glycolysis and OXPHOS by inhibiting
the activities of the key enzymes in the process of glucose metabolism,
such as hexokinase 2 (HK2), pyruvate kinase M2 (PKM2) and lactate
dehydrogenase A (LDHA). At the same time, it can further damage
mitochondria functions by depriving the TCA cycle of energy substances
and metabolites. Consequently, this multifunctional ZIAMH nanoplatform
is expected to combine the multiple therapeutic effects of bioenergetic
therapy with PTT/CDT/CT therapy, providing an effective strategy for
tumor treatment through precise energy deprivation and targeted
therapy.
Scheme 1.
[90]Scheme 1
[91]Open in a new tab
Schematic diagram of the synthesis process of ZIAMH and its therapeutic
mechanism of interference tumor glucose metabolism (glycolysis, OXPHOS
and TCA cycle) through enhanced bioenergetic therapy integrated with
PTT/CDT/CT therapy
Materials and methods
Materials and animals
Iron (III) chloride (FeCl[3]) and 2-methylimidazole (2-MI) was
purchased from Sigma-Aldrich (St. Louis, MO, USA). Zinc nitrate
hexahydrate [Zn (NO[3])[2]·6H[2]O] were purchased from Aladdin
Biochemical Technology Co., Ltd. (Shanghai, China). Gossypol (Gp) was
obtained from Jinan Daigang Biomaterial Co., Ltd. (Jinan, China).
Thiazolyl blue tetrazolium bromide, Annexin V-FITC, JC-1 and propidium
iodide (PI) were provided by the Beyotime Institute of Biotechnology
(Shanghai, China). Cytochrome C, caspase-9, caspase-3, Bcl-2, Bax and
GAPDH were all obtained from Abcam (Cambridge, UK). PKM2, LDHA, SDHA,
HSP70 and HSP90 were provided by Cell Signaling Technology
(Massachusetts, USA). Dithiothreitol, glycine and Tris-base were from
Biosharp (Anhui, China). PMSF, TEMED, bromophenol blue and acrylamid
were purchased from Amresco (WA, USA). DMEM, FBS, PBS buffer and
trypsin–EDTA solution (0.25%) were bought from Gibco (Carlsbad, CA).
All other chemical reagents were of analytical or HPLC grade.
HepG2 cells were provided by Cell Resource Center, Peking Union Medical
College (PCRC) (Beijing, China). Male BALB/c nude mice (6–8 weeks,
19–23 g) were purchased from Beijing Vital River Laboratory Animal
Technology Co. Ltd (Beijing, China). All animal experiment procedures
were approved by the Institutional Animal Care and Use Committee of the
Institute of Chinese Materia Medica, China Academy of Chinese Medical
Sciences, and followed the Guide for the Care and Use of Laboratory
Animals.
Preparation and characterization of ZIAMH nanocomposites
Synthesis of ZIF-8@ArBu&ICG
40 mg Zn (NO[3])[2]·6H[2]O, 0.77 g 2-methylimidazole, 1 mg ArBu and
1 mg ICG were dissolved in 5 mL methanol, stirring at room temperature
for 5 min. After centrifugation, desired precipitate was washed with
water and dried for further use.
Synthesis of ZIF-8@ArBu&ICG@MPN (ZIAM)
The material ZIF-8@ArBu &ICG, Gp and FeCl[3]·6H[2]O were mixed in
defined ratio (1:0.75:0.36 w/w). After being stirred overnight at room
temperature, the mixture was dialyzed in deionized water, and the
dialysis melium was interplaced by fresh deionized water every 2 h.
Nanoparticles were collected by centrifugation and washed three times
with deionized water. ZIF-8@MPN of control group was synthesized in
similar way.
Synthesis of ZIAMH
The HA solution (1.0 mg/mL) was stirred with ZIAM at a ratio of 1:50 in
deionized water for 2 h, and ZIAMH was obtained by centrifugation,
resuspended in PBS, and kept at 4 °C for further experiments.
Characterization of ZIAMH
The morphology of the nanoparticles was observed by transmission
electron microscope (HITACHI, Tokyo, Japan). The particle size,
dispersion index (PDI) and Zeta potential of the prepared ZIAMH
nanoparticles were measured by a Malvern NanoZS90 (UK). The phase
composition and microstructure of the nanoparticles were further
analyzed using Powder X-ray diffraction (PXRD), The element
distribution was analyzed by Energy Dispersive Spectrometer (EDS). The
surface area of the nanoparticles was measured by
Brunauer − Emmett − Teller (BET) method. The loading content (LC) and
encapsulation efficacy (EE) of the ArBu and ICG in the nanoparticles
was measured by UV − vis spectra, and was determined according to the
following equations.
[MATH:
LC=weightofdruginthenanoparticl
esweightofdrugloadedmicelles×100% :MATH]
[MATH:
EE=weightofdruginthenanoparticl
esweightoftheaddeddrug×100% :MATH]
Performance investigation of ZIAMH nanomaterials
In vitro drug release
The in vitro ArBu and ICG release from nanoparticles was determined at
37 °C in PBS containing 0.5% Tween-80 (pH 4.0, pH 5.6 or pH 6.5). The
quantities of released ArBu and ICG drug were determined by UV–vis
spectrophotometer, respectively. Each sample was measured for three
times.
Detection of ·OH
Methylene blue (MB) was introduced to detect the hydroxyl radical (·OH)
generated by ZIAMH. Using methylene blue (MB) as an indicator, the
generation of ·OH was detected by UV–Vis spectrophotometer. 100 μg and
200 μg of ZIAMH was mixed with MB (10 μg/mL) and H[2]O[2] (150 μM),
respectively, to investigate the effect of sample size on the chemical
kinetic catalytic ability.
The photothermal properties of ZIAMH
The temperature elevation curve and photothermal stability of ZIAMH
were measured under 808 nm NIR laser irradiation in vitro. Firstly, the
thermal stability of ZIAMH was established. The temperature variation
curves of ZIAMH solution at different concentrations (125, 250, 500,
1000 μg/mL) were measured under the irradiation of 808 nm NIR with a
power density of 1.5 W/cm^2 according to the previous study [[92]44].
