Abstract
Tumor metastases are considered the leading cause of cancer-associated
deaths. While clinically applied drugs have demonstrated to efficiently
remove the primary tumor, metastases remain poorly accessible. To
overcome this limitation, herein, the development of a theranostic
nanomaterial by incorporating a chromophore for imaging and a
photosensitizer for treatment of metastatic tumor sites is presented.
The mechanism of action reveals that the nanoparticles are able to
intervene by local generation of cellular damage through photodynamic
therapy as well as by systemic induction of an immune response by
immunotherapy upon inhibition of the mTOR signaling pathway which is of
crucial importance for tumor onset, progression and metastatic
spreading. The nanomaterial is able to strongly reduce the volume of
the primary tumor as well as eradicates tumor metastases in a
metastatic breast cancer and a multi-drug resistant patient-derived
hepatocellular carcinoma models in female mice.
Subject terms: Metastasis, Nanomedicine, Cancer therapy
__________________________________________________________________
Approaches to combine photodynamic therapy (PDT) with immunotherapy are
emerging, allowing the treatment of primary and metastatic tumors. Here
the authors report the design and characterization of a nanomaterial
for theranostic imaging and photodynamic therapy eliciting anti-tumor
immune response in preclinical cancer models.
Introduction
Tumor metastases are accountable for approximately 90% of all
cancer-associated deaths^[36]1,[37]2. Studies have shown that
metastases occur when cancer cells stemming from the primary tumor
invade the surrounding tissue and then enter the microvasculature of
the lymph and blood system. The cancer cells can then be transported
through the bloodstream to a distant site where the cells colonize and
form secondary or metastatic tumors^[38]3,[39]4. Over the last decades,
traditional treatment modalities have been optimized to eradicate
primary tumors. Despite recent improvements in chemotherapy,
radiotherapy, and immunotherapy, clinical treatments of metastatic
tumors remain highly challenging^[40]5,[41]6. This is attributed to the
diffuse spreading of the cancer cells towards various organs in the
human body as well as the genetic and epigenetic modification of the
cancer cells, causing drug resistance^[42]7. Among other approaches,
increasing research interest has been devoted to the use of
nanomaterials to identify or treat tumor metastases. Particles in the
nanoscale are intrinsically associated with tumor-targeting properties
based on their ability to accumulate in leaky, highly permeable
vasculature and poor lymphatic tissue characteristics also referred to
as the enhanced permeability and retention (EPR) effect^[43]8–[44]11.
Capitalizing on this, nanoparticle formulations of Doxorubicin (Doxil,
Caelyx, and Myocet), Paclitaxel (Abraxane, Genexol PM), and Irinotecan
(Onivyde) have been clinically approved for the treatment of metastatic
tumors^[45]12,[46]13.
As an emerging medicinal method for tumor treatment, increasing
attention has been devoted to photodynamic therapy (PDT). During this
therapeutic method, the patient is locally or systemically injected
with a photosensitizer and after a certain incubation time the tumor
site is exposed to irradiation. Upon light activation, the
photosensitizer is able to photo-catalytically generate reactive oxygen
species (ROS) and cause oxidative damage to the surrounding tissue,
presenting a localized tumor treatment^[47]14–[48]19. Despite its
clinical application, the clinically approved photosensitizers, which
are based on tetrapyrrolic compounds (i.e., porphyrin, chlorin,
bacteriochlorin, phthalocyanine), are associated with similar drawbacks
including poor cancer selectivity, poor water solubility, and poor
(photo-)stability^[49]20–[50]23. Capitalizing on this, research efforts
have been devoted to the modification of the ligand scaffold. Among
other promising compounds, dipyrrometheneboron difluoride (BODIPY)
complexes have emerged based on their strong photophysical properties
and high (photo-)stability in a biological environment^[51]24–[52]28.
Theoretical and experimental studies have indicated that the
functionalization of the BODIPY complex with iodine atoms can promote
the intersystem crossing process due to the heavy atom effect, yielding
a higher ROS production^[53]29. To enhance the water solubility and
provide cancer selectivity, research efforts have been devoted to the
incorporation of photosensitizers into nanomaterials^[54]30–[55]37.
Despite the strong therapeutic response of PDT agents upon irradiation,
the treatment is localized towards the previously identified tumor
site, preventing the treatment of metastatic tumors. To overcome this
limitation, over the last years, the combination of PDT with
immunotherapy has been proposed. During this multimodal treatment, the
therapeutic agent is able to generate cytotoxic species causing
localized cell damage as well as trigger a systemic immune response of
the organism through immunogenic cell death (ICD) which could present
useful in the treatment of tumor metastases^[56]38–[57]45. Recent
studies have indicated that the light activated PDT treatment is able
to alter the cellular, stromal, and/or vascular properties of the tumor
microenvironment, a process that is termed as photodynamic priming.
These influences can make the tumorous tissue more susceptible for
additional chemo- or immunotherapeutic treatments^[58]46,[59]47. Based
on this, research efforts have been focused on the discovery of
compounds with a combined PDT and immunotherapeutic profile. Previous
studies have indicated that the induction of an immune response inside
a body could trigger severe side effects^[60]38–[61]40. Capitalizing on
this, there is a high demand for tumor-targeted therapeutic strategies.
For an effective PDT treatment, various parameters including the
localization of (secondary) tumor sites, localization and concentration
of the photosensitizer, necessary light dose, oxygen concentration, and
heterogeneity of the tumor microenvironment need to be considered.
Modern imaging techniques are able to provide this information through
various methods. Alternatively, many research efforts have been
invested in the development of multi-functional theranostic systems for
combined cancer diagnosis and therapy. Based on the unique ability of
nanomaterials to encapsulate therapeutic agents, nanoparticles have
received much attention as multifunctional chromophore and drug
carriers^[62]48–[63]52.
In this work, the preparation, characterization, and in-depth
biological evaluation of nanoparticles for theranostic imaging and
treatment of tumors by multimodal photodynamic therapy and
immunotherapy are presented. The previously reported and highly
efficient chromophore
4,4′-(6,7-bis(4-hexylphenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-d
iyl)bis(N,N-diphenylaniline) (M1)^[64]53 and the photosensitizer
2,2′-((((1E,1′E)-(5,5-difluoro-2,8-diiodo-1,9-dimethyl-10-phenyl-5H-4λ^
4,5λ^4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinine-3,7-diyl)bis(ethene
-2,1-diyl))bis(4,1-phenylene))bis(oxy))bis(ethan-1-ol) (M2)^[65]54 are
structurally modified through functionalization with hydroxy groups,
allowing for the incorporation into a polymer backbone through
polyurethane formation (Fig. [66]1a). To provide a higher therapeutic
efficiency and ensure the excretion of the material after the
treatment, thioacetal moieties, that readily dissociates in the
presence of ROS^[67]55, are included in the polymer. Based on the
amphiphilic nature of the polymers, these self-assemble in an aqueous
solution into nanoparticles. For a theranostic application, the two
nanomaterials are equimolar mixed and the composite nanoparticles
(Comp-NPs) are formed (Fig. [68]1b). Insights into the mechanism of
action reveal that the nanoparticles are able to intervene combined by
the local generation of cellular damage through photodynamic therapy as
well as by the systemic induction of an immune response through
immunotherapy. Based on the intrinsic ability to the nanoparticles to
accumulate at tumor sites as well as the necessity to be activated by
light irradiation, this treatment is associated with a double
selectivity. Proteomics analyses indicate that the nanoparticles reduce
the mTOR signaling pathway, which is involved in tumor evolution and
reoccurrence, and in the development of metastases (Fig. [69]1c). The
nanomaterial demonstrates to reduce the volume of the primary tumor as
well as to prevent the development of tumor metastases in a metastatic
breast cancer mouse model in female mice.
Fig. 1. Structures and mechanism of action of Comp-NPs for the diagnosis by
imaging and treatment of tumors by multimodal photodynamic therapy and
immunotherapy.
[70]Fig. 1
[71]Open in a new tab
a Chemical structures of a polymer incorporating a chromophore for
imaging upon irradiation at 808 nm (P1) or a photosensitizer for PDT
upon irradiation at 650 nm (P2). b Self-assembly of the polymers into
the nanoparticles NP1 and NP2. The theranostic nanoparticle formulation
Comp-NPs is generated by mixing NP1 and NP2. c Biological mechanism of
action of Comp-NPs by combined photodynamic therapy and immunotherapy.
Results
Preparation and characterization
The phosphorescent monomer (M1) for imaging upon irradiation at 808 nm
and photosensitizer monomer (M2) for imaging and photodynamic therapy
upon irradiation at 650 nm were independently synthesized (synthetic
scheme Supplementary Figs. [72]1, [73]2). The compounds were
characterized by ^1H-NMR and ^13C-NMR spectroscopy as well as mass
spectrometry (Supplementary Figs. [74]3–[75]15). M1 was found with a
maximum absorption centered at 645 nm and phosphorescence in the NIR-II
region upon irradiation at 808 nm (Fig. [76]2a). M2 showed a maximum
absorption centered at 650 nm and phosphorescence in the NIR-I region
centered at 735 nm upon irradiation at 650 nm (Fig. [77]2b). The
monomers M1/M2, l-lysine diisocyanate (LDI), and the ROS sensitive
linker 2,2′-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol) (DSB)
were mixed to generate a polymeric material. The hydroxy groups of
M1/M2 and DSB can readily react with the isocyanate groups of LDI,
resulting in a linkage of the respective moieties through LDI in the
polymer. It is important to mention that M1/M2 and DSB are randomly
distributed within the polymeric material. The terminal ends were
functionalized with polyethylene glycol to generate the polymers P1 and
P2 (Supplementary Figs. [78]16, [79]17). Using gel permeation
chromatography, P1 was characterized with an average molecular weight
of approximately 24,000 (M[z]/M[w] = 1.55) and P2 of 30,000
(M[z]/M[w] = 1.89). Using ^1H-NMR spectroscopy, P1 was found with an
approximate average of 28 M1-LDI units and 1.5 DSB-LDI units and P2 was
found with an approximate average of 40 M2-LDI units and 2.0 DSB-LDI
units. Based on the amphiphilic nature of the polymers, these
self-assembled in an aqueous solution into the nanoparticles NP1 and
NP2. Notably, the composition of NP1 and NP2 only differs in the use of
a therapeutic or diagnostic agent in the lipophilic region of the
polymeric chain. As the lipophilic parts of the polymers are in the
center of the nanoparticles and the hydrophilic regions of the
polymeric chain are found on the outer surface of the nanoparticles, it
is expected that NP1 and NP2 have similar pharmacological properties.
The absorption spectra of the nanoparticles showed transitions in the
same range as the polymers, indicating the preservation of the
photophysical properties (Supplementary Fig. [80]18). For theranostic
applications, NP1 and NP2 were mixed in a 1:1 ratio and the theranostic
nanoparticle formulation Comp-NPs was generated. It is important to
highlight that Comp-NPs are not specific nanoparticles, but instead a
drug formulation containing an equimolar mixture of NP1 and NP2. As
expected, Comp-NPs showed absorption peaks of NP1 and NP2 and were
found to be highly phosphorescent in the NIR-II region (Fig. [81]2c).
