Abstract
Hydrogen gas (H[2]) therapy, recognized for its inherent biosafety,
holds significant promise as an anti‐cancer strategy. However, the
efficacy of H[2] treatment modalities is compromised by their reliance
on systemic gas administration or chemical reactions generation, which
suffers from low efficiency, poor targeting, and suboptimal
utilization. In this study, living therapeutics are employed using
photosynthetic bacteria Rhodobacter sphaeroides for in situ H[2]
production combined with near‐infrared (NIR) mediated photothermal
therapy. Living R. sphaeroides exhibits strong absorption in the NIR
spectrum, effectively converting light energy into thermal energy while
concurrently generating H[2]. This dual functionality facilitates the
targeted induction of tumor cell death and substantially reduces
collateral damage to adjacent normal tissues. The findings reveal that
integrating hydrogen therapy with photothermal effects, mediated
through photosynthetic bacteria, provides a robust, dual‐modality
approach that enhances the overall efficacy of tumor treatments. This
living therapeutic strategy not only leverages the therapeutic
potential of both hydrogen and photothermal therapeutic modalities but
also protects healthy tissues, marking a significant advancement in
cancer therapy techniques.
Keywords: hydrogen therapy, hydrogen‐photothermal therapy, living
bacterial therapies, photosynthetic bacteria
__________________________________________________________________
The development of hydrogen gas (H[2]) therapy is highly challenging by
reason of low efficiency and suboptimal utilization due to its high
diffusibility, nonpolarity, and low solubility of H[2] in physiological
conditions, which prevent long‐term release and reduce therapeutic
efficiency. In light of these challenges, the research offers a novel
approach that uses living photosynthetic bacteria, R. sphaeroides, to
generate and release H[2] for enhanced effective time under the control
of NIR light, and also converts NIR light to thermal energy
efficiently. This strategy providing a synergistic effect that
significantly enhances tumor suppression while protecting normal
tissues, which makes it a distinct candidate for anti‐tumor therapies.
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1. Introduction
Cancer continues to pose the most significant health threat globally,
but traditional treatments such as chemotherapy and radiotherapy often
demonstrate limited efficacy and substantial non‐specific toxicity to
normal tissues, thereby inflicting considerable discomfort on
patients.^[ [54]^1 ^] Rapid advancements in nanobiotechnology have led
to the development of a variety of therapeutic platforms that enhance
the precision and effectiveness of cancer treatment.^[ [55]^2 ^] These
platforms facilitate the development of versatile nanomedicines,
including gas‐based therapeutics that specifically eliminate cancer
cells while protecting healthy cells, thereby minimizing the adverse
effects commonly associated with conventional cancer therapies.^[
[56]^3 ^] Several types of physiologically gaseous molecules such as
oxygen (O[2]),^[ [57]^4 ^] nitric oxide (NO),^[ [58]^5 ^] hydrogen
sulfide (H[2]S),^[ [59]^6 ^] carbon monoxide (CO),^[ [60]^7 ^] sulfur
dioxide (SO[2]), and hydrogen gas (H[2])^[ [61]^8 ^] have the function
of regulating vasodilatation, neurotransmission, anti‐inflammatory, and
anti‐oxidative reactions in both physiological and pathophysiological
processes. Its specific therapeutic effects have been documented in
diseases ranging from cardiovascular ailments to neurodegenerative
disorders and cancers, thanks to its ability to modulate physiological
functions.
H[2], the lightest molecule in nature, constitutes ≈0.5 parts per
million of the Earth's atmosphere. Research dating back to 1975 has
highlighted the potential of H[2] in cancer treatment, particularly due
to its selectively anti‐oxidative properties, which allows for the
modulation of reactive oxygen species (ROS) levels within cancer
cells.^[ [62]^9 ^] Currently, hydrogen therapy excels over other
gas‐based treatments due to its selective antioxidative properties,
excellent safety profile, broad therapeutic applications, synergistic
potential with other therapies, and minimal disruption to normal
cellular processes.^[ [63]^10 ^] However, current H[2] delivery methods
are limited by its high diffusibility, nonpolarity, and low solubility
in physiological conditions, which prevent long‐term release and reduce
therapeutic efficiency.^[ [64]^11 ^] In situ H[2] generation
technologies, such as self‐decomposition of nanosystems and
light‐mediated water splitting, often suffer from low catalytic
efficiency.^[ [65]^12 ^] Addressing these delivery and long‐term in
situ generation challenges is crucial for maximizing the clinical
benefits of hydrogen therapy. Moreover, the integration of hydrogen
therapy with conventional treatment modalities like chemotherapy and
radiotherapy has demonstrated significant synergistic therapeutic
effects.^[ [66]^13 ^] Biohydrogen production offers sustainable,
environmentally friendly, and efficient energy generation from
renewable energy, providing versatile and economically viable
application,^[ [67]^14 ^] including the potential for long‐term
hydrogen release to enhance therapeutic efficiency.^[ [68]^15 ^]
Photosynthetic bacteria, such as purple non‐sulfur bacteria (PNSB), are
favored for their ability to efficiently produce and release H[2] under
illumination via hydrogenase and their capacity to metabolize a wide
range of substrates,^[ [69]^16 ^] which is potentially act as living
therapeutics. The advantages of living therapeutics, particularly in
terms of efficacy, precise control, and safety, highlight its
transformative potential to redefine and expand the scope of future
therapeutic strategies. This innovative approach offers the ability to
harness living systems for more targeted, adaptive, and sustainable
treatments, positioning it as a pivotal advancement in the next
generation of medicine.^[ [70]^17 ^]
In this work, we develop living therapeutics using photosynthetic
bacteria, Rhodobacter sphaeroides, for long‐term H[2] production
coupled with hyperthermia induction under near‐infrared (NIR) light
irradiation for tumor hydrogen‐photothermal therapy. R. sphaeroides, a
facultative anaerobic bacterium, is well‐suited for growth in hypoxic
conditions and demonstrates phototaxis.^[ [71]^18 ^] These unique
traits enhance its ability to adapt to the tumor microenvironment and
migrate toward illuminated areas, making it an ideal candidate for
tumor targeting and therapy. Living R. sphaeroides excels as
photosynthetic hydrogen producers and show remarkable NIR absorption,
efficient conversion of NIR light to heat, high photothermal stability,
with negligible cytotoxic effects. Consequently, we hypothesize that
the R. sphaeroides can simultaneously produce hydrogen gas and generate
heat under 808 nm laser irradiation, enabling a dual‐mode therapy that
combines hydrogen production with photothermal effects. Utilizing this
bacterium as a dual supplier for hydrogen and photothermal effects
allows for more precise and controllable delivery of hydrogen, while
also demonstrating relatively high biocompatibility. Importantly, R.
sphaeroides‐mediated synergistic hydrogen‐photothermal therapy has no
discernible impact on normal cells, underscoring its potential to
protect healthy normal cells from hyperthermia damage while selectively
targeting tumor cells. Further investigations reveal that this
synergistic strategy notably increases ROS generation within cancer
cells, disrupting their redox homeostasis and impairing mitochondrial
function, which ultimately leads to apoptosis (Figure [72]1 ). By
utilizing the inherent biological capabilities of R. sphaeroides, this
study offers a potentially efficient living therapeutic strategy to
mitigate tumor progression while safeguarding healthy cells. This
dual‐action strategy not only targets malignant cells with precision
but also minimizes collateral damage to surrounding non‐cancerous
tissues, thus presenting a promising avenue for advancing cancer
therapy.