FLIR Thermal CAM (E50) was used to record the real-time thermal image
of the sample, and FLIR Examiner software was used to quantify and draw
the temperature elevation curve. Secondly, the thermal stability of
ZIAMH was investigated. 1 mL of ZIAMH solution was irradiated with
808 nm NIR laser for 600 s and cooled down to room temperature,
followed by three cycles of laser irradiation. The temperature change
of the solution was recorded in real time curve.
Hemolysis Assay
The hemolysis of nanoparticles was determined by co-culture with human
red blood cells according to previously reported method. Red blood
cells were isolated by centrifugation at 5000 rpm, then were rinsed
with tris buffer and finally diluted to 5% blood cell stock solution.
The concentration of the material was started from 1000 μg/mL, which
was diluted using a double dilution method. The material was then
incubated with an equal amount of red blood cell samples in a 37% CO[2]
incubator for 1 h. The PBS group was used as negative control. 1%
Triton X-100 solution was defined as positive control (which induced
100% hemolysis). After centrifugation, the supernatant was collected
and its absorbance was measured at 540 nm. Triplicates were made for
each test, the data were recorded as mean ± standard deviation (SD,
n = 3). The hemolytic percentage was estimated by the following
equation:
[MATH: Hemolysis%=Am-<
msub>An/Ap-<
msub>An×100%
:MATH]
where A[m] is the absorbance of cells treated by ZIAMH, A[n] is the
absorbance of the negative control, and A[p] is the absorbance of the
positive control.
In vitro cellular uptake
HepG2 cells were inoculated in confocal laser scanning microscope
(CLSM) plates at a density of 1 × 10^5 cell/ well for 24 h incubation.
Free ICG (0.5 μg/mL), ZIF-8@ArBu&ICG@MPN (ZIAM, 0.625 mg/mL) and ZIAMH
(0.625 mg/mL) were added to the cells before incubation for 0, 0.5, 1,
2 or 4 h. HepG2 cells were washed with PBS and fixed with 4%
paraformaldehyde for 30 min. Pre-cooled TritonX-100 was added to
destroy the cell membrane. After washing with PBS, HepG2 cells were
stained with DAPI for 10 min. The fluorescence intensity was imaged and
analyzed by laser confocal microscopy at an excitation wavelength of
750 ± 10 nm and an emission wavelength of 810 ± 20 nm.
Cytotoxicity evaluation
HepG2 were seeded in 96-well plates with 5 × 10^3 cell/ well for 24 h,
and then different gradient concentrations of ZIAMH were added to
investigate the cytotoxicity. Next, ZIF-8@MPN@HA (ZMH), ArBu,
ArBu&ICG + NIR and ZIAMH + NIR were added and incubated for 24 h. The
cell viability value was detected using MTT colorimetry and calculated
as follows:
[MATH: Cell viability%=OD(treatment group)/OD(control group)×100% :MATH]
Flow cytometry analysis of apoptosis
The cell apoptosis mechanism was explored by flow cytometry using
Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences). HepG2 cells
were inoculated into 6-well plate for 24 h, follow by 24 h incubation
with PBS、ZMH (0.625 mg/mL), ArBu (90 μM), ArBu (90 μM)&ICG (50 μg) and
ZIAMH (0.625 mg/mL), during which ArBu&ICG group and ZIAMH group were
irradiated by 1.5 w/ 5 min/ well of 808 nm laser post 4-h incubation.
Collected cell pellets were washed with cool PBS and resuspended up to
1 × 10^6 cells/mL with binding buffer. 100 μL of the cell suspensions
and 5 μL of AnnexinV-FITC were then gently mixed in falcon tube,
followed by 30 min dark incubation at room temperature, with 5 μL of PI
added in the middle of incubation. The suspension was then replenished
with 400 μL of binding buffer and analyzed immediately.
Live/Dead assay
HepG2 cells were planted in 6-well plates at a density of 3 × 10^5
cells/ well, and then treated with PBS, ZMH (0.625 mg/mL), ArBu
(90 μM), ArBu (90 μM)&ICG (5 0 μg), ZIAMH (0.625 mg/mL) for 24 h,
during which ArBu&ICG group and ZIAMH group were irradiated with 1.5 W/
well 808 nm laser for 5 min after 4 h of treatment. The harvested cells
were stained with 10 nM of calcein AM and PI dye for 10 min at 37℃.
Cells were washed with PBS before analysis by confocal microscopy.
Measurement of mitochondrial membrane potential
HepG2 cells were treated with PBS, ZMH (0.625 mg/mL), ArBu (90 μM),
ArBu&ICG + NIR (90 μM ArBu & 50 μg ICG), ZIAMH + NIR (0.625 mg/mL) for
24 h, during which ArBu&ICG group and ZIAMH group were irradiated with
1.5 W/ well 808 nm laser for 5 min after 4 h of treatment. The
harvested cells were then incubated with 1 mL JC-1 working solution for
20 min. After washing, the cells were resuspended with 500 μL of JC-1
working solution for analysis by BD FACSCalibur flow cytometer.
Western blot analysis
Total protein was extracted from HepG2 cells or nude mouse tumors using
RIPA lysis buffer (Beyotime, China) with 1% protease inhibitor PMSF
(Amresco, China). The protein content was determined using a BCA
protein assay kit (Beyotime, China). 30 μg of total protein were taken
for SDS-PAGE electrophoresis, and wet transferred to polyvinylidene
fluoride (PVDF) membrane (Merck, Germany), based on the protein
quantification results. After blocking with 5% skimmed milk/TBST
solution at room temperature for 1 h, the PVDF membrane was incubated
with the indicated primary antibodies: caspase-9 (1:500), caspase-3
(1:1000), cytochrome C (1:1000), Bcl-2 (1:1000), Bax (1:1000), PKM2
(1:500), LDHA (1:1000), SDHA (1:500), HSP70 (1:1000) and HSP90
(1:1000), and was shaken overnight at 4 °C. After washing with TBST,
the PVDF membrane was incubated with the corresponding secondary
antibody (1:10,000) for 45 min at room temperature. The protein band
was acquired by Tanon 5200 Chemiluminescence Imaging System (Shanghai,
China) and GAPDH was used as internal control.