The emissive properties of NP1 and NP2 were individually assessed and
compared with Comp-NPs. The results showed that Comp-NPs had the exact
additive emission properties of the individual nanoparticles, ruling
out the presence of fluorescence resonance energy transfer processes
associated processes. The luminescence quantum yield of Comp-NPs was
determined to be 2.0% upon excitation at 808 nm. Transmission electron
microscope images of NP1, NP2, and Comp-NPs demonstrated the spherical
morphology of the nanomaterials (Supplementary Fig [82]19,
Fig. [83]2d). Elemental mapping of the nanoparticles verified the
uniform distribution of oxygen, sulfur, fluorine, and nitrogen in the
nanoparticles (Fig. [84]2e). Using dynamic light scattering
measurements, the hydrodynamic diameters of the nanoparticles NP1, NP2
and Comp-NPs were found to be in the range of 90–100 nm (Supplementary
Table [85]1, Fig. [86]2f), which is considered ideal for drug delivery.
Fig. 2. Physical and photophysical characterization of Comp-NPs.
[87]Fig. 2
[88]Open in a new tab
a–c Absorption and phosphorescence spectra of M1 in DCM (a,
λ[ex] = 650 nm), M2 in DCM (b, λ[ex] = 808 nm) and Comp-NPs in
phosphate-buffered saline (c, λ[ex] = 808 nm) (n = 3 independent
samples). d Representative transmission electron microscope image of
Comp-NPs. (n = 3 independent samples). The experiment was repeated
independently 3 times with similar results. scale bar = 200 nm. e
Representative scanning transmission electron microscopy coupled with
energy-dispersive X-ray spectroscopy images of Comp-NPs for oxygen,
sulfur, fluorine, and nitrogen. scale bar = 50 nm. (n = 3 independent
samples). f Size distribution of Comp-NPs in water determined by
dynamic light scattering measurements. g Electron paramagnetic
resonance spectra of Comp-NPs with or without laser irradiation
(wavelength: 650 nm, power: 0.1 W cm^−2, 60 J cm^−2 time: 10 min)
(n = 3 independent samples). h, i Gel permeation chromatography (GPC)
measurements of P1 or P2 upon the addition of H[2]O[2]. (n = 3
independent samples). j TEM images of Comp-NPs and upon addition of
H[2]O[2]. k Average particle size (left) and particle size distribution
(right) of Comp-NPs in the presence or absence of 10 mM H[2]O[2]
measured by dynamic light scattering (n = 3 independent samples). scale
bar = 200 nm. Error bars represent mean ± SD. Source data are provided
as a Source Data file.
The identity of the generated ROS by Comp-NPs upon exposure to light
was studied using electron paramagnetic resonance spectroscopy with
2,2,6,6-tetramethylpiperidine as a singlet oxygen (^1O[2]) scavenger
and 5,5-dimethyl-1-pyrroline-N-oxide as a ^•OOH or ^•OH radical
scavenger. While no signal for the generation of ^•OOH or ^•OH radical
species was observed, the characteristic ^1O[2]-induced triplet signal
for 2,2,6,6-tetramethylpiperidinyloxyl was measured (Fig. [89]2g). For
a deeper insight into the ability of Comp-NPs to generate ^1O[2] upon
irradiation, its production was time-dependently monitored by
absorption spectroscopy using 1,3-diphenylisobenzofuran (DPBF) as a
^1O[2] specific probe. Upon irradiation for 10 min, the absorption of
DPBF decreased from approximately 0.9 to 0.4 (Supplementary
Fig [90]20), indicating the efficient generation of ^1O[2].
In order to study the ROS sensitivity, the polymers P1 and P2 were
dissolved in dimethylformamide and exposed to hydrogen peroxide
(H[2]O[2]) as a model for the production of ROS upon irradiation. While
no changes in the average weight for P1 and P2 upon incubation in
organic solvents were observed, a quick degradation was noticed upon
the addition of H[2]O[2] into a mixture of oligomers with lower
molecular weights (1000–9000) (Fig. [91]2h, i). To study the stability
of the nanoparticles, the size and morphology of NP1, NP2, and Comp-NPs
were monitored by transmission electron microscopy (TEM). Upon the
addition of H[2]O[2], the degradation of the nanomaterials was observed
(Fig. [92]2j and Supplementary Fig [93]19). Complementary, the dye Nile
Red was encapsulated into Comp-NPs and the release of the chromophore
was time-dependently monitored by phosphorescence spectroscopy. Nile
Red is known to be highly luminescent in apolar environments such
inside nanoparticles, but is quenched inside an aqueous
solution^[94]56. While no changes in the spectra were observed upon
incubation of Comp-NPs in the dark, quenching and therefore release of
the dye from the nanomaterial was noticed upon irradiation of the
solution (Supplementary Fig [95]S21). Complementary, the decomposition
of the nanoparticles upon exposure to H[2]O[2] was further studied by
dynamic light scattering measurements. In the presence of H[2]O[2] the
size distribution and the polydispersity were drastically enhanced
(Fig. [96]2k) due to the release of small molecular therapeutic agents
and the aggregation of the lipophilic hydrophobic fragments. Overall,
these results indicate that Comp-NPs remains stable under physiological
conditions but are quickly degraded in the presence of ROS or upon
irradiation.
Biological PDT effect in a 4T1 cancer cell model
The biological properties of Comp-NPs were in-depth studied in mouse
breast cancer (4T1) cells. As a crucial property for a biological
effect, the cellular uptake was investigated upon time-dependent
monitoring of the phosphorescence of the nanoparticles inside the
cancer cells by confocal laser scanning microscopy (CLSM). With the
prolongation of the incubation time, an increasing amount of
phosphorescence was detected inside the cancer cells, indicating the
efficient and time-dependent cellular uptake of Comp-NPs (Fig. [97]3a).
Using flow cytometry, the cellular uptake was verified (Supplementary
Fig [98]22a-b). Followingly, the uptake of the nanoparticles was
investigated in multicellular tumor spheroids (MCTS) as a tissue
culture model for the delivery of compounds into multicellular
architectures. This is in particular important as many anticancer
agents have failed the translation from a monolayer cancer cell model
to animal models due to compromised drug delivery^[99]57,[100]58.
Therefore, herein, 4T1 MCTS with a diameter of ~700 μm were used as a
mimic to study the penetration of three-dimensional cellular
architectures. Upon incubation of Comp-NPs for 7 h, z-stack CLSM showed
a strong red phosphorescence signal at every section depth
(Supplementary Fig [101]22c), indicative of the complete penetration of
the MCTS.
Fig. 3. Cellular uptake, ROS generation, and cell death mechanism of Comp-NPs
in a 4T1 monolayer cancer cell model or 4T1 multicellular tumor spheroids.
[102]Fig. 3
[103]Open in a new tab
a CLSM images of 4T1 cells incubated with Comp-NPs at 37°C for 1 h, 4 h
and 7 h. The experiment was repeated independently 3 times with similar
results. scale bar = 10 μm. b CLSM images of 4T1 MCTS with a diameter
of ~700 μm incubated with Comp-NPs at 37 °C for 7 h and the ROS
specific probe for 30 min, followed by exposure to irradiation (650 nm,
0.1 W cm^−2, 30 J cm^−2, 5 min). The experiment was repeated
independently 3 times with similar results. scale bar = 100 μm. c CLSM
images of 4T1 cells incubated with the CRT fluorescent probe (CRT,
green) and DAPI (blue) upon various treatments in the light (650 nm,
0.1 W cm^−2, 2 min). scale bar = 20 μm. d, e Quantification of the
translocation of CRT to the cell surface of 4T1 cells and
quantification of extracellular adenosine triphosphate (ATP)upon
various treatments in the light (650 nm, 0.1 W cm^−2, 2 min) by flow
cytometry (n = 3 biologically independent samples). f CLSM images of
4T1 cells incubated with the HMGB1 protein fluorescent probe (HMGB1,
green) and DAPI (blue) upon various treatments in the light (650 nm,
0.1 W cm^−2, 2 min). scale bar = 20 μm. g Quantification of the release
of the HMGB1 protein from (f) (n = 5 biologically independent cells). h
Cell migration wound healing assay of 4T1 cells upon various treatments
in the light (650 nm, 0.1 W cm^−2, 30 J cm^−2, 5 min). The experiment
was repeated independently 3 times with similar results. scale bar =
50 μm. i, j Quantification of the maturation of DCs upon various
treatments in the light (650 nm, 0.1 W cm^−2, 30 J cm^−2, 5 min) (n = 3
biologically independent samples). Statistical analysis was performed
by one-way ANOVA with Tukey’s multiple comparisons test. All data are
presented as mean ± SD. Source data are provided as a Source Data file.
The ability of the nanoparticles to generate ROS inside the MCTS was
studied using the ROS specific probe 2′,7′-dichlorofluorescein
diacetate. While the probe is non-luminescent in a biological milieu,
in the presence of ROS it is oxidized and the fluorescent
2′,7′-dichlorofluorescein product is formed. While no green
fluorescence of the probe was observed upon incubation of the MCTS with
the nanoparticles in the dark, a strong green fluorescence signal upon
exposure of the MCTS to light was observed, suggesting the production
of ROS (Fig. [104]3b). Using flow cytometry, the ROS production inside
the cells was verified (Supplementary Fig [105]23).
The cytotoxic effect of the nanoparticles NP1, NP2, and Comp-NPs
towards murine breast 4T1 cancer cells, human ovarian cancer (SKOV3),
cisplatin sensitive ovarian cancer (A2780), cisplatin-resistant ovarian
cancer (A2780DDP) and human hepatocellular carcinoma (JHH7) cells was
investigated in the dark as well as upon irradiation (650 nm,
0.1 W cm^−2, 60 J cm^−2, 10 min). All nanoparticle formulations were
found to be non-toxic in the dark, which is an important requirement
for an imaging and PDT agent (IC[50,dark] > 6 μg/mL BODIPY). While
imaging upon irradiation at 808 nm nanoparticles NP1 did not show a
cytotoxic effect upon exposure to light, a strong phototoxic effect for
NP2 (IC[50,light] = 0.46 ± 0.13 μg/mL) and Comp-NPs
(IC[50,light] = 0.47 ± 0.03 μg/mL BODIPY, Supplementary Table [106]2,
Supplementary Fig [107]24) was observed. Remarkably, Comp-NPs showed to
eradicate 80% of the cancer cell population at a concentration of
0.5 μg/mL BODIPY upon exposure to light. For a deeper insight into the
biological properties, nanoformulations of Comp-NPs with various ratios
of NP1 and NP2 (1:2, 1:3, 2:1, 3:1) were prepared and their
cytotoxicity against 4T1 cells was assessed. Using an equal amount of
the photosensitizer BODIPY, a cytotoxic effect inside the same range
was observed (IC[50,light] = 0.38 – 0.76 μg/mL BODIPY) (Supplementary
Table [108]3, Supplementary Fig [109]25). These findings indicate that
the ratio between NP1 and NP2 could be fine-tuned in future studies to
generate only the desired therapeutic response.