Figure 1.
Figure 1
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Schematic illustration showing the process of biohydrogen production
and photothermal conversion of R. sphaeroides for synergistic
hydrogen‐photothermal therapy. BChl, bacteriochlorophyll; RC, Reaction
center; PSB, Photosynthetic bacteria; ICM, Intracytoplasmic membrane.
2. Results and Discussions
2.1. Combined Hydrogen Production and Photothermal Effects of R. Sphaeroides
R. sphaeroides, the most widely used model of purple nonsulfur
photosynthetic bacteria, demonstrates versatile metabolic capabilities,
including aerobic and anaerobic respiration, photosynthesis, and
fermentation.^[ [74]^18 ^c] We cultured the R. sphaeroides in Luria
Broth (LB) medium and performed the characterization. Scanning electron
microscopy (SEM) and transmission electron microscopy (TEM) revealed
its ovoid shape with a diameter approximately in 2 µm (Figure
[75]2a,b). After cultured for 12 h in the LB medium, R. sphaeroides
turned red color (Figure [76]2c). The UV‐vis spectra indicated a wide
range of absorption with peaks at 808 nm and 850 nm (Figure [77]2e),
characteristic absorption peak of bacteriochlorophyll (BChl). BChl
functions as a light‐harvesting antenna to use light energy for
photosynthesis.^[ [78]^16 ^] As shown in Figure [79]2d,e, the intensity
of coloration and absorbance of the bacterial suspension were
concentration‐dependent. To confirm the absorption property was derived
from BChl, it was extracted from R. sphaeroides and its absorption
spectra were analyzed, revealing absorption peaks in the NIR region
align with those of R. sphaeroides (Figure [80]2f). Within the
intracytoplasmic membrane (ICM), BChl absorbs photons and channels
energy to the photosynthetic reaction center (Figure [81]2g),
initialing the photosynthetic electron transfer chain, which generates
high‐energy electrons subsequently utilized by hydrogenase.^[ [82]^19
^] Hydrogenase captures electrons derived from ferredoxin, facilitating
the reduction of protons translocated across the membrane by
adenosine‐triphosphate (ATP) synthase, culminating in H[2]
production.^[ [83]^20 ^]
Figure 2.
Figure 2
[84]Open in a new tab
Characterization of R. sphaeroides. a) SEM image of the R. sphaeroides.
b) TEM image of the R. sphaeroides. c) Photograph of the R. sphaeroides
cultured in LB medium. d) Photographs of the R. sphaeroides suspended
in PBS at different concentrations. e) Absorption spectra of the R.
sphaeroides at different concentrations (0, 0.9×10^9, 1.8×10^9,
3.6×10^9, 7.2×10^9, and 1.4×10^10 CFU mL^−1). f) Absorption spectra of
the R. sphaeroides and its BChl. g) Schematic illustrating the process
of H[2] production and photothermal conversion in R. sphaeroides under
NIR light irradiation. (Q pool, Ubiquinone pool; Fd, Ferredoxin; Cyt
bc1, Cytochrome bc1; Δp, Proton motive force; Pi, Inorganic phosphate).
h) Schematic representation of H[2] production as monitored using MB as
the probe. i) H[2] released from R. sphaeroides suspensions at
different concentrations (0, 0.9×10^9, 1.8×10^9, 3.6×10^9, 7.2×10^9,
and 14×10^9 CFU mL^−1) under irradiation of an 808 nm LED at
0.06 W cm^−2 for 30 min (n = 3 biologically independent samples). j)
Infrared thermal images of R. sphaeroides suspensions at different
concentrations exposed to an 808 nm laser at 1.0 W cm^−2 for 5 min,
with an initial temperature at 27 °C. k) Photothermal temperature
curves of R. sphaeroides suspensions at different concentrations (0,
0.9×10^9, 1.8×10^9, 3.6×10^9, 7.2×10^9, and 14×10^9 CFU mL^−1) under
808 nm laser irradiation at the power density of 1.0 W cm^−2 for 5 min
(n = 3 biologically independent samples). Data are presented as means ±
standard deviation (SD).
To measure the capability of R. sphaeroides to produce H[2] under the
control of NIR light, we collected the R. sphaeroides cells and
resuspended them into phosphate‐buffered saline (PBS) with various
bacterial concentrations and illuminated them under 808 nm light
emitting diode (LED) light (0.06 W cm^−2). The methylene blue (MB)
reduction assay was used to quantify the H[2] production
(Figure [85]2h). The released H[2] production was enhanced with an
increase in bacterial cell concentration (Figures [86]S1 and [87]S2,
Supporting Information). The accumulated H[2] production reached
38.9 µM after 30 min with the bacteria concentration of 1.4×10^10
CFU mL^−1 (Figure [88]2i), showing 4‐fold increase compared with
bacteria concentration of 9.0×10^8 CFU mL^−1, and 17‐fold increase
compared with the PBS control (Figure [89]2i). The maximum H[2]
released rate was achieved during the first 10 min with the cell
concentration of 1.4×10^10 CFU mL^−1, and reached 1.3 µM min^−1 and
0.3 µM min^−1 with the cell concentrations of 1.4×10^10 CFU mL^−1 and
9.0×10^8 CFU mL^−1, respectively (Figure [90]S3, Supporting
Information). Those results indicated the R. sphaeroides could generate
and release H[2] efficiently under the control of NIR irradiation.