Seahorse cell energy metabolic analysis
The oxygen consumption rate (OCR) and extracellular acidification rate
(ECAR) of HepG2 cells were detected by Seahorse XF-96 system (Agilent,
Santa Clara, USA) according to the instructions of Seahorse XF Cell
Mito Stress Test Kit and Glycolysis Stress Test Kit. The HepG2 cells
were seeded into Seahorse XF cell culture plates and were treated with
PBS, ZMH, ArBu, ArBu&ICG + NIR and ZIAMH. The two indicators OCR and
ECAR were measured to evaluate the energy metabolism status and
mitochondrial function of cells, and the experimental data was analyzed
using wave software and report generator.
Cellular ROS analysis
Cellular reactive oxygen species (ROS) levels were determined using a
2,7-dichloro-fluorescein diacetate (DCFH-DA) assay kit (Beyotime,
#S0033M) following the manufacturer's protocol. HepG2 cells were seeded
into confocal small dish cultured for 24 h at 37 °C and incubated with
ZMH, ArBu, ArBu&ICG + NIR and ZIAMH + NIR for 4 h, respectively.
Subsequently, the cells were washed with PBS three times and incubated
with 10 μM DCFH-DA for 30 min. Then, the cells were observed by CLSM.
In vivo NIR fluorescence imaging of ZIAMH
For NIR fluorescence imaging and biodistribution analysis, HepG2
tumor-bearing mice were intravenously injected with ICG or ZIAMH. The
tumor-bearing mice were imaged using an animal imaging system (Xtreme,
Bruke) at 0.5, 4, 8, 24, and 48 h post-injection. Mice were sacrificed
48 h after administration, and tumor tissues and main organs (including
heart, liver, spleen, lung and kidney) were isolated for fluorescence
imaging and semi-quantitative analysis. The excitation wavelength of
ICG was 750 ± 10 nm and the emission wavelength was 810 ± 20 nm.
In vivo photothermal imaging of ZIAMH
To evaluate the in vivo photothermal effect of ICG-loaded ZIAMH
nanoparticles, PBS and ZIAMH were intravenously injected to
tumor-bearing mice, the thermal change of tumor irradiated at 808 nm
NIR laser was monitored. Moreover, 24 h after intravenous injection was
selected as the irradiation time and ICG accumulation reached its peak
at this time. The mice were anesthetized with isoflurane, at
predetermined time, and then 808 nm NIR laser (1.5 W/cm^2) was injected
to the tumor site. The mice were thermally imaged with an infrared
thermal imager (FLIR E50) every 0.5 min for a total of 5 min.
Quantitative analysis on imaging results and thermal variation data was
performed by FLIR tools.
In vivo tumor inhibition evaluation
To establish a xenograft tumor nude mouse model, 6 × 10^6 HepG2
cells/well were subcutaneously injected into male BALB/c nude mice
based on a previously described study with some minor changes [[93]16].
After the tumor volume reached approximately 100 mm^3, HepG2
tumor-bearing mice were randomly divided into five groups (n = 3): PBS
group, ZMH group, ArBu group, ArBu + ICG + NIR group, PD-L1 group,
ZIAMH + NIR group and ZIAMH + PD-L1 + NIR group, afterward the mice
were intravenously injected with the above drug formulations. For
photothermal therapy, 24 h after injection, the tumor site was
irradiated with 808 nm NIR laser (1.5 W/cm^2) for 5 min. The tumor
volume and mouse body weight (bw) were measured every two days for a
total of 10 days, and the tumor volume was calculated according to the
following formula: V (mm^3) = length × (width)^2/2. The tumor
inhibition ratio percentage = (bw control group -bw treatment group)/
bw control group × 100%. The tumor-bearing mice were sacrificed 21 days
after intravenous administration, and the heart, liver, lung, spleen
and kidney of the mice were obtained for hematoxylin and eosin (H&E)
staining to evaluate the morphology of cancer cells.
Histochemical staining
For hematoxylin and eosin (H&E) staining, tumor tissues and main organs
(heart, liver, spleen, lung and kidney) of HepG2 tumor-bearing mice
were fixed in 4% formaldehyde solution and then placed in different
concentrations of ethanol for dehydration. Subsequently, the tissues
were cleared with xylene, immersed and embedded in molten paraffin. The
wax blocks were cut into sections and dewaxed with xylene, then
rehydrated and rinsed with water, and stained with hematoxylin and
eosin. The coverslips were covered with neutral balsam mounting medium
for sealing. The tumor and major tissue sections were observed and
photographed with a microscope. For CD4 immunohistochemical staining,
the tissue fixation and section steps are similar to H&E staining,
expected that a CD4 monoclonal antibody was added to specifically
identify immune cell subpopulations that express CD4 molecules.
RNA-sequencing analysis
The tumor tissues were ground into powder using liquid nitrogen and
total RNA was extracted by Trizol (Thermo Scientific, USA). RNA
integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent
Technologies, USA). The transcriptome library was constructed using a
TruSeq Stranded mRNA LTSample Prep Kit (Illumina, USA), and
RNA-sequencing analysis was conducted by Shanghai OE Biotech Co., Ltd
(Shanghai, China).
Fastq software was used to perform quality control analysis on the
preprocessed data. The clean data were mapped to Homo sapiens
(GRCh38.p13) by HISAT2. The Fragments Per Kilobase Million (FPKM) value
of each gene was calculated by Cufflinks, and the sequence counts for
per gene were acquired by HTSeq-count. Differentially expressed genes
(DEGs) analysis was carried out using an R package, DESeq2. Data with
P < 0.05 and log[2] |(fold change)|> 1 was considered as significant
DEGs. Principal Component Analysis (PCA), heatmap analysis, Gene
Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
enrichment analysis was conducted using Omicshare cloud tools
([94]https://www.omicshare.com/tools/).
Targeted metabolomics of glucose metabolic pathways
The tumor tissues in each group were homogenized with threefold volume
of saline solution. 100 μL of the homogenate were added with 0.2%
formic acid-acetonitrile, then were vortexed for 60 s and sonicated for
10 min in an ice-cold sonication bath. The mixture was centrifuge at
12,000 r/min for 15 min at 4 °C. The supernatant was taken for
UPLC-MS/MS targeted metabolomic analysis.