For a deeper understanding of the phototoxic effect, the cell death
mechanism of the nanoparticles was studied. The ability to trigger
apoptotic cell death was investigated by flow cytometry using the dual
probes FITC-Annexin V/Propidium Iodide. While no cellular death upon
treatment of Comp-NPs in the dark was observed, the majority of the
cancer cell population showed apoptotic cell death upon exposure of the
cells to light (Supplementary Fig [110]26). Western Blot analysis
indicated that the treatment with Comp-NPs and exposure to light
triggered the activation of the expression of caspase-3 (Supplementary
Fig [111]27), indicative that cell death is induced by apoptosis using
the caspase-3 pathway. Using light microscopy, the shrinkage of the
cancer cells and the fragmentation into membrane-bound apoptotic bodies
is observed upon treatment with Comp-NPs and exposure to light as this
is typically observed for apoptotic death cell (Supplementary
Fig [112]28). Recent studies have indicated that apoptotic dying cells
could release damage-associated molecular patterns (DAMPs) which are
able to trigger an immune response inside the organism^[113]59,[114]60.
Capitalizing on this, the cancer cells were analyzed upon treatment
towards specific hallmarks for immunogenic cell death (ICD). Using
CLSM, the translocation of the endoplasmic reticulum resident
calreticulin (CRT) to the cell surface upon treatment with Comp-NPs in
the light was measured (Fig. [115]3c), supporting the interaction of
macrophages for tumor antigen presentation. This effect was further
quantified using flow cytometry (Fig. [116]3d). The secretion of
adenosine triphosphate (ATP) was studied which promotes the attraction
of tumor-specific T-cells. The results showed a highly increased amount
of ATP upon treatment with Comp-NPs and exposure to light
(Fig. [117]3e). The release of the nuclear high-mobility group box 1
(HMGB1) protein, which triggers the myeloid differentiation signaling
cascade required for antigen presentation to T-cells, was observed by
CLSM using a specific nuclear HMGB1 protein chromophore
(Fig. [118]3f-g). Complementary, the migration of the nuclear HMGB1
protein was observed by immunofluorescence CLSM (Supplementary
Fig [119]29). Combined these results indicate that Comp-NPs is able to
efficiently trigger cell death combined by apoptosis and ICD. The
ability to trigger an immune response could allow for the treatment of
distant tumor site or metastatic tumors. As a preliminary model, a cell
migration wound healing assay was performed. The incubation with
Comp-NPs in the light showed the least amount of cell migration
(Fig. [120]3h and Supplementary Fig [121]30), indicative of the ability
to inhibit cell migration and treat distant cancer cells. For a deeper
insight into the activation of the immune response, 4T1 cells were
treated with NP1, NP2 or Comp-NPs in the dark as well as upon
irradiation and afterwards dendritic cells (DC2.4) were added. Using
flow cytometry, the maturation of the DCs was assessed. While no
significant immune response was observed during the treatment in the
dark, strong activation of the immune system (up to ~34% of DC
maturation) was monitored upon incubation with Comp-NPs and exposure to
irradiation (Treatment in the dark: Supplementary Fig [122]31,
Treatment in the light: Fig. [123]3i-j). Overall, these results
demonstrated the ability of Comp-NPs to be efficiently taken up by
cancer cells, generate ROS, and cause a strong cytotoxic effect upon
irradiation by a combination of apoptosis and ICD.
Biological imaging and PDT effect in a 4T1 tumor-bearing mouse model
Based on the promising properties in a cancer cell model, the imaging
and therapeutic PDT effects were further studied inside a 4T1
tumor-bearing mouse model. For an evaluation of the biosafety of the
nanoparticles, Comp-NPs were intravenously injected into the tail vein
of the animal model and the behavior of the mouse was monitored. As no
signs of stress, discomfort, or change in weight of the animal were
observed over a period of two weeks after the injection, the high
biocompatibility of the nanomaterial is indicated. The biodistribution
of the nanoparticles inside the living mouse model was studied upon
NIR-I (λ[ex] = 650 nm, λ[em] = 745 nm) and NIR-II (λ[ex] = 808 nm,
λ[em] = 950 nm) phosphorescence imaging. With the prolongation of the
circulation time an increasing amount of Comp-NPs accumulated at the
tumor site, suggesting tumor-targeting properties of the nanomaterial
(Fig. [124]4a). For quantification, organs as well as the tumor were
collected, and the biodistribution was determined by phosphorescence
imaging of the respective tissues. The results showed that Comp-NPs
were found with the highest accumulation inside the liver, as this is
typically the case for nanomaterials, and the tumor (Fig. [125]4b-c).
These findings indicate the potential application of Comp-NPs as an
imaging agent for the identification of possible tumor sites.
Fig. 4. Imaging and PDT properties of Comp-NPs were evaluated in a 4T1
tumor-bearing mouse model.
[126]Fig. 4
[127]Open in a new tab
a Biodistribution of Comp-NPs inside the living animal model determined
upon irradiation at 808 nm and 650 nm. b Phosphorescence images of the
major organs and the tumor after the sacrifice of the mouse model which
was 48 h before intravenously injected in the tail vein with Comp-NPs.
λ[ex] = 650 nm, λ[em] = 735 nm. c Quantification of the accumulation of
Comp-NPs from (b) (n = 3 mice). Error bars represent mean ± SD. d Tumor
growth inhibition curves (n = 5 mice) upon treatments with Comp-NPs
(6 mg BODIPY kg^−1) or cisplatin (0.5 mg Pt kg^−1) in the dark or upon
irradiation (650 nm, 0.1 W cm^−2, 60 J cm^−2, 10 min). Error bars
represent mean ± SD. Statistical analysis was performed by two-tailed
unpaired t test. e Body weight of the mice (n = 5 mice) upon various
treatments with Comp-NPs (6 mg BODIPY kg^−1) or cisplatin (0.5 mg Pt
kg^−1) in the dark or upon irradiation (650 nm, 0.1 W cm^−2,
60 J cm^−2, 10 min). Error bars represent mean ± SD. Statistical
analysis was performed by one-way ANOVA with a Tukey’s multiple
comparisons test. f Photographs of the tumors after various treatments.
(n = 5 mice). g Weight of the tumors from (f) (n = 5 mice). Error bars
represent mean ± SD. Statistical analysis was performed by one-way
ANOVA with Tukey’s multiple comparisons test. h Photographs of
metastatic nodules (highlighted white circles) in the lungs and H&E
stain of tissue slices of this organ. scale bar = 100 μm. i
Quantification of the amount of metastatic lung nodules after various
treatments (n = 5 mice). Error bars represent mean ± SD. Statistical
analysis was performed by two-tailed unpaired t test as a Source Data
file.
The therapeutic PDT effect inside 4T1 tumor-bearing mice was studied
upon intravenous injection of Comp-NPs (6 mg BODIPY kg^−1) into the
tail vein and monitoring of changes in the tumor volume upon treatment
in the dark or exposure to irradiation (650 nm, 0.1 W cm^−2,
60 J cm^−2, 10 min). Promisingly, upon injection of the animal model
with Comp-NPs and exposure to light, a strong tumor growth inhibition
effect was observed within a single treatment. The tumor remission was
found to be slightly but not statically significantly stronger than
upon treatment with the anticancer drug cisplatin (Fig. [128]4d). While
the body weight of the animals treated with the nanoparticles remained
unchanged, a significant reduction of the body weight upon treatment
with cisplatin was observed (Fig. [129]4e), indicative for severe side
effects. As key parameters for the biosafety of the treatment, the
levels of ALT, AST, BUN and CR inside the animal model were analyzed.
While no changes in the biochemical levels of all parameters during the
treatment with Comp-NPs were observed, a strong increase of the BUN and
CR factor for the treatment with cisplatin was observed, verifying the
toxicity of cisplatin (Supplementary Fig [130]32a). Complementary, the
major organs of the animals treated with Comp-NPs in the dark or upon
irradiation did not show any pathological alternations in a hematoxylin
and eosin (H&E) stain of the respective tissues (Supplementary
Fig [131]32b). After the respective treatments, the tumors were
collected and weighted (Fig. [132]4f: Photographs of the tumor,
Fig. [133]4g: Tumor weight). While the mice injected with saline showed
an average tumor weight of ~1.7 g, the animals treated with Comp-NPs
and exposed to light were found with an average tumor weight of ~0.4 g,
highlighting the strong tumor remission. Based on the metastatic nature
of 4T1 breast cancer tumors^[134]61, various metastases were observed
in the lungs of the untreated animal model. It is important to mention
that metastases are considered the leading cause of (breast)
cancer-associated human deaths^[135]62. Strikingly, the mice treated
with Comp-NPs and exposed to irradiation did not show any signs of
metastases (Fig. [136]4h-i), indicating the ability to prevent the
development of metastases. Overall, these findings suggest that
Comp-NPs is able to act combined as a NIR-I and NIR-II imaging agent
for identification of tumor sites, PDT agent with a strong tumor
remission within a single dose and light treatment, and anti-metastatic
agent to prevent the development of metastases at distant tissues in
the animal model.
Immunogenic PDT effect in a (Metastatic) 4T1 tumor-bearing mouse model
Capitalizing on the strong antimetastatic effect of the nanoparticles,
the immune response inside the animal model was evaluated
(Fig. [137]5a). Based on the ability of the nanomaterial to trigger ICD
in monolayer cancer cells, this biological mechanism was investigated
inside the animal model upon biochemical assessment of tissue slices of
the tumorous tissue for specific hallmarks. Using a CRT-specific probe
and immunofluorescence CLSM, the migration of CRT to the cell surface
was observed as indicated by the red fluorescence in the microscopy
images upon treatment with Comp-NPs and exposure to light (Fig. [138]5b
top panel). Quantification of the fluorescence signal demonstrated that
the treatment with the nanoparticles caused an approximately 8.3 times
higher migration of CRT than the treatment with saline (Supplementary
Fig [139]33a). Followingly, the localization of the HMGB1 protein was
investigated by CLSM using a HMGB1 protein-specific probe. While the
HMGB1 protein was primarily found inside the nucleus upon treatment
with saline, as indicated by the congruency with the fluorescence
signal of the nucleus stain DAPI, the tumors treated with Comp-NPs and
exposure to irradiation showed the migration of the HMGB1 protein
(Fig. [140]5b middle panel). Quantification of the ratio of the
fluorescence of the HMGB1 protein with the fluorescence of nuclear
stain DAPI showed that the treatment with Comp-NPs and exposure to
irradiation resulted in the migration of the HMGB1 protein of
approximately 40% from the cell nucleus (Supplementary Fig [141]33b).