BChl exhibits an obvious absorption band in the 800 – 810 nm range,
indicating R. sphaeroides could convert NIR light into thermal energy
efficiently (Figure [91]2g). The photothermal conversion performance of
R. sphaeroides was assessed by exposing different concentrations of
bacterial cells to an 808 nm laser (1.0 W/cm^2) and monitoring the
changes of temperatures using a thermographic camera. As shown in
Figure [92]2j‐k, we found all tested cell concentrations displayed
notable temperature incasement, while there was no obvious temperature
increased by the PBS. The photothermal conversion effect positively
correlates with bacterial concentration. Notably, the changes of
temperature (ΔT) increased by 16.8 °C and 17.8 °C and the temperature
can reach 43.8 °C and 44.8 °C when the R. sphaeroides at the
concentration of 7.2 × 10^9 CFU mL^−1 and 1.4 × 10^10 CFU mL^−1
(Figure [93]2j,k; Figure [94]S4, Supporting Information). Thus, the
photothermal conversion efficiency was similar at the concentration
between 1.4 × 10^10 and 7.2 × 10^9 CFU mL^−1, and we selected
concentration of 7.2 × 10^9 CFU/mL for the following experiments. As
shown in Figure [95]S5 (Supporting Information), the accelerated
temperature increased with laser power intensity lower than
1.5 W cm^−2, and temperature was similar under the power of 1.5 W cm^−2
(52.3 °C) and 2.0 W cm^−2 (53.5 °C). Therefore, the laser power of
1.5 W/cm^2 was selected for photothermal experiments. Furthermore,
following 4 repeated on‐and‐off cycles of 808 nm laser irradiation, R.
sphaeroides shows no significant deterioration, highlighting its
photothermal stability (Figure [96]S6, Supporting Information). The
photothermal conversion efficiency (η) of R. sphaeroides was calculated
to be 7.4% (Figure [97]S7, Supporting Information).^[ [98]^21 ^]
Remarkably, R. sphaeroides displays a high degree of thermotolerance,
proliferating even at 55 °C (Figure [99]S8, Supporting Information),
with no significant differences in proliferation under 808 nm laser
irradiation compared to the conditions without laser irradiation. In
contrast, for the bacteria without BChl, such as Synechococcus
elongatus, Bacillus thuringiensis, and Escherichia coli, those stains
showed no photothermal conversion ability upon exposure to 808 nm laser
irradiation with the same power and cell concentration (Figure [100]S9,
Supporting Information), indicating the curial role of BChl in the
photothermal conversion. Taking together, our results underscore the
unique capabilities of R. sphaeroides in combining hydrogen production
and photothermal effects under the control of NIR light, making it an
effective candidate agent for cancer therapy.
2.2. In Vitro Hydrogen‐Photothermal Therapy
We first explore the efficiency of R. sphaeroides and BChl for
producing H[2] and heat simultaneously under the control of NIR light.
As shown in Figure [101]S10 (Supporting Information), the bacteria
irradiated with 808 nm laser (1.5 W cm^−2, the experimental group)
could produce 42.2 µM H[2] and increase the ΔT to 24.9 °C. While the
control group, including the bacteria irradiated with 808 nm LED light
(0.08 W cm^−2), produces H[2] (46.6 µM), which is comparable to the
experimental group and represents hydrogen therapy. The BChl irradiated
with 808 nm laser (1.5 W cm^−2) only generates heat (ΔT = 23.0 °C)
which represents photothermal therapy (PTT), and neither H[2] nor heat
was produced by the BChl irradiated with 808 nm LED light
(0.08 W cm^−2). Consequently, hydrogen therapy, PTT, and synergistic
hydrogen‐photothermal therapy were implemented in vitro.
We evaluated the cytotoxicity of R. sphaeroides using Cell Counting
Kit‐8 (CCK‐8) assay on a cancer cell line (4T1 cells) and a normal cell
line (HEK‐293T cells). Without 808 nm laser/LED irradiation, R.
sphaeroides exhibited negligible cytotoxicity, even at a concentration
of 7.2×10^9 CFU mL^−1, demonstrating its biosafety in vitro (Figure
[102]3a). Hydrogen therapy (G4) using the bacteria with 808 nm LED
light selectively killed cancer cells (Figure [103]3b). The BChl with
808 nm laser group, assigned for PTT (G5), was lethal to both cancer
cells and normal cells. Notably, the bacteria with 808 nm laser
irradiation (assigned hydrogen‐photothermal therapy, G6) demonstrates
enhanced lethality to cancer cells compared to the PTT group, while
minimizing damage to normal cells. Live/dead cell staining further
confirmed that G6 induces the highest cell mortality among all groups
in 4T1 cells, while maintain the activity of HEK‐293T cells,
demonstrating the efficacy of the synergistic therapy (Figure [104]3c).
In contrast, G5 led to cell death in HEK‐293T cells, indicating the
potential of damage to normal cells caused by PTT. Flow cytometry
following dual staining with fluorescein isothiocyanate (FITC)‐labeled
Annexin V and propidium iodide (PI) revealed notably enhanced apoptosis
in 4T1 cells at 64.6% for the G6 of hydrogen‐photothermal therapy,
while only 8.0% apoptosis in HEK‐293T cells (Figure [105]3d). Those
results indicated hydrogen‐photothermal therapy enhances anti‐cancer
effects while minimizing damage to normal cells, indicated the
selectivity and effectiveness of the anti‐cancer effects.
Figure 3.
Figure 3
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In vitro hydrogen‐photothermal therapy using R. sphaeroides. a) Cell
viabilities of 4T1 cells and 293T cells incubated with R. sphaeroides
at different concentrations (0, 0.18×10^8, 1.8×10^8, 18×10^8, 36×10^8,
and 72×10^8 CFU mL^−1) (n = 3 biologically independent samples). b)
Cell viabilities of 4T1 cells and 293T cells after different treatments
(n = 3 biologically independent samples). c) Confocal microscopy images
of 4T1 cells and 293T cells stained with Calcein‐AM and PI after
different treatments (green: living cells; red: dead cells). Scale
bars, 100 µm. d) Flow cytometry analysis of 4T1 cells and 293T cells
stained with PI and Annexin V‐FITC. e) Confocal microscopy images of
4T1 cells and 293T cells stained with 2′,7′ – Dichlorofluorescein
diacetate (DCFH‐DA) after different treatments. Scale bar, 100 µm. f)
Confocal microscopy images of 4T1 cells stained with JC‐1 after
different treatments. Scale bars, 50 µm. g) ATP activity in 4T1 cells
after different treatments. (n = 3 biologically independent samples).
h) Schematic illustration showing mechanism underlying R. sphaeroides
mediated hydrogen‐photothermal therapy. Data are presented as mean
values ± SD.
Intracellular ROS levels were assessed to evaluate redox homeostasis.
4T1 cells showed significant increases in ROS following hydrogen
therapy and combined hydrogen‐photothermal therapy, indicating that
H[2]induces oxidative stress in cancer cells (Figure [107]3e).