The UPLC-6500 + triple quadrupole MS/MS system (SCIEX, CA, USA) was
applied for quantitative analysis of TCA cycle metabolites in the above
tumor tissues. A Waters UPLC^® HSS PFP column (2.1 mm × 100 mm, 1.8 μm)
was used for sample separation at 35 ℃. The mobile phase consisted of
(A) water containing 0.05% formic acid and (B) acetonitrile containing
0.05% formic acid with a flow rate of 0.3 mL/min. The following
gradient program of chromatographic condition was performed: 0–4 min,
2% B; 4–6 min, 2%–98% B; 6–10 min, 98% B; 10–10.1 min, 98%–2% B;
10.1–14 min, 2% B. The injection volume was 3 µL. The MS spectrum
conditions were optimized as follows: ESI source negative ion mode;
multiple reaction monitoring (MRM) detection mode; spray
voltage, ± 4500 V; atomization temperature, 550 ℃; curtain gas, 35 psi;
collision air pressure, 9 psi; atomization gas pressure (N[2]) and gas
pressure (N[2]) were both 55 psi. The parameters of metabolites
including Q1 Mass, Q3 Mass, collision energy (CE) and decluttering
potential (DP) were listed in Table S1.
Statistical analysis
All data were analyzed by GraphPad Prism software (Version 9.0,
SanDiego, USA) and expressed as means ± standard deviation (SD).
One-way ANOVA and t-test statistical analysis were used for all
experimental data. The significant differences were defined as
*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Results and discussion
Synthesis and characterization of ZIAMH
In order to fabricate the nanocomposites, ArBu and ICG were firstly
loaded into ZIF-8 nanocarriers, gossypol and Fe^3+ were then added to
establish a metal polyphenol network on the surface of ZIF-8
nanoparticles. HA was encapsulated on the outer layer of the
nanoparticles to form the final structure of ZIAMH. As observed by
transmission electron microscopy (TEM) (Fig. [95]1a), the morphology of
ZIF-8, ZIF-8@MPN and ZIAMH exhibited regular polyhedral structures, and
the particle size distribution was between 150–190 nm. The elemental
mapping of ZIAMH nanoparticles were analyzed using a TECNAI F20
transmission electron microscope equipped with an energy dispersive
X-ray spectrometer (EDX). The results clearly showed that Zn, Fe, C, O
and N elements were uniformly distributed in ZIAMH (Figure S1), which
confirmed the metal polyphenol network on the surface of ZIF-8
nanoparticles. Dynamic light scattering (DLS) test showed that the
sizes of three particles fluctuated by about 10 nm around 190 nm,
indicating that the outer layer of ZIF-8 did not alter its basic
morphology (Fig. [96]1b). The Zeta potential of ZIAM was
16.5 ± 1.28 mV, while the Zeta potential of ZIAMH shifted
to − 21.9 ± 1.78 mV after HA coating (Table S2). This change from
positive to negative charge emphasizes the influence of the HA layer on
the physical characteristics of ZIAMH. The PDI values of three
particles were all less than 0.2. The above results demonstrated the
assembled nanoparticles have uniform morphology and good size
distribution.
Fig 1.
[97]Fig 1
[98]Open in a new tab
Synthesis and characterization of ZIAMH. a TEM images of ZIF-8,
ZIF-8@MPN and ZIAMH (scale bar: 100 nm). b DLS results of ZIF-8,
ZIF-8@MPN and ZIAMH. c XPS pattern of ZIAMH. d PXRD patterns of
ZIF-8@ICG&Arbu, ZIF-8@MPN and ZIAMH. e UV–vis spectra of MB, MB after
reaction with ZIAMH in different concentrations. f The cumulative
release of ArBu and ICG from ZIAMH at pH 6.5. g 10-min photothermal
temperature curves of ZIAMH dispersed in water at various
concentrations. h Photothermal conversion curves of ZIAMH under three
cycles of irradiation
The assay on surface elements through X-ray photoelectron spectroscopy
(XPS) also confirmed the desired construction of the material
(Fig. [99]1c). Derived from the analysis of the phase composition and
microstructure of nanoparticles using PXRD, it was found that the
signature peaks of all nanoparticles were almost identical to those
simulated of ZIF-8, the crystal structure of the nanoparticles remained
intact post subsequent modifications (Fig. [100]1d). The evaluation on
material's specific surface area, pore volume, and pore structure was
conducted by N[2] adsorption measurement (Figure. S2). According to the
BET model, the specific surface area of ZIAMH nanoparticles was
calculated to be 76.7554 m^2/g, the pore volume and pore diameter were
0.0394 cm^3/g and 2.604 nm, respectively. The sharp increase in N[2]
absorption by ZIAMH nanoparticles under low relative pressure (< 0.01)
indicates the existence of mesoporous structure. Additionally,
methylene blue (MB) was used as an indicator to test whether Fe^2+ in
the material can effectively generate ∙OH through Fenton reaction. As
shown in Fig. [101]1e, with the increase amount of material sample, the
absorbance of MB significantly decreased, indicating that the amount of
material sample was proportional to the catalytic chemical kinetic
energy.
The UV spectrophotometer assay was applied to determine that the
material could load with 5.7% ArBu and 7.2% ICG, and it was also used
to investigate the release of nanoparticles loaded with the two drugs
at different pH values (Fig. [102]1f and S3). It can be seen that the
cumulative release drug amount could be accelerated at lower pH. It was
hypothesized that the structures of MPNs and ZIF-8 within the
nanomaterials can be decomposed under acidic environments, which leaded
to the breakage of the material structure and release of the
incorporated drugs. TEM images confirmed that ZIAMH composites
gradually decomposed in acidic PBS (pH 5.6 and 6.5), producing
small-sized ZIAMH fragments, while they retained the regular morphology
in neutral PBS (pH 7.4) within 8 h, which is consistent with the
literature reports (Figure. S4).