Based on these hallmarks for an immune response of the organism, the
levels of CD8^+ T-cells were assessed by CLSM using a CD8^+ T-cells
specific fluorescent probe. While only a minimal amount of CD8^+
T-cells were noticed in the microscopy images during the treatment with
saline, a significant amount of immune CD8^+ T-cells were observed upon
treatment with Comp-NPs and exposure to light as indicated by the red
fluorescence signal (Fig. [142]5b bottom panel). A comparison of the
fluorescence signals suggested that an approximately 5 times higher
amount of CD8^+ T-cells was found in the tissue treated with Comp-NPs
and exposure to light than in the control group (Supplementary
Fig [143]33c).
Fig. 5. Mechanism of the immunogenic PDT response inside a 4T1 tumor-bearing
mouse model upon treatment with Comp-NPs and exposure to irradiation.
[144]Fig. 5
[145]Open in a new tab
a Schematic diagram of immune response test after Comp-NPs upon the
650 nm laser. b CLSM images of tumor slices obtained from variously
treated 4T1 mouse xenograft models incubated with DAPI (blue,
λ[ex] = 410 nm, λ[em] = 506 nm) and top: CRT specific fluorescent probe
(CRT, red, λ[ex] = 488 nm, λ[em] = 525 nm), middle: HMGB1 protein
fluorescent probe (HMGB1, red, λ[ex] = 488 nm, λ[em] = 525 nm), bottom:
CD8^+ T-cells fluorescent probe (red, λ[ex] = 488 nm, λ[em] = 525 nm).
(The images are representative of 3 mice per group). The experiment was
repeated independently 3 times with similar results. scale bar = 20 μm.
c–d Analysis of the levels of activated dendritic cells (CD80^+,
CD86^+) in tumor slices obtained from variously treated 4T1 mouse
models (n = 3 mice). Error bars represent mean ± SD. Statistical
analysis was performed by two-tailed unpaired t test. e, f Analysis of
the levels of activated dendritic cells (CD80^+, CD86^+) in lymph nodes
slices obtained from variously treated 4T1 mouse models. (n = 3 mice).
Error bars represent mean ± SD. Statistical analysis was performed by
two-tailed unpaired t test. g, h Analysis of the levels of effector
T-cells (activated CD8^+ T-cells, activated CD4^+ T-cells) in tumor
slices obtained from variously treated 4T1 mouse models. (n = 3 mice).
Error bars represent mean ± SD. Statistical analysis was performed by
two-way ANOVA with Šídák’s multiple comparisons test. Source data are
provided as a Source Data file.
The ability of the nanoparticles to stimulate and promote the systemic
host immune response was studied upon the determination of the levels
of T-cells and matured DC cells. During ICD, DAMPs are released which
are able to promote the transmission of phagocytic signals to DC-based
antigen-presenting cells and therefore enhance the immune response of
the organism. The treatment with Comp-NPs and exposure to irradiation
demonstrated to enhance the population of matured DC cells (CD80^+,
CD86^+) up to approximately 21%, corresponding to an approximate 3
times augmentation of the levels found in the animal models
(Fig. [146]5c, d). The proportion of matured DC cells in the lymph
nodes was increased to approximately 39%, indicating a doubling of the
amount of DC cells in this organ (Fig. [147]5e, f). In agreement with
this finding, the proportion of T-cells (CD4^+, CD8^+) in the tumor
tissues approximately also doubled up to 40% (Fig. [148]5g, h).
Overall, these results demonstrate the immunogenic effect upon PDT
treatment with Comp-NPs and rationalize the strong tumor remission as
well as inhibition of the development of tumor metastases.
To provide a deeper understanding, the tumor growth inhibition effect
as well as induction of the immune response of Comp-NPs was compared to
the clinically approved ICD inducing anticancer drug oxaliplatin inside
a 4T1 tumor-bearing mouse model. Comp-NPs and oxaliplatin were injected
into the tail vein and the changes in the tumor volume upon treatment
in the dark or exposure to irradiation (650 nm, 0.1 W cm^−2,
60 J cm^−2, 10 min) were monitored. The weight of the mice did not
significantly change, indicative of the biocompatibility of the
treatment. The tumor of the animals treated with Comp-NPs grew
exponentially in a similar manner as the control group. The treatment
with oxaliplatin showed a remission of the tumor. The tumor of the mice
treated with Comp-NPs and exposed to irradiation was fully eradicated
(Supplementary Fig [149]34a: Cumulative tumor growth inhibition curves,
Fig S[150]34b: Photographs of the animals after the treatment,
Supplementary Fig [151]34c: Changes in body weight, Supplementary
Fig [152]34d: Individual tumor growth inhibition curves). Using flow
cytometry, the induction of the immune response upon treatment was
quantified. The treatment with Comp-NPs in the dark was found not to
elevate the levels of DCs inside the animal model. The treatment with
Comp-NPs and exposure to irradiation demonstrated to enhance the
population of matured DC cells (CD80^+, CD86^+) up to approximately
22%, corresponding to an approximately 2.2 times higher level than upon
treatment with oxaliplatin (level of matured DC cells of ~10%) and
approximately 4.4 times higher level than in the control group (level
of matured DC cells of ~5%) (Supplementary Fig [153]34e, f). The
proportion of T-cells (CD4^+, CD8^+) in the tumor tissue was found to
be highly elevated upon treatment with oxaliplatin or Comp-NPs. The
levels of CD4^+ T-cells upon treatment with Comp-NPs reached
approximately 49%, corresponding to an approximately 1.3 times higher
level than upon treatment with oxaliplatin (level of CD4^+ T-cells of
~39%) and approximately 2.9 times higher level than in the control
group (CD4^+ T-cells of ~17%). In agreement, the levels of CD8^+
T-cells upon treatment with Comp-NPs reached approximately 31%,
corresponding to an approximately 1.4 times higher level than upon
treatment with oxaliplatin (level of CD4^+ T-cells of ~22%) and
approximately 2.8 times higher level than in the control group (CD4^+
T-cells of ~11%) (Supplementary Fig [154]34g, h). The proportion of
effector T lymphocyte interferon‐gamma IFN‐γ^+CD8^+ T cells inside the
tumor was assessed. The treatment with Comp-NPs and exposure to
irradiation demonstrated to enhance the population of IFN‐γ^+CD8^+ T
cells up to approximately 16%, corresponding to an approximately 2.0
times higher level than upon treatment with oxaliplatin (level of
matured DC cells of ~8%) and approximately 5.3 times higher level than
in the control group (level of matured DC cells of ~3%) (Supplementary
Fig [155]34i-j). Overall, these results suggest that the treatment with
Comp-NPs showed a stronger tumor growth inhibition effect as well as
stronger immune response than the treatment with the anticancer drugs
oxaliplatin.
The ability of the immunogenic response to treat distant tumors was
studied in a mouse model with two separated 4T1 tumors. The primary
tumor was grown on the right side of the animal model and the mouse
twice treated by intravenous administration of Comp-NPs and exposure to
light. Afterwards, cancer cells were injected on the left of the mouse
to form a secondary tumor and the tumor growth monitored in both places
(treatment schedule: Fig. [156]6a). In agreement with the previous
assessment (Fig. [157]4), a strong primary tumor remission was observed
upon administration of the nanoparticles and exposure to light
(Fig. [158]6b–e). Strikingly, the growth of the secondary tumor was
also strongly inhibited upon injection of Comp-NPs and exposure to
light (Fig. [159]6f–i). Since the mice, which were injected with the
second dose of cancerous cells, were kept in the dark and the
nanoparticles were found not to influence the tumor growth in the dark,
the remission of the secondary tumor is attributed to the immunogenic
response in the animal model. The monitoring of the long-term survival
indicated a strong enhancement of the life expectancy upon treatment.
While the non-treated or in the dark treated animals died after 19–21
days, all mice models injected with Comp-NPs and exposed to irradiation
were still alive after 30 days (Fig. [160]6j). These results strongly
indicate the ability of the nanoparticles to eradicate
distant/secondary tumor sites through an immunogenic response as well
as enhance the survival of the animal model.
Fig. 6. PDT properties of Comp-NPs evaluated in a mouse model bearing a
primary and secondary distant tumor.
[161]Fig. 6
[162]Open in a new tab
a Schematic illustration of the establishment of a dual primary and
secondary tumor mouse model, administration and treatment schedules
with Comp-NPs (6 mg BODIPY kg^−1) in the dark or upon irradiation
(650 nm, 0.1 W cm^−2, 60 J cm^−2, 10 min). Phosphate-buffered saline
(PBS) was used as a control. b Mean tumor growth inhibition curves
(n = 5 mice) of the primary tumor upon treatment. Error bars represent
mean ± SD. Statistical analysis was performed by two-tailed unpaired t
test. c–e Individual tumor growth inhibition curves (n = 5 mice) of the
primary tumor upon treatment. f Mean tumor growth inhibition curves
(n = 5 mice) of the secondary tumor upon treatment. Error bars
represent mean ± SD. Statistical analysis was performed by two-tailed
unpaired t test. g–i Individual tumor growth inhibition curves of the
secondary tumor upon treatment. j Long-term survival of the animal
models (n = 5 mice) upon treatment. Source data are provided as a
Source Data file.
Therapeutic PDT effect in patient-derived tumor-bearing mouse models
To assess the potential of the nanoparticles to treat clinically
challenging tumors, the therapeutic properties of Comp-NPs towards
multi-drug resistant patient-derived breast carcinoma tumors inside a
mouse model were investigated. The cancer cells were obtained from a
patient with reoccurring tumors despite multiple rounds of surgery and
chemotherapy. The tumors of the animals treated with nanoparticles in
the dark showed a similar growth as the control group. However, when
the animals were injected with Comp-NPs and exposed to irradiation, the
tumor was fully eradicated after a single treatment. Due to the drug
resistance of the tumor to various chemotherapeutic drugs, the
treatment with oxaliplatin showed only a minimal effect on the tumor
remission. These findings are highly promising and suggest that
Comp-NPs have the potential to effectively treat multi-drug resistant
and clinically challenging tumors (Fig. [163]7a: Cumulative tumor
growth inhibition curves, Supplementary Fig [164]35: Photographs of the
animals after the treatment, Fig. [165]7b–e: Individual tumor growth
inhibition curves). It is noteworthy that the animals treated with
Comp-NPs did not exhibit any signs of pain, discomfort, or weight loss
(Fig. [166]7f). For a deeper insight, the tumorous tissue was
histologically analyzed. The tumor slices of the mice treated with
Comp-NPs and exposed to irradiation showed cellular and tissue damage
(Fig. [167]7g), indicative of the efficient PDT treatment.
Fig. 7. PDT properties of Comp-NPs (6 mg BODIPY kg^−1) in comparison to
clinically approved chemotherapeutic drug oxaliplatin (Oxa, 0.5 mg Pt kg^−1)
in the dark or upon irradiation (650 nm, 0.1 W cm^−2, 60 J cm^−2, 10 min)
evaluated in a multi-drug resistant patient-derived breast carcinoma inside a
mouse model.