Conversely, HEK‐293T cells exhibited increased ROS production only
after PTT with BChl and 808 nm laser irradiation. Notably, H[2] helps
restore normal cellular redox homeostasis, suggesting that the
synergistic therapy may protect normal cells against oxidative damage
caused by heat. Considering the naturally higher levels of ROS in
cancer cells, hydrogen can initially lower the ROS levels because of
its reducing properties. However, given the capacity for redox
homeostasis in cells, this reduction might prompt a compensatory
increase in ROS, potentially resulting in the death of cancer cells.^[
[108]^10 , [109]^22 ^] In contrast, H[2] alleviates oxidative damage in
normal cells induced by photothermal effect.^[ [110]^3 , [111]^8 ,
[112]^23 ^]
We also investigated the impact of hydrogen‐photothermal therapy on
mitochondrial membrane potential (MMP) and the intracellular ATP levels
to elucidate the anti‐tumor mechanism. As shown in Figure [113]3f,g,
hydrogen‐photothermal therapy caused a prominent decrease in MMP, and
significant reductions in ATP levels were observed in 4T1 cells,
primarily due to hyperthermia‐induced impairments. The relationship
between excess ROS and mitochondrial damage is complementary. The
excessive ROS leads to oxidative damage in mitochondria, which, when
dysfunctional, releases substantial ROS in return.^[ [114]^24 ^] Thus,
hydrogen therapy induces slight mitochondrial impairment in cancer
cells due to excessive ROS generation, while PTT results in subtle ROS
upregulation within cancer cells, possibly from the release of damaged
mitochondria (Figure [115]3h). Furthermore, we delved into the effects
of hydrogen‐photothermal therapy on other cellular organelles in cancer
cells. The results indicated that this strategy induces endoplasmic
reticulum (ER) stress and lysosomal damage in 4T1 cells (Figures
[116]S11 and [117]S12, Supporting Information). These findings
underscore the dual functionality of R. sphaeroides in combining
hydrogen production and photothermal effects, offering a highly
effective and selective approach for targeted cancer therapy.
2.3. Mechanism of Hydrogen‐Photothermal Therapy
To elucidate the potential mechanisms underlying the anti‐tumor effects
driven by R. sphaeroides in 4T1 cells, RNA sequencing (RNA‐seq)
analysis was conducted. A total of 16083 expressed genes were
identified (Figure [118]S13, Supporting Information), with subsequent
differential expression analysis revealing significant variances among
different treatment groups (Figure [119]4a,b; Figure [120]S14,
Supporting Information). Specifically, the hydrogen‐photothermal
synergy therapy group exhibited 2408 differentially expressed genes
(DEGs) compared to the control group (without treatment), with 979
genes up‐regulated and 1429 genes down‐regulated (| Fold change | ≥ 1,
p < 0.05, Figure [121]4c). In contrast, only 949 DEGs were observed
when compared with the PTT group alone (599 up‐regulated, 350
down‐regulated, Figure [122]4d). Furthermore, a comparison between the
control group and PTT group revealed 1922 differential genes (623
up‐regulated, 1299 down‐regulated, Figure [123]4e), indicating that
heat notably contributes to gene expression alteration in tumor cells
within the context of synergistic therapy.
Figure 4.
Figure 4
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Transcriptional mechanism of R. sphaeroides mediated
hydrogen‐photothermal therapy. a) Venn diagram of the transcriptomic
profiles among control group, PTT group, and synergy therapy group. b)
Different change genes statistics among control group, PTT group, and
synergy therapy group. Volcano plots showing the identified upregulated
and downregulated genes on 4T1 cells between c) synergy therapy group
and control group, d) synergy therapy group and PTT group, e) PTT group
and control group. f) GO pathway enrichment analysis of the identified
DEGs between control group and synergy therapy group. g) KEGG pathway
enrichment analysis of the identified DEGs between control group and
synergy therapy group. h) Heat map illustrating the DEGs related to the
mitochondrial respiratory chain, mitochondrial damage and ROS metabolic
processes among control group, PTT group and synergy therapy group.
Gene Ontology (GO) pathway enrichment analysis and Kyoto Encyclopedia
of Genes and Genomes (KEGG) analysis were performed on the DEGs to
assess their involvement in metabolic pathways. The synergy therapy
group displayed significant enrichment in pathways related to
mitochondria function and signaling (Figure [125]4f). KEGG analysis
further highlighted significant enrichment in pathways governing redox
homoeostasis and mitochondria function, such as ROS production,
oxidative phosphorylation, p53, mitogen‐activated protein kinase (MAPK)
signaling pathway, and apoptosis (Figure [126]4g). The DEGs are
enriched in the PI3K/Akt signaling pathway and the NF‐κB signaling
pathway, suggesting that this synergistic strategy might induce cell
apoptosis through these critical pathways.^[ [127]^25 ^] These findings
suggest that the synergy therapy profoundly impacts redox homoeostasis
and mitochondria function in tumor cells.
Analysis of DEGs within these pathways in heat maps revealed that genes
such as cytochrome c oxidase assembly protein 15 (Cox15), associated
with mitochondrial respiratory chain, were down‐regulated, while genes
such as lon peptidase 1 (Lonp1), linked to mitochondrial damage, were
up‐regulated after both PTT and synergistic therapy (Figure [128]4h).
Thus, in this strategy, photothermal therapy might lead to
mitochondrial dysfunction by aberrantly activating
mitochondrial‐related signaling pathways or promoting atypical
expression of certain mitochondrial proteins.^[ [129]^26 ^]
Additionally, genes involved in ROS metabolic processes, such as
neutrophil cytosolic factor 1 (Ncf1), were predominantly up‐regulated
after synergistic therapy, suggesting that disruption of mitochondrial
function is primarily attributed to heat, whereas alternations in ROS
are mainly influenced by H[2].^[ [130]^27 ^] Furthermore, up‐regulated
genes such as growth arrest and DNA damage inducible alpha (Gadd45a),
associated with stress signaling and injury response,^[ [131]^28 ^] and
other significantly altered genes such as B‐cell lymphoma‐2 (Bcl‐2),
associated with tumor suppression,^[ [132]^29 ^] indicated that the
synergistic strategy might trigger the cell apoptosis process by
regulating these downstream regulators (Figure [133]S15, Supporting
Information). The RNA‐seq results comprehensively demonstrate that R.
sphaeroides‐mediated hydrogen‐photothermal therapy compromises
mitochondrial function and disrupts redox homeostasis in cancer cells,
leading to oxidative stress and ultimately resulting in apoptosis of
tumor cells (Figure [134]S16, Supporting Information).
2.4. In Vivo Hydrogen‐Photothermal Therapy
To evaluate the in vivo therapeutic efficacy of hydrogen‐photothermal
treatment, we initially investigated the biocompatibility of R.
sphaeroides by administering it intravenously to healthy BALB/c mice.
Over a 21‐day observation period, no mortality occurred, and no
significant changes in body weight were detected, indicating the
absence of systemic toxicity from R. sphaeroides (Figure [135]5c, and
Table [136]S1, Supporting Information). Furthermore, comprehensive
blood routine and blood biochemistry assessed at the end of this period
show that all indexes were within normal ranges, even at the
concentration of 7.2×10^9 CFU mL^−1 (Figure [137]5a; Figure [138]S17,
Supporting Information), suggesting benign blood compatibility and no
discernible toxicity to liver or kidney functions. Histopathological
analyses via Hematoxylin and Eosin (H&E) staining of major organs
including heart, liver, spleen, lung, and kidney, corroborated the high
hemocompatibility, with no visible organ damage, confirming the
compatibility of R. sphaeroides (Figure [139]5b). Consequently, the use
of R. sphaeroides as a therapeutic agent has been shown to be safe in
mice, yet more extensive safety evaluations on large animal models or
humans are required for clinical application. Subsequently, 4T1 breast
cancer tumor‐bearing mice model were constructed, and bacteria was
peritumorally injected into the mice. During 808 nm laser irradiation
at the tumor region, the temperature in the tumor region reached up to
55 °C for 10 min, which was sufficient to effectively ablate tumor
cells (Figure [140]5d,e; Figure [141]S18, Supporting Information),
indicating the potential of R. sphaeroides for effective cancer cells
ablation in vivo.