ICG can convert light energy into thermal energy under NIR laser
irradiation due to its photothermal characteristic [[103]45]. In this
study, different concentrations (0, 125, 250, 500, and 1000 μg/mL) of
ZIAMH nanoparticles were exposed to 808 nm NIR light (1.5 W/cm^2). As
shown in Fig. [104]1g, the temperature rise amplitude is positively
correlated with the concentration and illumination time of the
nanoparticles. The temperature of the aqueous solution rapidly
increased from 27.1 ℃ to 52.5 ℃ after 600 s illumination even at a
nanomaterial concentration as low as 125 μg/mL, suggesting that the
ZIAMH nanomaterials had good photothermal conversion effects. Moreover,
the thermal performance test also showed the material exhibited
sustained photothermal ability at least post three cycles
(Fig. [105]1h).
To evaluate the biocompatibility of ZIAMH nanomaterials in vitro,
hemolysis experiments were conducted using red blood cells. The results
suggested that the hemolysis rate of the nanoparticles at all
concentrations (15.6, 31.25, 125, 250, and 500 μg/mL) was less than 5%,
which demonstrated that ZIAMH nanoparticles have wonderful
hemo-compatibility (Figure. S5).
In vitro cellular uptake and anti-tumor effects of ZIAMH nanoparticles
HA is a tumor-targeting ligand and can actively target liver tumors by
binding to the CD44 receptor [[106]46–[107]48]. HepG2 cell was applied
as the tumor model to explore the cancer targeting performance of HA
coated nanoparticles. The in vitro HepG2 cells uptake of ZIAMH
nanoparticles was observed using confocal laser scanning microscopy. As
shown in Fig. [108]2a, the blue fluorescence represented DAPI stained
nuclei, while the red ones represented ICG molecules internalized into
the cytoplasm of the nanoparticles. There was barely red signal in the
cytoplasm after co-incubation with free ICG for 1 h, indicating the
lower cellular uptake of free ICG by the tumor cells. ICG has strong
hydrophobicity and poor dispersibility in water, making it hardly to
permeate through the cell membrane through endocytosis. As a
comparison, strong red fluorescence appeared in the cytoplasm treated
with ZIAM nanoparticles, proving that the size effect and good water
solubility of nanoparticles promoted the cellular internalization of
ICG (Figure. S6). And due to the addition of HA, stronger ICG signal
was observed in the cytoplasm of the ZIAMH group (Fig. [109]2b and c),
suggesting that the presence of HA could help to enhance the targeting
ability of the nanoparticles and further promote the internalization of
ICG.
Fig 2.
[110]Fig 2
[111]Open in a new tab
The cellular uptake assay and in vitro anticancer activity of various
drug formulations. a The CLSM images of HepG2 cells treated with ICG. b
The CLSM images of HepG2 cells treated with ZIAMH. (Blue channel: DAPI,
Red channel: ICG, scale bar: 50 µm). c The mean fluorescence intensity
of ICG in HepG2 cells treated with ICG, ZIAM or ZIAMH for 0–4 h
(n = 3). d The viability of the HepG2 cells incubated with ZMH, ArBu,
ArBu&ICG + NIR or ZIAMH + NIR for 24 h (n = 6). e, f Apoptosis analysis
of HepG2 cells after different treatments (n = 3). All quantitative
data are presented as mean ± SD. ***P < 0.001, ****P < 0.0001
The cytotoxicity of the carrier material was investigated using MTT
assay. The cellular viability of HepG2 cells were measured after
co-incubation with different concentrations of ZIAMH for 24 h. The
exposure of ZIAMH to 808 nm NIR laser irradiation induced elevated
temperature due to the photothermal effect of the loaded ICG, leading
to the apoptosis of cancer cells. The survival rate of HepG2 cells
gradually decreased with the increase of the concentration of ICG.
These results demonstrated that ZIAMH nanoparticles can significantly
inhibit the growth of cancer cells under NIR light irradiation. In
addition, the cytotoxicity of HepG2 cells in different treatments was
also evaluated (Fig. [112]2d and S7).
The mechanism study of cell apoptosis
Furthermore, the cell apoptosis of each treatment group was studied by
Annexin V-FITC/PI assay (Fig. [113]2e and f). The cell apoptosis rate
of ZIAMH nanoparticles increased to 26.7% after 24 h incubation, as
compared with the 14.1% apoptosis rate of free ArBu. This result
demonstrated that the ZIAMH nanoparticles could be effectively targeted
for delivery to tumor cells due to the presence of HA molecules, which
was consistent with previous studies on the tumor targeting ability of
HA [[114]49, [115]50].
The apoptosis of HepG2 cells was also investigated after treatment with
various therapeutic agents combined with NIR light irradiation,
accompanied by calcein AM (2 μM) and PI solution (4 μM) staining. As
shown in Fig. [116]3b, the absence of red signal in cells of PBS group
demonstrated that the single laser irradiation treatment could not
damage the cancer cells. As for the blank material group ZMH, the red
signal in the cells after irradiation was shown as scattered and weak,
indicating the partial apoptosis of cancer cells. The metal-polyphenol
network in the blank material exhibited chemodynamic effect inducing
cellular apoptosis, however, the therapeutic efficacy of chemodynamic
catalysis was less remarkable than that of other groups. Interestingly,
compared with the ICG group and free ArBu group, the ZIAMH group showed
massive death of cancer cell, as reflected by the strong red signal.
The results demonstrated that ZIAMH nanoparticles exhibited optimized
in vitro anticancer effect under NIR light irradiation.
Fig 3.
[117]Fig 3
[118]Open in a new tab
In vitro anticancer activity and mechanism of cell apoptosis. a
Mitochondrial membrane protein JC-1 red/green florescence ratio
(n = 3). b Live (green) and dead (red) cells staining after treatment
with PBS, ZMH, ArBu, ArBu&ICG + NIR or ZIAMH + NIR. scale bar: 50 µm. c
Western blot imaging of proteins caspase-9, caspase-3, cytochrome C,
Bcl-2, Bax and GAPDH after incubation with PBS, ArBu and ZIAMH. d The
relative expression level of caspase-9, caspase-3, cytochrome C, Bcl-2
and Bax by western blot analysis (n = 3). All quantitative data are
presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001
The cellular apoptosis is closely related to mitochondrial dysfunction
[[119]51]. In order to investigate the mechanism of the tumor cell
apoptosis, JC-1 staining was used to observe the change of
mitochondrial membrane potential in each dosing group. As shown in
Fig. [120]3a, ZIAMH nanoparticles exhibited significant anti-tumor
effect, which could be seen by the highest green-to-red ratio after
24 h incubation, hinting the extensive mitochondrial damage of the
cells.