[168]Fig. 7
[169]Open in a new tab
a Cumulative tumor growth inhibition curves (n = 6 mice) upon
treatment. Error bars represent mean ± SD. Statistical analysis was
performed by two-tailed unpaired t test. b–e Individual tumor growth
inhibition curves (n = 6 mice) upon treatment. f Body weight of the
mice (n = 6 mice) upon treatment. Error bars represent mean ± SD. g
Terminal deoxynucleotidyl transferase dUTP nick end labeling (green)
and DAPI (blue) stain of tumor tissue slices after various treatments
(the images are representative of 6 mice per group). scale bar = 1 mm.
Source data are provided as a Source Data file.
Hepatocellular carcinoma, also referred to as liver cancer, has one of
the highest clinical incidences^[170]63. While there exist effective
treatments in the clinics, certain parts of the liver are poorly
accessible or associated with drug resistance, limiting the therapeutic
options for patients. To overcome these drawbacks, there is a need for
the development of therapeutic drugs and methods. To evaluate the
ability of the here reported nanoparticles to treat hepatocellular
carcinoma tumors, the biological properties of Comp-NPs were studied
against human hepatocellular carcinoma (JHH7) cells in the dark as well
as upon irradiation (650 nm, 0.1 W cm^−2, 60 J cm^−2, 10 min). While
being non-toxic in the dark (IC[50,dark] > 6 μg/mL BODIPY), Comp-NPs
had a strong cytotoxic effect upon exposure to irradiation
(IC[50,light] = 0.45 ± 0.02 μg/mL BODIPY, Supplementary Fig [171]36a).
The ability to induce ICD in JHH7 cells upon treatment with the
nanoparticles and exposure to light was studied through monitoring for
ICD specific hallmarks. The translocation of CRT to the cell surface
upon treatment with Comp-NPs in the light was noticed by flow cytometry
(Supplementary Fig [172]36b). Using immunofluorescence CLSM, the
migration of HMGB1 from the nucleus into the cytoplasm was observed
(Supplementary Fig [173]36c). The treatment with the nanoparticles
triggered the release of ATP (Supplementary Fig [174]36d). Combined
these findings indicate the induction of ICD in JHH7 cells upon
treatment with Comp-NPs and exposure to irradiation. Based on these
promising preliminary findings, the therapeutic properties of Comp-NPs
were further studied against multi-drug resistant patient-derived
hepatocellular carcinoma tumors inside a mouse model. The cancer cells
were obtained from a patient with reoccurring tumors despite multiple
rounds of surgery and chemotherapy. While the tumors of the animals
injected with the nanoparticles in the dark grew exponentially in a
similar manner as the mice injected with saline, a strong tumor
reduction for the animal injected with Comp-NPs and exposed to
irradiation was observed within a single drug and light treatment
(Fig. [175]8a). Due to the drug resistance of the tumor, the treatment
with cisplatin showed only a minimal effect on tumor remission. These
findings are very promising and indicate the potential of Comp-NPs to
treat multi-drug resistant and clinically challenging tumors
(Fig. [176]8b). Importantly, the animals treated with Comp-NPs did not
show any signs of pain or discomfort as well as did not lose body
weight (Fig. [177]8c). Following the treatment period, their major
organs analyzed. As no pathological alternations were noticed
(Supplementary Fig [178]37), the high biocompatibility of the treatment
is indicated. In addition, the tumors were collected and weighted
(Fig. [179]8d: Photographs of the tumor, Fig. [180]8e: Tumor weight).
While the animals injected with saline or treated with Comp-NPs in the
dark showed an average tumor weight of ~1.8 g, the mice treated with
Comp-NPs and exposed to irradiation showed a strongly reduced tumor
weight of ~ 0.6 g, highlighting the strong tumor growth inhibition
effect. For a deeper insight, the tumorous tissue was histologically
analyzed. The tumor slices of the mice treated with Comp-NPs and
exposed to irradiation showed cellular and tissue damage
(Fig. [181]8f), indicative of the efficient PDT treatment. Overall,
these results demonstrated the ability of Comp-NPs to cause a strong
tumor reduction of a multi-drug resistant clinically challenging
hepatocellular carcinoma inside the animal model, suggesting promising
properties for clinical development.
Fig. 8. PDT properties of Comp-NPs evaluated towards a multi-drug resistant
patient-derived hepatocellular carcinoma inside a mouse model.
[182]Fig. 8
[183]Open in a new tab
a Schematic illustration of the establishment of the mouse model as
well as the subsequent administration and treatment schedules with
Comp-NPs. Phosphate-buffered saline (PBS) and cisplatin (CisPt) were
used as controls. b Tumor growth inhibition curves (n = 5 mice) upon
treatments with Comp-NPs (6 mg BODIPY kg^−1) or cisplatin (0.5 mg Pt
kg^−1) in the dark or upon irradiation (650 nm, 0.1 W cm^−2,
60 J cm^−2, 10 min). Error bars represent mean ± SD. Statistical
analysis was performed by two-tailed unpaired t test. c Body weight of
the mice (n = 5 mice) upon various treatments with Comp-NPs (6 mg
BODIPY kg^−1) or cisplatin (0.5 mg Pt kg^−1) in the dark or upon
irradiation (650 nm, 0.1 W cm^−2, 60 J cm^−2, 10 min). Error bars
represent mean ± SD. d Photographs of the tumors after various
treatments. e Weight of the tumors from (d) (n = 5 mice). Error bars
represent mean ± SD. Statistical analysis was performed by two-tailed
unpaired t test. f H&E as well as terminal deoxynucleotidyl transferase
dUTP nick end labeling stain of tumor tissue slices. (The images are
representative of 5 mice per group). The experiment was repeated
independently 3 times with similar results. Tunel: scale bar = 50 μm.
H&E: scale bar = 100 μm. Source data are provided as a Source Data
file.
Biochemical mechanism of action in a hepatocellular carcinoma patient-derived
tumor-bearing mouse model
For a deeper insight into the mechanism of action of the nanoparticles,
proteomics studies have been performed. These investigations allow for
an understanding of signaling pathways, metabolism, and cellular
responses to the treatment. For this purpose, hepatocellular carcinoma
patient-derived tumor xenograft models were treated with
phosphate-buffered saline or Comp-NPs in the dark as well as upon
irradiation and the up- or down-regulation of specific proteins studies
by mass spectrometry. While in total 4272 differential proteins were
detected, among these 2344 proteins were found in all mice models
(Fig. [184]9a). An analysis of the protein expression levels between
the treatment with phosphate-buffered saline or Comp-NPs in the dark
indicated that 33 proteins were up-regulated and 1 protein was
down-regulated (Fig. [185]9b). Contrary, the comparison of the
expression levels of the xenograft models treated with
phosphate-buffered saline and Comp-NPs in the light showed that 1146
proteins were up-regulated and 31 proteins were down-regulated
(Fig. [186]9c), highlighting the different cellular response upon
exposure to light. For an understanding of the PDT treatment, the
protein expression levels of the mice models treated with Comp-NPs in
the dark or in the light were compared. The results demonstrated that
948 proteins were up-regulated and 12 proteins were down-regulated
(Log[2]FC > 0.8 and p < 0.05, Fig. [187]9d).
Fig. 9. Biochemical mechanism of action of the treatment of multi-drug
resistant patient-derived hepatocellular carcinoma inside a mouse model with
Comp-NPs.
[188]Fig. 9
[189]Open in a new tab
a Venn diagram of the number of proteins detected upon the respective
treatment (n = 3 mice). b–d Volcanic plot of the differences in protein
expression levels inside the animal models upon the respective
treatment. The up-regulated proteins are highlighted in blue,
down-regulated proteins are highlighted in red, and the unchanged
proteins are marked in gray (Log[2]FC > 0.8 and p < 0.05) (n = 3 mice).
Statistical analysis was performed by two-tailed unpaired t test. e
Proteins enrichment GOcircos analysis upon treatment with Comp-NPs and
exposure to irradiation (n = 3 mice). f KEGG pathway enrichment
analysis upon treatment with Comp-NPs and exposure to irradiation
(n = 3 mice). Statistical analysis was performed by two-tailed unpaired
t test. g Positive and negative correlation of differential pathway
analysis upon treatment with Comp-NPs and exposure to irradiation
(n = 3 mice). Statistical analysis was performed by two-tailed unpaired
t test. h Expression of mTOR pathway-related proteins (n = 3 mice).
The signaling pathways involved in the regulation of differential
proteins upon treatment with Comp-NPs and exposure to light were
studied using a GOcircos analysis. The results showed that the p53
mediated signal transduction, cell cycle regulation, cell-substrate
adherens junction, R-SMAD binding, and cell adhesion molecule binding
pathways were miss regulated (Fig. [190]9e). Previous studies have
identified these pathways to be involved in cancer cell growth^[191]64,
adhesion^[192]65, and metastasis^[193]66. The KEGG enrichment analysis
revealed that the treatment with Comp-NPs and exposure to light
strongly influenced the mTOR signaling, eIF4 and p70S6K signaling, eIF2
signaling and RNA signaling pathways (Fig. [194]9f). Of particular
interest is the modification of the mTOR signaling pathway
(Fig. [195]9g, Supplementary Fig [196]38) which is closely related to
tumorigenesis^[197]67, metastasis development and chemotherapy
resistance^[198]68. Further analyses revealed that the mTOR signaling
pathway is influencing the expression of the EIF3 protein family and
RAC2 protein (Fig. [199]9h). An inhibition of the mTOR signaling
pathway correlates to a miss regulation of the expression of the elF4
and P70S6K proteins. Studies have shown that the EIF3 protein family is
associated with the development of metastases^[200]69 and evolution of
tumors by regulation of the cell cycle, RNA synthesis, and DNA
repair^[201]70. RAC2 regulates cell responses, secretion processes and
apoptotic cellular processes^[202]71. For additional verification of
the involvement of the mTOR signaling pathway in the therapeutic
mechanism of the nanoparticles, the expression levels of mTOR and
P70S6K proteins in 4T1 cells was studied using Western Blot. The
results show a strong reduction of mTOR and P70S6K proteins upon
treatment with Comp-NPs and exposure to light (Supplementary
Fig [203]39), highlighting the inhibition of the mTOR signaling
pathway. As such the miss regulation of the mTOR signaling pathway
could present a target for anticancer drug development. Overall, these
results indicate the ability of Comp-NPs upon exposure to light to
interact in various cellular processes. The influence of the mTOR
signaling pathway could present beneficial for a highly efficient
treatment as well as present a mechanism for therapeutic intervention.