Figure 5.
Figure 5
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In vivo biocompatibility and photothermal performance investigation of
R. sphaeroides. a) Blood routine and blood chemistry of the mice after
treatment with R. sphaeroides at different concentrations (0, 1.8×10^9,
3.6×10^9, and 7.2×10^9 CFU mL^−1) (n = 5 biologically independent
animals). b) Histological analyses of main organs from mice injected
with R. sphaeroides at different concentrations (0, 1.8×10^9, 3.6×10^9
and 7.2×10^9 CFU mL^−1). c) The body weight changes of mice were
observed over 21 days after they were intravenously injected with
different concentrations (0, 1.8×10^9, 3.6×10^9, and 7.2×10^9
CFU mL^−1) of R. sphaeroides (n = 5 biologically independent animals).
d) Photothermal temperature curves of 4T1 tumors region under 808 nm
laser irradiation (1.5 W cm^−2, 10 min) in untreated mice (Laser only)
and R. sphaeroides injected mice (R. sphaeroides + Laser). e) Thermal
images of 4T1 tumors under 808 nm laser irradiation (1.5 W cm^−2,
10 min) in untreated mice and R. sphaeroides injected mice. Data are
presented as mean values ± SD.
The anti‐tumor efficacy of hydrogen‐photothermal therapy was
investigated in 4T1 tumor‐bearing mice, divided into four groups:
control without any treatment (Group I), treated with laser irradiation
(Group II), treated with R. sphaeroides (Group III), and treated with
R. sphaeroides under laser irradiation (Group IV) (Figure [143]6a).
Notably, the Group IV showed significant tumor suppression
(Figure [144]6b‐e), with tumor inhibition rate approaching 97%
(Figure [145]6f). Furthermore, there were no obvious weight changes in
all four treatment groups (Figure [146]6g), underscoring the biosafety
of the therapeutic strategies. Mice were then euthanized, and the
anti‐tumor activity was further studied by H&E staining, TdT‐mediated
dUTP‐biotin nick end labeling (TUNEL) staining and Ki‐67 staining
analysis (Figure [147]6h). Compared to other groups, H&E staining of
tumor tissues showed extensive tumor tissue destruction, cellular and
nuclear shrinkage, and complete nuclear disappearance in the Group IV,
with no morphological changes in major organs (Figure [148]6h; Figure
[149]S19, Supporting Information). TUNEL staining images of tumor
slices displayed a high proportion of apoptotic cells in the Group IV,
while no or lower proportion in other groups. Additionally, the tumor
proliferation and malignancy were significantly reduced in the Group IV
compared with other control groups by Ki‐67 staining (Figure [150]6h).
These findings collectively demonstrate the biocompatibility and
remarkable efficacy of the synergistic hydrogen‐photothermal therapy
using R. sphaeroides, promising significant advancements in cancer
treatment.
Figure 6.
Figure 6
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In vivo R. sphaeroides mediated hydrogen‐photothermal therapy. a)
Schematic illustration showing the treatment schedule. b) Individual
tumor growth curves on 4T1 tumor‐bearing mice after different
treatments (n = 5 biologically independent animals). c) Average tumor
growth curves on 4T1 tumor‐bearing mice after different treatments (n =
5 biologically independent animals). d) Photograph of the excised
tumors from 4T1 tumor‐bearing mice after different treatments (n = 5
biologically independent animals). e) Tumor weight of the excised
tumors from 4T1 tumor‐bearing mice after different treatments (n = 5
biologically independent animals). f) Tumor growth inhibition rate on
4T1 tumor‐bearing mice after different treatments (n = 5 biologically
independent animals). g) The body weights of 4T1 tumor‐bearing mice
after different treatments (n = 5 biologically independent animals). h)
H&E, TUNEL and Ki‐67 staining assays of the tumor from 4T1
tumor‐bearing mice after different treatments. Data are presented as
mean values ± SD. P values calculated by the Two‐tailed Student's t
test (****P< 0.0001, ** P<0.01).
3. Conclusion
This study introduces a distinct therapeutic modality that utilizes
living photosynthetic bacteria, specifically R. sphaeroides, to
concurrently generate H[2] and induce hyperthermia under NIR light
irradiation for targeted cancer therapy. The findings confirm that R.
sphaeroides possesses strong NIR absorption capabilities, enabling
sustained H[2] production and efficient conversion of light energy into
thermal energy directly at the tumor site. This dual functionality is
critical for the therapeutic efficacy observed in the experiments. Both
in vitro and in vivo results demonstrate that this
hydrogen‐photothermal therapy approach yields selective and synergistic
antitumor effects. Remarkably, the combination of H[2] production with
NIR‐triggered hyperthermia significantly enhances overall tumor
suppression. This dual‐action strategy not only targets malignant cells
with precision but also minimizes collateral damage to surrounding
healthy tissues. The enhanced tumor suppression observed is attributed
to the complementary effects of H[2] and hyperthermia. H[2] exerts its
therapeutic effects by modulating ROS levels, leading to oxidative
stress and apoptosis in cancer cells. Simultaneously, NIR‐induced
hyperthermia disrupts mitochondrial function and further increases ROS
levels, compounding the cytotoxic effects on tumor cells. Importantly,
the synergistic therapy offers protection to normal cells and tissues,
likely due to the bidirectional regulatory effects of H[2]. In normal
cells, H[2] enhances protective mechanisms, thereby mitigating
oxidative damage and maintaining cellular homeostasis. In contrast, in
tumor cells, H[2] augments cytotoxicity through oxidative stress,
demonstrating its selective action. This bidirectional regulation
underscores the potential of H[2] to differentiate between cancerous
and healthy cells, making it a versatile therapeutic agent. Our study
highlights the potential of hydrogen‐photothermal therapy as a
comprehensive and effective strategy for cancer treatment, offering a
promising alternative, especially for patients unresponsive to
conventional treatments. The biocompatibility and high efficacy of R.
sphaeroides in generating H[2] and inducing hyperthermia suggest that
this approach can be further developed and optimized for clinical
applications. Future studies should focus on detailed investigations of
the distribution, activity, and lifespan of R. sphaeroides in vivo to
ensure biosafety and maximize therapeutic outcomes. In conclusion, this
work establishes a representative approach utilizing living
photosynthetic bacteria for hydrogen‐photothermal therapy,
demonstrating significant advancements in targeted cancer treatment. In
addition, many photosynthetic bacteria demonstrate remarkable
photohydrogen production capabilities, which hold significant promise
for hydrogen therapy. For instance, cyanobacteria, primitive
prokaryotes, can perform photosynthesis and generate hydrogen under
specific conditions. We anticipate that further studies will explore
diverse photosynthetic bacteria in combination with other therapeutic
modalities, aiming to facilitate the clinical translation of living
therapeutics in the development of novel cancer treatment strategies.