To elucidate the levels of mitochondrial apoptosis proteins such as
anti-apoptotic Bcl-2 and pro-apoptotic Bax in HepG2 cells induced by
ZIAMH nanoparticles, western blot analysis was performed on HepG2 cells
treated with ArBu and ZIAMH nanoparticles. As exhibited in Fig. [121]3c
and d, compared with the western blot results of free ArBu, ZIAMH
nanomaterials significantly reduced the level of Bcl-2 in HepG2 cells,
and promoted expressions of apoptosis-related proteins
including Bax/Bcl-2 ratio, cytochrome C, caspase 9 and caspase 3.
Therefore, it can be concluded that there is indeed a close
relationship between tumor cell death and mitochondrial apoptosis
pathways.
In vitro mechanism study of tumor glucose metabolism by ZIAMH
Most of the energy required for cell life activities originates from
mitochondria, thus, the study of mitochondrial function is particularly
important to understand the energy metabolism of cells. The seahorse
cell energy metabolic analysis was performed in this study to explore
the regulation effects of ZIAMH nanomaterials on tumor glycolytic
capacity and mitochondrial OXPHOS pathway. Besides, ECAR and OCR were
used to reflect glycolysis and mitochondrial respiratory functions
respectively, thereby to quickly assess the energy metabolism status of
intracellular mitochondria.
As exhibited in Fig. [122]4a, the ECAR of each treatment group was
significantly reduced compared with the control group, indicating that
the glycolysis process was inhibited. As compared with the free drug
ArBu, the supplement of ICG and NIR irradiation further suppressed the
glycolysis process. In addition, it can be concluded from the OCR
result that the level of mitochondrial OXPHOS decreased significantly
in HepG2 cells after treatment with ZIAMH nanomaterials (Fig. [123]4b).
To sum up, the regulation of glucose metabolism by ZIAMH nanocomposites
combining with NIR irradiation was demonstrated in the dual inhibition
pathway of glycolysis and mitochondrial OXPHOS. It was speculated that
ArBu in the ZIAMH nanomaterial may inhibit the mitochondrial OXPHOS
through the mitochondrial apoptosis pathway, thereby exerting
anti-tumor effects.
Fig 4.
[124]Fig 4
[125]Open in a new tab
In vitro antitumor mechanism based on glucose metabolism. a The ECAR
(glycolysis indicator) of HepG2 cells by seahorse cell energy metabolic
analysis. b The OCR (OXPHOS indicator) of HepG2 cells by seahorse cell
energy metabolic analysis. c Western blot imaging of proteins PKM2,
LDHA, SDHA, HSP70, HSP90 and GAPDH after incubation with PBS, ZMH,
ArBu, ArBu&ICG + NIR or ZIAMH + NIR. d The relative expression level of
PKM2, LDHA, SDHA, HSP70 and HSP90 by western blot analysis (n = 3). e
The fluorescence images of intracellular ROS generation in HepG2 cells
treated with PBS, ZMH, ArBu, ArBu&ICG + NIR or ZIAMH + NIR using
DCFH-DA probe (Blue channel: Hoechst, Green channel: DCFH-DA, scale
bar: 50 µm) and the quantitative analysis of intracellular ROS
generation (n = 3). All quantitative data are presented as mean ± SD.
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Furthermore, the expression of related proteins in the glucose
metabolism pathway was detected to further investigate the regulatory
effect of ZIAMH on tumor glucose metabolism through western blot assay
(Fig. [126]4c and d). Compared with the control group, the protein
expression of each treatment group was varied to some extent,
especially in the ZIAMH + NIR group, where the proteins related to
glucose metabolism were all decreased. Among them, PKM2 and LDHA are
the main proteins related to the glycolysis pathway, which have the
functions of generating pyruvate and converting pyruvate into lactate,
respectively. The reduced expression of proteins PKM2 and LDHA
indicated that the glycolytic pathway was inhibited, and there is no
doubt that the content of lactate, the final product of glycolysis,
will be reduced. Notably, the expression of SDHA, a marker of
mitochondrial regulation, was also reduced. Decreased SDHA activity
suggested that mitochondrial OXPHOS was also affected, as it regulates
the cellular metabolic balance between OXPHOS and glycolytic
metabolism. Heat shock proteins (HSP70 and HSP90) has been shown to
control metabolic reprogramming of tumor cells [[127]52]. In terms of
tumor metabolism, overexpression of Hsp70 enhances the glycolytic
activity, leading to a shift in energy metabolism, while HSP90 is
involved in metabolic regulation through interaction with SDHA and PKM2
[[128]53].
Currently, studies have shown that mitochondria are an important source
of cellular ROS, and the production of ROS can break the mitochondrial
electron transport chain, thereby inhibiting mitochondrial OXPHOS
[[129]54, [130]55]. Based on this, intracellular ROS was detected by
the DCFH-DA probe to assess its impact on tumor mitochondrial OXPHOS.
As exhibited in Fig. [131]4e, compared with the PBS or free ArBu
groups, the level of ROS in cell mitochondria increased significantly
after treatment with ZIAMH. From this perspective, ZIAMH nanoparticles
indirectly inhibited OXPHOS in tumor cells by generating a large amount
of ROS, which is consistent with the OCR results by seahorse cell
energy metabolic analysis.
Biodistribution and in vivo antitumor efficacy of ZIAMH
In vivo NIR fluorescence imaging of ZIAMH
To investigate in vivo targeting ability of ZIAMH nanoparticles, PBS,
free ICG, and ZIAMH were intravenously injected into the respective
groups when HepG2 tumor size reached around 100 mm^3, followed by NIR
fluorescence imaging on living mice at predefined time points. As shown
in Fig. [132]5a, the fluorescence signal in tumor tissue of free ICG
group gradually increased in the first 8 h post-injection, but
decreased rapidly afterward. In contrast, the signal of that in the
ZIAMH group continued to increase up t a peak at 24 h after injection,
and then decreased slowly, which suggested that 24 h after injection is
the best time for subsequent NIR light therapy.