The proteomics results indicate that Comp-NPs could therapeutically
intervene inside the mouse model upon inhibition of the mTOR signaling
pathway. To verify these findings, 4T1 tumor-bearing mice models were
treated with Comp-NPs, Comp-NPs and Leucine, or a combination of
Comp-NPs, Leucine and αCD8 (treatment schedule: Fig. [204]10a). Leucine
is an mTOR pathway agonist that upregulates the mTOR protein
expression, thereby activating the mTOR signaling pathway and promoting
tumor growth. αCD8 is a CD8^+ T cell-specific antibody that
specifically binds to CD8^+ T cells in tumorous tissues, preventing
CD8^+ T cells from attacking tumor cells and therefore leading to
immune escape of the tumor cells. Importantly, the mice behave normally
with signs for pain or stress and did not lose or gain any weight,
indicating the high biocompatibility of the treatment (Fig. [205]10c).
The co-administration of Leucine and/or αCD8 with Comp-NPs demonstrated
to reduce the therapeutic efficiency of the treatment (Fig. [206]10b).
Since the co-administration with Leucine reduced the therapeutic
efficacy, it is strongly suggested that Comp-NPs therapeutically
intervenes through inhibition of the mTOR signaling pathway. In
addition, the co-administration with αCD8 further diminished the
therapeutic efficiency indicating that the part of the therapeutic
effect of Comp-NPs is caused by immune activation. For further
verification of reduced immunogenic effect upon co-treatment with
Leucine and αCD8, the spleens of the treated animals were collected at
day 23 and the levels of CD8^+ T cells analyzed by flow cytometry. The
results showed a drastic reduction of CD8^+ T cells from 21% to 7%
(Fig. [207]10d, e). Combined these findings suggest that Comp-NPs
therapeutically interacts inside the mouse model through inhibition of
the mTOR signaling pathway and activates the immune response inside the
animal model.
Fig. 10. PDT properties of Comp-NPs evaluated in a mouse model bearing a 4T1
tumor.
[208]Fig. 10
[209]Open in a new tab
a Schematic illustration of the establishment of the tumor mouse model,
administration and treatment schedules with Comp-NPs (6 mg BODIPY
kg^−1), Leucien, and/or αCD8 in the dark or upon irradiation (650 nm,
0.1 W cm^−2, 60 J cm^−2, 10 min). Phosphate-buffered saline (PBS) was
used as a control. b Mean tumor growth inhibition curves (n = 6 mice)
of the tumor upon treatment. Error bars represent mean ± SD.
Statistical analysis was performed by one-way ANOVA with Tukey’s
multiple comparisons test. c Body weight of the mice (n = 6) upon
treatment. Error bars represent mean ± SD. d, e Analysis of the levels
of CD8^+ T-cells inside the spleens of the treated mouse models (n = 3
mice). Error bars represent mean ± SD. Statistical analysis was
performed by two-tailed unpaired t test. Source data are provided as a
Source Data file.
Discussion
In summary, the development of a theranostic multifunctional
nanomaterial for applications in the diagnosis and treatment of tumors
is presented. The nanoparticles were found to be highly stable under
physiological conditions but quickly dissociate in the presence of ROS.
The incorporation of a photosensitizer into the polymer backbone allows
for the efficient treatment of breast cancer monolayer cells,
multicellular tumor spheroids as well as tumor-bearing mice. The use of
a chromophore irradiation at 808 nm presents the ability to detect
tumorous tissue. Insights into the mechanism of action revealed that
the phototoxic effect of the nanomaterial is caused by immunogenic cell
death in cancer cells as well as the animal model, presenting a
multimodal treatment by localized cellular damage and the inducting of
an immune response of the organism. Based on this combined therapeutic
effect, the nanoparticles were able to strongly reduce the volume of
the primary tumor as well as eradicate tumor metastases within a single
treatment. For a more realistic assessment, the therapeutic properties
of the nanoparticles were investigated towards a multi-drug resistant
clinically challenging hepatocellular carcinoma inside an animal model.
The nanomaterial was found with a strong tumor growth inhibition effect
within a single drug and light doses. Proteomics analyses revealed that
the nanoparticles interact mTOR signaling pathway, which is involved in
tumor evolution and reoccurrence, and in the development of metastases.
We are confident that the here described approach of targeting the mTOR
signaling pathway could present a promising target for anticancer
agents. The ability of the nanoparticles to intervene through the
localized generation of oxidative stress and the induction of an immune
response presents a promising medicinal method to prevent or treat
tumor metastases as well as tumor reoccurrences. The combination of
diagnosis and therapy in a single nanomaterial could represent an
alternative tool for theranostic drug development.
Methods
Ethical Approval
All samples from patients were obtained following written consent and
all experiments were conducted in compliance with the ethical
regulations for human samples. The experiments received approval from
the People’s Liberation Army (PLA) General Hospital and the Clinical
Trial Registry (ChiCTR2100047481). In our study, patient-derived
material has been only used for the generation of the PDX model with
informed consent from the patient. All animal experiments were
conducted in compliance with the ethical regulations for animal testing
and received approval from the Peking University Institutional Animal
Care and Use Committee (LA2021316). The mice were euthanized in
accordance with the animal ethics guidelines of animal welfare
regulations when the tumor volume reached 2000 mm^3. Mice were
euthanized via CO[2] inhalation at the end point of the study.
Materials
Methanol, toluene, dichloromethane, ethyl acetate, tetrahydrofuran and
petroleum ether were purchased from Concord (Tianjin, China).
Methoxyl-poly(ethylene glycol) (mPEG[5000]) and hydrogen peroxide
(H[2]O[2]) were purchased from Energy Chemical (Shanghai, China).
4,9-dibromo-6,7-bis(4-hexylphenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline
(DPTQ6-2Br) was purchased from Alfa (Zhengzhou, China). Cisplatin
(purity 99%) was purchased from Shandong Boyuan Pharmaceutical Co.,
Ltd. (Shandong, China).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and
sodium dodecyl sulfate (SDS) were purchased from Beyotime (Shanghai,
China). Cell culture vessels were purchased from Corning (Corning, NY,
USA). Roswell Park Memorial Institute -1640 (RPMI-1640) medium, fetal
bovine serum (FBS), 0.25% trypsin-EDTA, and penicillin/streptomycin
(P/S) were purchased from Gibco (Gran Island, NY, USA).
2-(4-Amidinophenyl)-1H-indole-6-carboxamidine (DAPI) was purchased from
Sigma-Aldrich (Shanghai, China). 2’,7’-dichlorodihydrofluorescein
diacetate (DCFH-DA), Annexin V-FITC/Propidium iodide (PI) apoptosis
detection kit, Hoechst 33258 and Calcein-AM/PI double stain kit, ATP
assay kit were purchased from Beyotime Institute of Biotechnology
(Jiangsu, China). Female Balb/c mice and female Balb/c nude mice (4–6
weeks) are used in the animal experiments.
Instrumentation and methods
^1H- and ^13C-NMR spectra were recorded on a 300 MHz or 400 MHz NMR
spectrometer (Bruker). Chemical shifts (δ) are reported in parts per
million (ppm) referenced to tetramethylsilane (δ 0.00) ppm using the
residual proton solvent peaks as internal standards. Absorption spectra
were measured on a spectrophotometer (TU-1901). Photofluorescence (PL)
spectra were recorded on a fluorescence spectrometer (FLS980). Gel
permeation chromatography (GPC) measurements were performed on a
GPC-1515 instrument (USA) [Column type: Styragel HT6E^.2, HT2, Column
length: 7.8^. 30 mm, Detector: 2414, pump 1515, Test temperature:
40 °C, flow: 1 mL min^−1, eluent: N,N-dimethylformamide]. Transmission
electron microscopy (TEM) images were recorded on a Hitachi HT-7700
TEM. Dynamic light scattering measurements were performed on a
Zetasizer (Nano ZS, UK).
Synthesis of ethyl (E)-3-(4-(diphenylamino)phenyl)acrylate (2)
4-Diphenylbenzaldehyde (5.46 g, 20 mmol) and
ethoxylmethyltriphenylphosphine (20 mmol) were dissolved in toluene
(60 mL) and stirred at room temperature for 48 h under nitrogen
atmosphere. After this time, the solvent was removed under reduced
pressure. The crude product was purified by chromatography on silica
gel using petroleum ether /dichloromethane (v/v = 10/1) as the eluent.
The fractions containing the product were united and the solvent
removed under reduced pressure. The compound was dried under high
vacuum. Yield: 85%. ^1H-NMR (400 MHz, CDCl[3]): δ = 7.61 (d,
J = 15.9 Hz, 1H), 7.35 (d, J = 8.7 Hz, 2H), 7.28 – 7.24 (m, 5H),7.10 –
7.04 (m, 6H), 6.98 (dd, J = 9.1, 2.2 Hz, 2H), 6.27(d, J = 15.9 Hz,1H),
4.21(q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H) ppm. The obtained
analytic data was found to be in agreement with the previous
literature^[210]72.
Synthesis of ethyl 3-(4-(diphenylamino)phenyl)propanoate (3)
A solution of ethyl (E)-3-(4-(diphenylamino)phenyl)acrylate (6.86 g,
20 mmol) in methanol (60 mL) was suspended with Pt/C (1 g, 5 %) under a
hydrogen atmosphere for 48 h. After this time, the solution was
filtered over celite and the solvent was removed under reduced
pressure. The compound was dried under high vacuum. Yield: 96%. ^1H-NMR
(400 MHz, CDCl[3]), δ 7.24 – 7.19 (m, 4H), 7.09 – 6.95 (m, 10H),4.71
(q, J = 22.0 Hz, 2H) 2.93 (t, J = 7.8 Hz, 2H), 2.63 (t, J = 7.8 Hz,
2H),1.27 (t, J = 14.2 Hz, 3H) ppm. The obtained analytic data was found
to be in agreement with the previous literature^[211]72.
Synthesis of 3-(4-(diphenylamino)phenyl)propan-1-ol (4)
Ethyl 3-(4-(diphenylamino)phenyl)propanoate (6.92 g, 20 mmol) was
dissolved in anhydrous tetrahydrofuran (40 mL) and the solution cooled
down to 0 °C. Three portions of lithium aluminum hydride (1520 mg,
40 mmol) were slowly added over a time period of 1 h. The ice bath was
removed and the mixture was stirred at room temperature for 6 h. Sodium
hydroxide solution (10 %) was added dropwise to the solution until no
gas formation was observed. The solution was then filtered over celite
and the solvent was removed by a rotary evaporator. The crude product
was purified by chromatography on silica gel using petroleum ether
/dichloromethane (v/v = 10/1) as the eluent. The fractions containing
the product were united and the solvent removed under reduced pressure.
The compound was dried under high vacuum. Yield: 95%. ^1H-NMR (400 MHz,
CDCl[3]), δ 7.25 (dd, J = 8.3, 7.6 Hz, 4H), 7.07 - 6.95 (dd,
J = 42.9 Hz, 9H), 3.75 (t, J = 21.8 Hz, 3H), 2.69 – 2.64 (m, 2H),
1.92(dt, J = 13.0, 19.4 Hz, 2H) ppm. The obtained analytic data was
found to be in agreement with the previous literature^[212]72.