4. Experimental Section
Strains, Cells, and Materials
R. sphaeroides strain was obtained from the CAS Center for Excellence
in Molecular Plant Sciences (CEMPS) and stored at −80 °C. The 4T1
murine breast cancer cell lines, and HEK‐293T embryonic kidney cells
were sourced from Procell Life Science & Technology Co., Ltd (Wuhan,
China). MB was purchased from Macklin (Shanghai, China). Roswell Park
memorial institute‐1640 (RPMI 1640), Dulbecco's modified Eagle's medium
(DMEM) and PBS were both obtained from VivaCell (Shanghai, China).
Fetal bovine serum (FBS) and penicillin‐streptomycin were purchased
from Gibco (Shanghai, China). CCK‐8 was obtained from Beyotime
(Shanghai, China). Calcein‐AM and PI live/dead double stain kit was
obtained from Solarbio (Beijing, China). DCFH‐DA assay kit, MMP assay
kit with JC‐1, ATP assay kit, and Annexin V‐FITC apoptosis detection
kit were purchased from Beyotime (Shanghai, China). The FastPure
Cell/Tissue Total RNA Isolation Kit V2 was purchased from Vazyme
(Nanjing, China). The H&E staining was purchased from Servicebio
(Wuhan, China). The Ki‐67 polyclonal antibody was purchased from
Proteintech (Wuhan, China). The one step TUNEL apoptosis assay kit was
purchased from Beyotime (Shanghai, China).
Bacterial Culture Condition
For bacterial culture, R. sphaeroides was streaked on LB solid plates
and incubated at 37 °C for 24 h under sterile conditions. Colonies were
selected and cultured overnight in LB liquid medium in a shaker at
37 °C and 220 rpm. The culture was then diluted 50‐fold into fresh
medium and allowed to grow further. The bacteria were harvested by
centrifugation at 4000 rpm for 5 min, and the pellet was resuspended in
PBS for subsequent experiments.
Characterization of R. Sphaeroides
SEM image of R. sphaeroides was acquired using a Phenom Pharos G2
FEG‐SEM (Eppendorf, Hamburg, Germany). Initially, the samples were
fixed in 2.5% (v/v) glutaraldehyde in PBS (0.1 M, pH 7.0) overnight.
Following fixation, the samples were washed three times with PBS
(0.1 M, pH 7.0). The samples then underwent sequential dehydration with
increasing concentrations of ethanol (30, 50, 70, 80, 90, and 95%)
(v/v). Final dehydration was performed using a NP‐ZL3‐1K vacuum
centrifugal concentrator (Gejian, Shanghai, China). Subsequently, the
dehydrated samples were coated with gold‐palladium using an ISC 150 ion
sputter (SuPro, Shenzhen, China) for 4 to 5 min and then observed under
the Phenom Pharos G2 FEG‐SEM. UV‐Vis spectra were recorded using a
GENESYS 150 spectrophotometer (Thermo Fisher, Waltham, USA). TEM images
were captured using a HITACHI H‐7650 field emission transmission
electron microscope at 120 Kv (Hitachi, Ltd, Japan).
Extraction of the BChl
To extract the BChl from R. sphaeroides, 500 mL of fresh bacterial
culture at the logarithmic (log) growth phase was collected and
harvested by centrifugation at 8000 rpm for 5 min at 4 °C. Then, the
pellet was washed with PBS for three times and resuspended in 20 mL of
60% (w/v) sucrose solution. The mixture was stirred for 24 h in the
dark to extract the BChl. Finally, store the BChl at 4 °C in the
dark.^[ [152]^30 ^]
Measurement of Hydrogen Production using MB Probe
Hydrogen production was measured using a MB probe. In the presence of
hydrogen, MB is reduced to colorless leucomethylene blue (MBH[2]).^[
[153]^31 ^] In the presence of hydrogen, the blue color of MB is
reduced to colorless leucomethylene blue (MBH[2]).^[ [154]^31 ^] The MB
solution exhibits a characteristic absorption peak at 664 nm, and the
release of hydrogen results in a decrease in this absorbance.
Therefore, hydrogen release was measured based on the color change of
the MB solution. Different concentrations of R. sphaeroides (0,
9.0×10^8, 1.8×10^9, 3.6×10^9, 7.2×10^9, and 1.4×10^10 CFU mL^−1) were
dispersed in 4 mL of MB solution (9 µg mL^−1). Subsequently, 1 µg mL^−1
platinum (Pt) solution was added. The mixture was irradiated with NIR
light (808 nm LED at 0.06 W cm^−2 for 30 min). The absorption peak at
664 nm was detected using spectroscopy. The amount of hydrogen produced
was calculated based on the decrease in the intensity of the absorbance
peak and the standard curve of MB.
Photothermal Performance Assessment
To evaluate the photothermal efficacy of R. sphaeroides at varying
concentrations, bacterial suspensions were prepared in the following
concentrations: 0, 9.0×10^8, 1.8×10^9, 3.6×10^9, 7.2×10^9, and
1.4×10^10 CFU mL^−1. 300 µL of each concentration was dispensed into
separate wells of a 96‐well plate. Samples were exposed to an 808 nm
laser (SLOC, Shanghai, China) with a power density of 1.0 W cm^−2 for
5 min. PBS served as the negative control. Temperature measurements
were recorded every second, and thermal images were captured at
one‐minute intervals using a thermographic camera (Fotric, Shanghai,
China). To assess the photothermal performance of R. sphaeroides under
different power densities, 300 µL of bacterial suspension at a
concentration of 7.2×10^9 CFU mL^−1 was irradiated for 5 min using an
808 nm laser at power densities of 0.5, 1.0, 1.5, or 2.0 W cm^−2.
Temperature changes were monitored using a Fotric 225S infrared thermal
imager. To determine the impact of repeated irradiation on the
photothermal efficiency of R. sphaeroides, bacterial suspension at a
concentration of 7.2×10^9 CFU mL^−1 was exposed to an 808 nm laser at
1.5 W cm^−2 for four cycles. Each cycle consisted of a 5‐minute
exposure to 808 nm laser light at a power density of 1.5 W cm^−2,
followed by a 7‐minute cooling period. Temperature measurements were
recorded every second using a thermographic camera to monitor the
thermal response accurately. To evaluate the effects of 808 nm laser
irradiation on bacterial viability, R. sphaeroides cultures were
exposed to laser at a power density of 1.5 W cm^−2 for 5 min. After
irradiation, the viability was assessed by enumerating colony‐forming
units (CFUs) on LB agar plates. Additionally, to examine the influence
of hyperthermia on bacterial survival, cultures were subjected to
various temperatures ranging from 30 to 60 °C for 10 min. Following
thermal treatment, CFUs were quantified on LB agar plates to determine
survival rates.