Fig 5.
[133]Fig 5
[134]Open in a new tab
The biodistribution and in vivo antitumor efficacy of ZIAMH
nanomedicine. A: Control (PBS); B: ZMH; C: ArBu, D: ArBu&ICG + NIR; E:
PD-L1; F: ZIAMH + NIR; G: ZIAMH + PD-L1 + NIR. a The in vivo
distributions of ICG visualized by using an IVIS Spectrum system after
intravenous administration. b The NIR thermographic images of
tumor-bearing mice after receiving exposure to an 808 nm laser at a
power density of 1.5 W/cm^2 in different groups, taken at 5 min. c
Schematic illustration for the therapeutic model. d Representative
images of the excised tumors after different treatments. e Tumor volume
after different treatments at the 21th day. (n = 6, ***P < 0.001). f
The body weight of mice among the different treatment groups presented
no differences. g CD4 staining images of the dissected tumor tissues
after sacrifice. Scale bar: 100 μm
The encapsulation of ICG in ZIAMH also facilitated drug concentration
in tumor site due to the active tumor-targeting ability of HA.
Subsequently, each group of mice was sacrificed 48 h post injection to
collect main organs (heart, liver, spleen, lungs, kidneys) and tumors.
The biodistribution and concentration of ZIAMH in collected samples was
detected using in vivo imaging technology. It has been shown that the
ZIAMH group demonstrated stronger fluorescence signal in tumors and
liver of the mice. Moreover, the ZIAMH nanoparticles may not induce
cardiotoxicity because no significant fluorescence was observed.
In vivo photothermal imaging of ZIAMH
To evaluate the photothermal effect in vivo, ZIAMH nanoparticles were
injected into tumor-bearing mice through tail vein injection, with the
PBS group as a control. Them the tumor region was exposed to 808 nm NIR
laser for 5 min after 24 h of injection. As manifested in Fig. [135]5b,
the temperature of the tumor areas in the PBS group scarcely rise
within 5 min, while that of the ZIAMH nanoparticles group increased
significantly. Moreover, the maximum temperatures reached by the tumor
sites in these two groups were 30.5 ℃ and 52.5 ℃, respectively.
Importantly, the accumulation of ZIAMH nanoparticles at the tumor site
was improved due to the active targeting effect of HA, thereby
promoting photothermal conversion within tumor, which was consistent
with the results of NIR imaging. These results demonstrated that ZIAMH
nanoparticles can not only efficiently accumulate in tumor area, but
can also generate sufficient heat to neutralize cancer cells under
irradiation, which is a promising photothermal agent.
In vivo anti-tumor evaluation
Recently, great progress has been made in tumor immunotherapy for
regulation of the patient's own immune cells to enhance the treatment
outcome of tumor cells [[136]56, [137]57]. Programmed cell death
1-ligand 1 (PD-L1) is currently the most widely used immune checkpoint
pathway in clinical practice, and the commercialized PD-L1 antibodies
have shown tremendous success, especially in the treatment of advanced
cancer [[138]58]. Thus, PD-L1 treatment group and ZIAMH&PD-L1 treatment
group were added to evaluate the in vivo antitumor effect.
The anti-tumor activity of ZIAMH with laser irradiation was studied by
establishing mouse HepG2 xenograft tumor model. PBS, ZMH, Arbu,
Arbu&ICG, ZIAMH, PD-L1 and ZIAMH&PD-L1 was intravenously injected into
mice, respectively (Fig. [139]5c). The PTT treatment was conducted 24 h
post administration, with tumor site being irradiated with 808 nm NIR
laser (1.5 W/cm^2) for 5 min. The body weight and tumor volume of mice
were recorded every other day and results were displayed in
Fig. [140]5d–f. The tumor growth trend of each group is basically
consistent regardless of irradiation and no significant reduction of
weight was observed. Under NIR irradiation, the ZIAMH group containing
ICG showed stronger antitumor effects on tumors than the free Arbu
group. It should be noted that the tumor volume in the ZIAMH group was
smaller than that of the PD-L1 group, indicating that the ZIAMH
nanoparticles had a better antitumor effect. In addition, the
combination of ZIAMH and PD-L1 therapy exhibited the most potent
antitumor efficacy among all of the treatment groups. These results
suggested that the combined use of ZIAMH and PD-L1 inhibitors may offer
a promising therapeutic approach for cancer treatment.
The tumor tissues in each group were further collected for histology
analysis. From immunofluorescence results in Fig. [141]5g, we found
that that CD4 cell was particularly high in ZIAMH added with PD-L1.
Interestingly, CD4 cell in ZIAMH nanomedicines was higher than that in
PD-L1, indicating that ZIAMH may be better than PD-L1 in immunotherapy
of tumors, because CD4 cells play an important role in the immune
response process. It can also be observed from the H&E staining results
that the tumor pathological sections in the treatment group were
severely damaged compared to the control group, especially in ZIAMH
nanomaterial (Figure. S8a). Considering that ZIAMH with synergistic
therapy may cause damage to the normal organs when exerting its
anti-cancer effect, the major organs (heart, liver, spleen, lung,
kidney) of the mice was extracted in the ZIAMH + NIR group for
histology analysis. It can be seen that the health status of these
organs in the ZIAMH + NIR group was basically the same as that in the
PBS group (Figure. S8b), indicating that the combination of PPT, CDT
and chemotherapy did not cause significant damage to the normal organs
of the mice.
In vivo mechanism study of tumor glucose metabolism by ZIAMH
Subsequently, transcriptomics analysis was performed and the results
indicated that the PBS group and ZMH, ArBu, ArBu&ICG + NIR, PD-L1,
ZIAMH + NIR, ZIAMH + PD-L1 + NIR groups clustered into different
categories in the PCA plot (Figure. S9), and a total of 686 common DEGs
were obtained among these groups compared with the PBS group (Figure.