Synthesis of 4-(3-((tert-butyldimethylsilyl)oxy)propyl)-N,N-diphenylaniline
(5)
3-(4-(diphenylamino)phenyl)propan-1-ol (3.03 g, 10 mmol),
tert-butyldimethylsilyl chloride (2.25 g, 15 mmol), and imidazole
(1.17 g, 15 mmol) were dissolved dimethylformamide (40 mL) and the
solution was stirred at room temperature for 3 h. After this time,
deionized water (100 mL) was added and the organic phase was extracted
three times with ethyl acetate (3×30 mL). The solvent was removed under
reduced pressure. The crude product was purified by chromatography on
silica gel using petroleum ether /ethyl acetate (v/v = 10/1) as the
eluent. The fractions containing the product were united and the
solvent removed under reduced pressure. The compound was dried under
high vacuum. Yield: 90%. ^1H-NMR (300 MHz, CDCl[3]), δ 7.23 – 7.19 (m,
4H), 7.078– 6.94 (m, 11H), 3.66 (t, J = 6.3 Hz,2H), 2.65 (t,
J = 7.7 Hz, 2H), 1.87 – 1.80 (m, 2H), 0.91 (s, 9H), 0.05 (s, 6H) ppm.
The obtained analytic data was found to be in agreement with the
previous literature^[213]72.
Synthesis of
4-bromo-N-(4-(3-((tert-butyldimethylsilyl)oxy)propyl)phenyl)-N-phenylaniline
(6)
4-(3-((Tert-butyldimethylsilyl)oxy)propyl)-N,N-diphenylaniline (4.18 g,
10 mmol) was dissolved in dichloromethane (60 mL) and the solution
cooled down to 0 °C. Three portions of N-bromsuccinimid (2.25 g,
15 mmol) were slowly added over a time period of 1 h. The ice bath was
removed and the mixture was stirred at room temperature for 4 h. The
solvent was removed under reduced pressure. The crude product was
purified by chromatography on silica gel using petroleum ether /ethyl
acetate (v/v = 10/1) as the eluent. The fractions containing the
product were united and the solvent removed under reduced pressure. The
compound was dried under high vacuum. Yield: 88%. ^1H-NMR (300 MHz,
CDCl[3]), δ 7.26 – 7.15 (dd, J = 31.5, 4H), 7.05 (ddd, J = 30.2, 21.2,
8.6 Hz, 9H), 3.61 (t, J = 6.2 Hz, 2H), 2.61 (t, J = 7.7 Hz, 2H), 1.82 –
1.73 (m, 2H), 0.85 (d, J = 9.9 Hz, 9H), 0.00 (s, 6H) ppm. The obtained
analytic data were found to be in agreement with the previous
literature^[214]72.
Synthesis of
4-(3-((tert-butyldimethylsilyl)oxy)propyl)-N-phenyl-N-(4-(4,4,5,5-tetramethyl
- 1,3,2-dioxaborolan-2-yl)phenyl)aniline (7)
Bis(triphenylphosphine)palladium(II) dichloride(129 mg, 0.175 mmol),
potassium acetate (413 mg, 4.21 mmol), bis(pinacolate)diboron (535 mg,
2.11 mmol) and
4-bromo-N-(4-(3-((tert-butyldimethylsilyl)oxy)propyl)phenyl)-N-phenylan
iline (129 mg, 0.175 mmol) were dissolved in dioxane (20 mL) under an
argon atmosphere. The reaction mixture was heated at 80 °C for 12 h.
After this time, deionized water (40 mL) was added and the organic
phase was extracted three times with ethyl acetate (3×50 mL). The
organic phase was dried over anhydrous magnesium sulfate and the
solvent was removed under reduced pressure. The crude product was
purified by chromatography on silica gel using petroleum ether /ethyl
acetate (v/v = 10/1) as the eluent. The fractions containing the
product were united and the solvent removed under reduced pressure. The
compound was dried under high vacuum. Yield: 60%. ^1H-NMR (400 MHz,
CDCl[3]), δ 7.61(d, J = 8.3 Hz, 2H), 7.19 – 7.17 (m, 2H), 7.04 – 7.02
(d, J = 7.1 Hz, 4H), 6.98 – 6.94 (m, 4H), 3.61-3.58 (m, 2H), 2.59 –
2.57 (m, 2H), 1.82 – 1.75 (d, J = 27.1 Hz, 2H), 1.27 (s, 12H), 0.86 (s,
9H), 0.00 (s, 6H) ppm. The obtained analytic data was found to be in
agreement with the previous literature^[215]72.
Synthesis of
4,4’-(6,7-bis(4-hexylphenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-diyl)bi
s(N-(4-(3-((tert-butyldimethylsilyl)oxy)propyl)phenyl)-N-phenylaniline) (9)
4-(3-((tert-butyldimethylsilyl)oxy)propyl)-N-phenyl-N-(4-(4,4,5,5-tetra
methyl- 1,3,2-dioxaborolan-2-yl)phenyl)aniline (407.5 mg, 0.75 mmol),
4, 9-dibromo-6, 7-bis (4-hexylphenyl)-[1,2,5] thiadiazole and [3,4-g]
quinoxaline (compound 8, 150.0 mg, 0.25 mmol), and
tetrakis-(triphenylphosphin)-palladium (462.2 mg, 0.40 mmol) were
dissolved in toluene (20 mL) under an argon atmosphere. An aqueous
solution of potassium carbonate (1 M, 3 mL) was added and the reaction
mixture was heated at 95 °C for 12 h. After this time, the solution was
cooled down and diluted with water (40 mL). The organic phase was
extracted with ethyl acetate (3×50 mL). The combined organic layers
were washed with water (2×20 mL), dried over anhydrous magnesium
sulfate, and the solvent removed under reduced pressure. The product
was isolated by column chromatography on silica gel using petroleum
ether:ethyl acetate (v/v = 20/1). The fractions containing the product
were united and the solvent was removed under reduced pressure. The
compound was dried under high vacuum. Yield: 50%. ^1H-NMR (300 MHz,
CDCl[3]): δ 7.98-7.95 (d, J = 9.0 Hz, 4H), 7.58-7.55 (d, J = 8.2 Hz,
4H),7.29-7.24 (d, J = 14.5, 8H), 7.22-7.07 (m, 18H), 3.71-3.66 (t,
J = 12.8,4H), 2.69-2.60 (m, 8H), 1.88-1.81 (m, 4H), 1.65- 1.58 (m, 4H),
1.31 (s, 12H), 0.92 (s, 18H), 0.89 (s, 6 H), 0.09 (s, 12H) ppm;
^13C-NMR (100 MHz, CDCl[3]): δ 162.8, 147.0, 143.7, 128.4, 127.2,
124.5, 123.7, 121.9, 120.2, 61.4, 30.7, 28.7, 27.8, 25.0, 21.5, 17.3,
13.1, -6.3 ppm; MALDI-TOF-MS: [M + H]^+ calcd. for
C[86]H[103]N[6]O[2]SSi[2]^+: 1339.7, found: 1339.2.; HR-ESI-MS: calcd.
for [M + H]^+ C[86]H[103]N[6]O[2]SSi[2]^+: 1339.7402, found: 1339.7403.
Synthesis of
4,4’-(6,7-bis(4-hexylphenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-diyl)bi
s(N,N-diphenylaniline) (M1)
4,4’-(6,7-Bis(4-hexylphenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-d
iyl)bis(N-(4-(3-((tert-butyldimethylsilyl)oxy)propyl)phenyl)-N-phenylan
iline) (30 mg, 0.27 mmol) and Amberlyst 15 (100 mg) were suspended in
dichloromethane (15 mL) and the mixture was stirred at room temperature
for 3 h. After this time, the solid was removed by filtration. The
solvent of the filtrate was removed under reduced pressure. The product
was isolated by column chromatography on silica gel using
dichloromethane: methanol (v/v = 20/1). The fractions containing the
product were united and the solvent was removed under reduced pressure.
The compound was dried under high vacuum. Yield: 40%. ^1H-NMR (300 MHz,
CDCl[3]): δ 7.98-7.96 (d, J = 8.4 Hz, 4H), 7.60-7.57 (d, J = 8.0 Hz,
4H), 7.30 (s, 8H), 7.18-7.12 (t, J = 18.3 Hz, 16H), 3.76-3.72 (t,
J = 12.5 Hz, 4H), 2.66-2.61 (t, J = 15.5 Hz, 4H), 1.99-1.90 (m, 4H),
1.63- 1.61 (d, J = 14.4 Hz,4H), 1.31-1.26 (d, J = 18.8 Hz, 16H),
0.89-0.87 (t, J = 14.0 Hz, 6H); ^13C-NMR (75 MHz, CDCl[3]):153.0,
143.4, 134.2, 134.1, 130.0, 128.3, 104.3, 62.4, 34.3, 31.5, 29.7, 29.0,
22.7, 14.2; MALDI-TOF-MS: [M + H]^+ calcd. for
C[74]H[75]N[6]O[2]S^+:1112.5, found: 1112.2. HR-ESI-MS: calcd. for
[M + H]^+ C[74]H[75]N[6]O[2]S^+: 1111.5672, found: 1111.5667.
Synthesis of
5,5-difluoro-1,3,7,9-tetramethyl-10-phenyl-5H–4λ^4,5λ^4-dipyrrolo[1,2-c:2’,1’
-f][1,3,2]diazaborinine (12)
Benzaldehyde (0.32 g, 3.0 mmol), 2,4-dimethylpyrrole (0.63 g,
6.6 mmol), and several drops of trifluoroacetic acid were dissolved in
tetrahydrofuran (90 mL). The mixture was stirred at room temperature
overnight. After this time, a solution of
2,3-dichloro-5,6-dicyano-p-benzoquinone (0.68 g, 3.0 mmol) in
tetrahydrofuran (120 mL) was added and the mixture was stirred for
another 4 h. After this time, a solution of bortrifluoriddiethyletherat
(18 mL, 0.15 mol) in diisopropylethylamine (22 mL, 0.13 mol) was added
dropwise to the mixture. The mixture was stirred at room temperature
overnight. After this time, undissolved solids were removed upon
filtration over celite. The solvent was removed under reduced pressure.
The residue was redissolved in dichloromethane (100 mL) and the
solution was washed with 5% aqueous sodium bicarbonate solution
(100 mL) and water (2 × 100 mL). The organic phase was dried over
anhydrous magnesium sulfate. The crude product was purified by
chromatography on silica gel using dichloromethane as the eluent. The
fractions containing the product were united and the solvent removed
under reduced pressure. The compound was dried under high vacuum.
Yield: 43%. ^1H-NMR (300 MHz, DMSO-d[6]): δ 7.57 (s, 3H), 7.38 (s, 2H),
6.17 (s, 2H), 2.45 (s, 6H), 1.34 (s, 6H). The obtained analytic data
was found to be in agreement with the previous literature^[216]73.