Calculation of the Photothermal Conversion Efficiency
To assess the photothermal performance of the R. sphaeroides under an
808 nm laser, 300 µL of bacteria suspension was exposed to an 808 nm
laser (1.5 W cm^−2). Real‐time temperature changes were monitored using
a thermographic camera with a precision of 1 s. The photothermal
conversion efficiency (η) was calculated using Equation ([155]1)
[MATH: η=h·ATMaxsample
−TSursample
−h·ATMaxwater−TSurwaterI1−10−A808
:MATH]
(1)
where T[max] represents the maximum steady‐state temperature, T[sur] is
the ambient temperature of the surrounding, A is the irradiated area, h
is the heat transfer coefficient, I is the laser power, and A [808] is
the absorbance of R. sphaeroides at 808 nm.
Cell Lines and Culture Conditions
4T1 murine breast cancer cells and HEK‐293T human embryonic kidney
cells were cultured under specified conditions. The 4T1 cells were
cultured in RPMI 1640 medium supplemented with 10% (v/v) foetal bovine
serum (FBS) and 1% (v/v) antibiotics (penicillin‐streptomycin, 10 000 U
mL^−1). HEK‐293T cells were cultured in DMEM supplemented with 10%
(v/v) FBS and 1% (v/v) antibiotics (penicillin‐streptomycin, 10 000 U
mL^−1). Both cell types were cultured in an incubator (Thermo
Scientific) at 37 °C in an atmosphere of 5% (v/v) CO[2] and 90% (v/v)
relative humidity. For cell digestion and subculturing, 0.25% (w/v)
trypsin was utilized.
Cell Cytotoxicity Assay
The in vitro cytotoxicity of bacteria against 4T1 murine breast cancer
cells and HEK‐293T human embryonic kidney cells was evaluated using the
CCK‐8 assay. For the toxicity test, cells were seeded in a 96‐well
plate at a density of 1×10^4 cells per well. After a 24‐hour
incubation, 100 µL of medium containing varying concentrations of
bacteria was added. Following a further 4‐hour incubation, the
supernatant in each well was discarded, and the cells were washed 3–5
times with PBS. Subsequently, 100 µL of medium mixed with 10 µL of
CCK‐8 solution was added to each well. The plates were incubated for an
additional 2–4 h before measuring the absorbance at 450 nm using a
BioTek SYNERGY H1 microplate reader. The anti‐cancer effects of
hydrogen‐photothermal therapy on 4T1 or HEK‐293T cells were evaluated
using CCK‐8 assay. Cells were plated at a density of 1×10^4 cells per
well in a 96‐well plate. After a 24‐hour incubation, 100 µL of medium
containing varying concentrations of bacteria was added. After a
further 4‐hour incubation, different treatments were applied according
to the designated groups: medium alone in darkness, an 808 nm laser at
1.5 W cm^−2 for 10 min, an 808 nm LED at 0.08 W cm^−2 for 10 min for
the three control groups; bacteria (7.2×10^9 CFU mL^−1) and 808 nm LED
(0.08 W cm^−2, 10 min) irradiation for the hydrogen therapy group; BChl
and 808 nm laser (1.5 W cm^−2, 10 min) irradiation for the PTT group;
and bacteria (7.2×10^9 CFU mL^−1) with 808 nm laser (1.5 W cm^−2,
10 min) irradiation for the hydrogen‐photothermal therapy group.
Following different treatments, the supernatant in each well was
removed, and cells were washed 3–5 times with PBS, and 100 µL of medium
with 10 µL CCK‐8 solution was added to each well. The plates were
incubated for an additional 2–4 h before the absorbance at 450 nm was
measured using a BioTek SYNERGY H1 microplate reader.
Live/Dead Cell Staining Assay
The anti‐cancer effects of the therapies on 4T1 cells or HEK‐293T cells
were further evaluated by a Calcein‐AM/PI dual staining assay. Cells
were seeded into a 12‐well plate at a density of 1×10^5 cells per well
and cultured for 24 h, after which 1 mL of medium containing the
experimental samples was added. Following another 4‐hour incubation,
treatments were consistent with the aforementioned grouping. After
different treatments, the supernatant in each well was discarded and
cells were washed 3–5 times with PBS, then stained with Calcein‐AM and
PI suspended in PBS. After a 30‐minute incubation at 37 °C, cells were
imaged using an inverted fluorescence microscope (Nikon, ECLIPSE Ti2).
Cellular ROS Assessment
The intracellular ROS levels in 4T1 and HEK‐293T cells were assessed
using the DCFH‐DA fluorescent probe. Cells were seeded in a 12‐well
plate at a density of 1×10^5 cells per well. After culturing for 24 h,
1 mL of the sample‐containing medium was added to each well. Following
an additional 4‐hour incubation, treatments corresponding to the
previously described groupings were applied. After these treatments,
the supernatant in each well was removed, and the cells were washed 3–5
times with PBS. Subsequently, the cells were stained with DCFH‐DA for
20 min. Fluorescence in the cells after different treatments was then
examined using an inverted fluorescence microscope.
Cell Apoptosis Measurement
The apoptosis of 4T1 or HEK‐293T cells was assessed using flow
cytometry. Cells were seeded into a 12‐well plate at a density of
1×10^5 cells per well and cultured for 24 h. After this period, 1 mL of
medium containing the samples was added. Following an additional 4‐hour
incubation, treatments corresponding to the previously described
groupings were applied. After the treatments, the supernatant from each
well was discarded and cells were washed 3–5 times with PBS.
Subsequently, all cells were collected and stained with Annexin
V‐FITC/PI for 15 min. The stained cells were then analyzed by flow
cytometry to determine the percentage of apoptotic cells.
Detection of Intracellular MMP and ATP Level
The levels of MMP in 4T1 cells were assessed using JC‐1 staining, which
differentiate between monomeric form (green fluorescence) and
aggregated form (red fluorescence). 4T1 cells were seeded into a
12‐well plate at a density of 1×10^5 cells per well and cultured for
24 h. After this period, 1 mL of medium containing the samples in each
well was added to each well. Following a further 4‐hour incubation,
treatments corresponding to the previously described groupings were
applied. After the treatments, the supernatant in each well was removed
and cells were washed 3–5 times with PBS. The cells were stained with
JC‐1 for 20 min, after which red and green fluorescence were detected
using a fluorescence microscope. For the assay of intracellular ATP
levels in 4T1 cells, the cells were lysed following the same treatment
protocols. ATP levels were then measured using an ATP bioluminescent
assay kit.