S10). To better understand the in vivo mechanisms of tumor metabolic
reprogramming by ZIAMH nanomedicines, we further investigate the
transcriptomics data. As shown in Fig. [142]6a, PCA plot revealed that
the ZIAMH + NIR group was significantly different from the PBS group.
Then, a volcano diagram was plotted to provide a basic description of
the DEGs between the ZIAMH + NIR group and the PBS group, and
|log[2](fold change)|> 1 and Q-value < 0.05 were adopted as the
filtering criteria for DEGs screening (Fig. [143]6b), 1470 genes were
downregulated and 1226 genes were upregulated. Further heatmap analysis
were conducted to uncover DEGs associated with glucose metabolism
(Fig. [144]6c). After ZIAMH + NIR treatment, the expression of key
enzymes in glycolysis, OXPHOS or the TCA cycle, such as genes HK2,
SDHA, LDHB, PFKM, PDK1, were significantly downregulated, which
suggested that ZIAMH + NIR treatment had a considerable influence on
glucose metabolism pathway and played a key role in inhibiting tumor
growth. Among them, gene HK2 plays an important role in allowing
glucose to enter the glycolysis and OXPHOS pathway [[145]59]. SDHA is
an enzyme involved in the TCA cycle and energy metabolism, which not
only plays a role in mitochondrial energy production, but also
regulates the cellular metabolic balance between the TCA cycle and
glycolysis [[146]60]. To gain further insight into the potential
targeting pathways of ZIAMH + NIR, GO and KEGG enrichment analysis were
carried out. As can be seen in Fig. [147]6d, it was observed that the
negative regulation of carboxylic acid metabolic process, cellular
metabolic process and other metabolic process were significantly
enriched after ZIAMH + NIR treatment. The KEGG enrichment analysis
results indicated that ZIAMH nanomedicines could downregulated
glycolysis and carbon metabolism, and achieve antitumor effects by
regulating carbohydrate metabolism and energy metabolism (Fig. [148]6e
and S11). Additionally, protein–protein interaction (PPI) network
analysis was performed using the genes associated with glucose
metabolism pathways that were significantly altered after the
ZIAMH + NIR treatment (Fig. [149]6f). Key proteins in the PPT network,
including ACLY, LDHB, ENO2, ADH6 and PFKM, were involved in a variety
of biological processes, for example, glycolysis, OXPHOS and tumor
growth.
Fig 6.
[150]Fig 6
[151]Open in a new tab
The in vivo biological mechanism of ZIAMH nanomedicines by
RNA-sequencing analysis. a The PCA plot of PBS (A) and ZIAMH + NIR (B)
groups. b The volcano map of PBS and ZIAMH + NIR groups. c The heatmap
analysis of DEGs associated with glucose metabolism in PBS (A) and
ZIAMH + NIR (B) groups. d GO enriched in PBS and ZIAMH + NIR groups. e
KEGG analysis of genes and genomes enriched in PBS and ZIAMH + NIR
groups (red box represent ZIAMH relative pathways). f PPI network
associated with glucose metabolism pathways
Glycolysis is a common pathway for glucose degradation in all living
organisms, a process that produces pyruvate, followed by
decarboxylating to generate acetyl CoA, which is completely degraded to
carbon dioxide and water through the TCA cycle, and releases large
amounts of energy. Glycolysis is closely linked with the TCA cycle, and
together they constitute an important aerobic oxidation process of
sugar. Thus, we further conducted targeted metabolomic analysis related
with glycolysis and the TCA cycle pathway to explore the underlying
sugar metabolism mechanism of intelligent ZIAMH nanomaterials in the
TME (Fig. [152]7a). To visualize these products, the parameters of 9
metabolites were optimized using UPLC-triple quadrupole MS/MS (Table
S1). As shown in Fig. [153]7b, compared with the PBS group, the content
of glycolysis metabolites pyruvate and lactate were both decreased
significantly. Furthermore, the levels of the TCA cycle intermediate
metabolites citrate, cis-aconitate, succinate, fumarate and malate were
all significantly reduced, indicating that ZIAMH treatment weakened the
activity of TCA cycle and may have a tumor suppression effect.
Fig 7.
[154]Fig 7
[155]Open in a new tab
Targeted metabolomic analysis of tumor tissues from HepG2 xenograft
mice. a The diagram of the TCA cycle. b The concentration of key
metabolites involved in glycolysis and the TCA cycle. The statistical
significance was analyzed by one-way ANOVA and data are presented as
means ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Conclusion
In summary, we successfully constructed an MOF-derived intelligent
ZIAMH nanoplatform that might promote energy deprivation by
synchronously interfering with glycolysis, OXPHOS and TCA cycle,
thereby achieving antitumor bioenergetic therapy integrated with
PTT/CDT/CT therapy. Encapsulated with ZIF-8, the ZIAMH nanomaterials
have considerably better antitumor efficacy than free ArBu, as well as
good biosafety and solubility. Furthermore, the ZIAMH nanoparticles
loaded with HA can efficiently be delivered to the tumor area by
targeting the CD44 receptor, followed by the generation of ·OH
triggered via the Fenton reaction and exhibiting enhanced CDT effects.
Owing to the photothermal properties of ICG, such ZIAMH nanoparticles
can effectively killed tumor cells under NIR laser irradiation without
obvious side effects. In addition, in vitro evaluation demonstrated
that the ZIAMH nanomaterials promoted cellular mitochondrial apoptosis
through facilitating the expression of apoptosis-related proteins, such
as Bax, Bax/Bcl-2, cytochrome C, caspase 9 and caspase 3. Importantly,
systematic in vivo and in vitro mechanism studies suggested that ZIAMH
nanomedicines can simultaneously inhibited glycolysis and OXPHOS in
tumor cells by suppressing the expression of HK2, PKM2, LDHA, SDHA and
PDK1. Moreover, they can further impair mitochondria function and
decrease the level of ATP by depriving the TCA cycle of energetic
substances and metabolites. Therefore, this work provides a new
strategy for tumor treatment through precise energy deprivation and
targeted therapy, integrating multiple pathways of bioenergetic therapy
for metabolic interference and PTT/CDT/CT therapy.
Supplementary Information
[156]Supplementary material 1^ (6.8MB, pdf)
Acknowledgements