Detection of ER Stress and Lysosomal Damage
The ER stress in 4T1 cells was assessed using the ER‐Tracker and the
lysosomal damage in 4T1 cells was assessed using the Lyso‐Tracker.
Cells were seeded in a 12‐well plate at a density of 1×10^5 cells per
well. After culturing for 24 h, 1 mL of the sample‐containing medium
was added to each well. Following an additional 4‐hour incubation,
treatments corresponding to the previously described groupings were
applied. After these treatments, the supernatant in each well was
removed, and the cells were washed 3–5 times with hank's balanced salt
solution (HBSS). For the ER stress experiment, the cells were stained
with ER‐Tracker for 15 min and then stained with Hoechst 33342 for
10 min. For the lysosomal damage experiment, the cells were stained
with Lyso‐Tracker for 25 min. Fluorescence in the cells after different
treatments was then examined using an inverted fluorescence microscope.
In Vivo Biocompatibility of Photosynthetic Bacteria
In the animal experiments, the animal care and handling procedures
adhered to the guideline of the Shenzhen Top Biotech Co.,Ltd
Institutional Animal Care and Use Committee, IACUC (Approval number:
TOP‐IACUC‐2022‐0158). Female BALB/c healthy mice (6 – 8 weeks old) were
purchased from Zhuhai BesTest Bio‐Tech Co. Ltd. Mice were housed in
ventilated cages under a 12‐hour light‐dark cycle (8:00 – 20:00 light;
20:00 – 8:00 dark), with constant room temperature (21 ± 1 °C) and
relative humidity (40 – 70%). The mice were provided with food and
water freely available at all times. To investigate the in vivo
biocompatibility of photosynthetic bacteria, 20 healthy BALB/c mice
were randomly divided into four groups. BALB/c healthy mice were
sequentially intravenously injected with 150 µL of photosynthetic
bacteria at varying doses (0, 1.8×10^9, 3.6×10^9, and 7.2×10^9
CFU mL^−1) dispersed in PBS. Mice injected with PBS served as the
control group. Behavioral changes and body weights were monitored over
a two‐week period. After two weeks, all mice were euthanized, and blood
was collected via the orbital bleeding method for complete blood count
(lymphocytes percentage, hemoglobin, red blood cells, hematocrit, means
corpuscular volume, means corpuscular haemoglobin concentration, white
blood cells, and red blood cell volume distribution width) and serum
biochemistry analysis (albumin, total protein, globulin,
albumin/globulin, alanine transaminase, alkaline phosphatase,
creatinine, blood urea nitrogen, and blood glucose) using the blood
analyzer BM830 (Baolingman, Beijing, China) and biochemical analyzer
SMT‐120VP (Seamaty, Chengdu, China). The main organs including heart,
liver, spleen, kidney, and lungs were preserved in a 10% (v/v) formalin
solution and stained with H&E for histological analysis to evaluate the
potential toxicity of the photosynthetic bacteria.
Animal Model
Female BALB/c nude mice (6‐8 weeks old) were purchased from Zhuhai
BesTest Bio‐Tech Co. Ltd. The husbandry conditions of mice were
consistent with the previously described method. To establish the
subcutaneous breast tumor model, 4T1 cells (1×10^7 cells mL^−1)
suspended in 100 µL of PBS were subcutaneously injected into the right
flank of each mouse. All animals were randomly assigned to experimental
groups before any treatments. All experimental protocols were conducted
in strict adherence to relevant ethical guidelines and regulations.
Photothermal Performance of Photosynthetic Bacteria In Vivo
4T1 tumor‐bearing mice received peritumoral injection with 150 µL of
photosynthetic bacteria suspension (7.2×10^9 CFU mL^−1) dispersed in
PBS. Subsequently, the tumor areas were irradiated with an 808 nm laser
at a power density of 1.5 W cm^−2 for 10 min. Temperature changes and
photothermal images during the treatment were recorded using a Fotric
225S infrared thermal imager.
Anti‐Tumor Treatment In Vivo
When the volume of tumors reached ≈80 mm^3, the 4T1 tumor‐bearing mice
were randomly divided into four groups (n = 5) for different
treatments: Group I, PBS; Group II, 808 nm laser (1.5 W cm^−2, 10 min);
Group III, photosynthetic bacteria (7.2×10^9 CFU mL^−1); Group IV,
photosynthetic bacteria (7.2×10^9 CFU mL^−1) + 808 nm laser
(1.5 W cm^−2, 10 min). The mice from Group III and Group IV were
peritumorally injected with 150 µL of bacteria on days 0, 2, and 5. The
mice from Group I and Group II were peritumorally injected with 150 µL
of PBS on days 0, 2, and 5. Laser irradiation was performed immediately
following the injection. During the therapeutic period, the tumor
volume and body weights of mice were monitored every other day. Tumor
volume (TV) was calculated using Equation ([156]2):
[MATH: TV=tumorwidth2×tumorlength÷2 :MATH]
(2)
After 14 days of treatment, the mice were euthanized, and all tumors
were collected. The tumor weights were measured, and the tumor
inhibition rate (TIR) was calculated using Equation ([157]3):
[MATH: TIR=meantumorweightofthecontrolgroup−meantumorweightofthetreatmentgroup/meantumorweightofcontrolgroup×100% :MATH]
(3)
Histological Analysis
On day 3 of therapy, one mouse from each group was randomly selected
and euthanized. The major organs (heart, spleen, lungs, liver, and
kidneys) and tumor tissues were harvested and immediately fixed in 10%
(v/v) neutral buffered formalin solution. Tissue sections were prepared
and subsequently stained with H&E for histological evaluation.
Immunohistochemical staining was performed by the Ki‐67 polyclonal
antibody to assess the expression of the proliferation marker Ki‐67.
Additionally, a TUNEL immunofluorescent assay was conducted by the one
step TUNEL apoptosis assay kit to detect apoptotic cells in the tissue
sections.
Biosafety of Hydrogen‐Photothermal Therapy In Vivo
The body weights of mice and the H&E‐stained histology of the main
organs were used to assess the biosafety of hydrogen‐photothermal
therapy in vivo. During the therapy, the body weights of mice were
recorded every other day. For histological assessment via H&E‐stained,
major organs including the heart, liver, spleen, lung, and kidney were
excised from mice after a 14‐day treatment. The harvested tissues were
immersion‐fixed in 4% (v/v) paraformaldehyde solution and subsequently
paraffin‐embedded for sectioning and H&E staining procedures.
Statistical Analysis
All results in this study were recorded as means ± SD. Statistical
analysis was performed by the Two‐tailed Student's t test to identify
significant differences. Differences were considered significant when
**P < 0.01, ***P < 0.001, and ****P < 0.0001. All data were analyzed by
and Graphpad Prism 8.0.
Conflict of Interest
The authors declare no conflict of interest.
Supporting information
Supporting Information
[158]ADVS-12-2408807-s001.docx^ (4.4MB, docx)
Acknowledgements