Synthesis of
5,5-difluoro-2,8-diiodo-1,3,7,9-tetramethyl-10-phenyl-5H-4λ^4,5λ^4-dipyrrolo[
1,2-c:2’,1’-f][1,3,2]diazaborinine (13)
5,5-Difluoro-1,3,7,9-tetramethyl-10-phenyl-5H--4λ^4,5λ^4-dipyrrolo[1,2-
c:2’,1’f][1,3,2]diazaborinine (0.32 g, 1.0 mmol) and iodine (0.63 g,
2.5 mmol) were dissolved in ethanol (25 mL). A solution of iodic acid
(0.35 g, 2 mmol) in water (5 mL) was added dropwise. The mixture was
heated at 60 °C for 2 h. After this time, undissolved solids were
removed upon filtration over celite. The solvent was removed under
reduced pressure. The crude product was purified by chromatography on
silica gel using dichloromethane/ethyl acetate (v/v = 2/1) as the
eluent. The fractions containing the product were united and the
solvent removed under reduced pressure. The compound was dried under
high vacuum. Yield: 88%. ^1H-NMR (300 MHz, DMSO-d[6]): δ 7.62 (s, 3H),
7.42 (s, 2H), 2.56 (s, 6H), 1.34 (s, 6H). The obtained analytic data
was found to be in agreement with the previous literature^[217]54.
Synthesis of
2,2’-((((1E,1’E)-(5,5-difluoro-2,8-diiodo-1,9-dimethyl-10-phenyl-5H-4λ^4,5λ^4
-dipyrrolo[1,2-c:2’,1’-f][1,3,2]diazaborinine-3,7-diyl)bis(ethene-2,1-diyl))b
is(4,1-phenylene))bis(oxy))bis(ethan-1-ol) (M2)
5,5-Difluoro-2,8-diiodo-1,3,7,9-tetramethyl-10-phenyl-5H-4λ^4,5λ^4-dipy
rrolo[1,2-c:2’,1’-f][1,3,2]diazaborinine (13) (0.17 g, 0.3 mmol),
glacial acetic acid (0.6 mL, 10.5 mmol),
4-(2-hydroxyethoxy)benzaldehyde (0.1 g, 0.6 mmol), and piperidine
(0.8 mL, 8.0 mmol) were dissolved in toluene (40.0 mL). The mixture was
heated at reflux for 2 h. During the reaction the generated water was
azeotropically removed using a Dean-Stark apparatus. After this time,
the solvent was removed under reduced pressure. The crude product was
purified by chromatography on silica gel using chloroform/methanol
(v/v = 50/1) as the eluent. The fractions containing the product were
united and the solvent removed under reduced pressure. The compound was
dried under high vacuum. Yield: 30%. ^1H-NMR (300 MHz, DMSO-d[6]): δ
8.11-8.05(d, 2H), 7.61-7.43(m, 7H), 7.46-7.41(d, 4H), 7.08-7.06(d, 4H),
4.93-4.89(t, 2H), 4.06-4.03(t, 4H), 3.77-3.72(q, 4H), 1.41(s, 6H). The
obtained analytic data was found to be in agreement with the previous
literature^[218]54.
Synthesis of P1
M1 (10 mg, 0.009 mmol), L-lysine diisocyanate (33.52 mg, 0.171 mmol),
and 2,2´-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol) (40.7 mg,
0.18 mmol) were suspended in anhydrous N,N-dimethylformamide (5 mL) for
24 h. After this time, mPEG[5000] (100 mg, 0.02 mmol) was added to the
reaction mixture. After this time, the reaction mixture was dialyzed
against N,N-dimethylformamide for 24 h (MWCO: 7000 Da) to remove the
unreacted monomers and oligomers. The obtained solution was
freeze-dried under reduced pressure to obtained polymer as a powder P1.
Synthesis of P2
M2 (7.85 mg, 0.009 mmol), L-lysine diisocyanate (33.52 mg, 0.171 mmol),
and 2,2´-(propane-2,2-diylbis(sulfanediyl))bis(ethan-1-ol) (40.7 mg,
0.18 mmol) were suspended in anhydrous N,N-dimethylformamide (5 mL) for
24 h. After this time, mPEG[5000] (100 mg, 0.02 mmol) was added to the
reaction mixture. After this time, the reaction mixture was dialyzed
against N,N-dimethylformamide for 24 h (MWCO: 7000 Da) to remove the
unreacted monomers and oligomers. The obtained solution was
freeze-dried under reduced pressure to obtained polymer as a powder P2.
Preparation of NP1
P1 (10.0 mg) was dissolved in N,N-dimethylformamide (2 mL) and the
solution was added dropwise into deionized water (5 mL) under
continuous stirring. After 10 min, the reaction mixture was dialyzed
against water for 48 h (MWCO: 7000 Da) to remove the organic solvent.
The nanoparticle solution was concentrated by centrifugation at
3000 rpm for 15 min and afterwards filtrated through a 0.22 μm PES
syringe driven filter (Sartorius). The nanoparticle solution was stored
at 4 °C.
Preparation of NP2
P2 (10.0 mg) was dissolved in N, N-dimethylformamide (2 mL) and the
solution was added dropwise into deionized water (5 mL) under
continuous stirring. After 10 min, the reaction mixture was dialyzed
against water for 48 h (MWCO: 7000 Da) to remove the organic solvent.
The nanoparticle solution was concentrated by centrifugation at
3000 rpm for 15 min and afterwards filtrated through a 0.22 μm PES
syringe driven filter (Sartorius). The nanoparticle solution was stored
at 4 °C.
Preparation of Comp-NPs
NP1 and NP2 were mixed in a 1:1 (P1 and P2 polymer mass concentrations)
to form the nanoparticle formulation Comp-NPs.
Singlet oxygen generation measured by absorption spectroscopy
Comp-NPs (2 μg/mL BODIPY) and 1,3-diphenylisobenzofuran (0.02 mM) were
dissolved in water. The solutions were aerated and irradiated (650 nm,
0.1 W cm^−2) using different time intervals. The absorbance of the
samples at 410 nm was measured during these time intervals with a
TU-1901 absorption spectrometer.
Identity of ROS species
Comp-NPs (2 μg/mL BODIPY) was incubated in phosphate-buffered saline
containing 20 mM 2,2,6,6–tetramethylpiperidine or 20 mM
5,5-dimethyl-1-pyrroline N-oxide. Capillary tubes were filled with the
solution and sintered by fire. The samples were kept in the dark or
exposed to irradiation with a 650 nm laser (0.1 W cm^–2) for 10 min.
The electron spin resonance spectrum was recorded with an EPR
spectrometer (A3000, BRUKER).
Fluorescence quantum yield of Comp-NPs
The fluorescence quantum yield of Comp-NPs was determined using the
fluorophore IR dye 26 as a reference. The optical absorbance was
measured for both a Comp-NPs suspension and an IR dye 26 solution at
808 nm. Then their fluorescence emission intensities were measured
under the same 808 nm excitation. Photofluorescence (PL) spectra were
recorded on a fluorescence spectrometer (FLS980) in the 900–1700 nm
region. Using the measured optical density (OD) and spectrally
integrated fluorescence intensity (F), one can calculate the quantum
yield of Comp-NPs according to the following formula:
[MATH: Φxλ=Φstλ⋅
FxFst⋅Astλ
Axλ=Φstλ⋅
FxFst
⋅1−10−ODstλ1−10−
ODxλ
=0.5%⋅678713.5cps5690769.5cps⋅
1−10−2.535
51−10
−0.1477=2.0% :MATH]
The fluorescence quantum yield of Comp-NPs was determined to be 2.0%
upon excitation at 808 nm.
H[2]O[2] sensitivity of the polymers measured by GPC
P1 and P2 (7 mg/mL) were dissolved in N,N-dimethylformamide. Hydrogen
peroxide (10 mM) was added to the solution and the polymers were
analyzed by gel permeation chromatography.
Stability of the nanoparticles observed by phosphorescence spectroscopy
The dye Nile Red (20 μg/mL) was encapsuled with Comp-NPs (2 μg/mL
BODIPY) and the nanoparticles dissolved in water. The solution was
exposed upon irradiation (650 nm, 0.1 W cm^−2) and the phosphorescence
spectra (600–750 nm) time-dependently (0–60 min) monitored with a
SpectraMax microplate reader.
H[2]O[2] sensitivity of the nanoparticles measured by GPC
NP1 (10 mg/mL P1), NP2 (10 mg/mL P2) and Comp-NPs (5 mg/mL P1 and
5 mg/mL P2) were dissolved in water. H[2]O[2] (10 mM) was added to the
solution and the nanoparticles were analyzed by TEM.
Cell culture
The mouse breast cancer (4T1), human ovarian cancer (SKOV3), cisplatin
sensitive ovarian cancer (A2780) and cisplatin-resistant ovarian cancer
(A2780DDP) cells were purchased from the American Type Culture
Collection (Manassas, VA, USA). The 4T1, A2780, A2780DDP cells were
cultivated in RPMI-1640 media. The SKOV3 cells were cultivated in DMEM
media. The JHH7 cells were cultivated in DMEM media. The cell lines
were complemented with 10% of fetal calf serum and a100 U/mL
penicillin-streptomycin mixture. The cells were cultivated in a
humidified atmosphere at 37 °C with 5% of CO[2] and 21% O[2]. All cell
lines were verified to be free of mycoplasma.
Cellular uptake measured by confocal laser scanning microscopy
4T1 cells were incubated with the nanoparticles (1 μg/mL BODIPY) for
varying time points (1, 4, 7 h) at 37 °C. The cells were incubated with
the fluorescent probe Alexa Fluor® 488 phalloidin (Thermo Fisher
Scientific) as a stain for cell actin cytoskeleton for 30 min at 37 °C
in the dark. The cells were washed three times with PBS. Confocal
images were taken with a CLSM (LSM-800, ZEISS, Germany). Comp-NPs:
λ[ex] = 650 nm, λ[em] = 745 nm, Alexa Fluor 488: λ[ex] = 495 nm,
λ[em] = 519 nm.
Cellular uptake measured by flow cytometry
4T1 cells were incubated with the nanoparticles (1 μg/mL BODIPY) for
varying time points (1, 4, 7 h) at 37 °C. The cells were washed three
times with PBS. The cellular uptake was measured with a flow cytometry
(Cytomics FC500, Beckman). Comp-NPs: λ[ex] = 650 nm, λ[em] = 745 nm,
Alexa Fluor 488: λ[ex] = 495 nm, λ[em] = 519 nm.
Generation of multicellular tumor spheroids
A suspension of 0.75% agarose in PBS was heated inside a high-pressure
autoclave. The hot emulsion was transferred into a 96 well plate (50 μL
per well). The plates were exposed for 3 h to UV irradiation and
allowed to cool down. After this time, a cell suspension of 3^.10^3
cells was seeded on top of the agarose ground layer. Within two-three
days MCTS were formed from the cell suspension. The multicellular tumor
spheroids were cultivated and maintained at 37 °C in a cell culture
incubator at 37 °C with 5% CO[2] atmosphere. The culture medium with
serum was replaced every two days. The formation, integrity, and
diameter of the multicellular tumor spheroids was monitored with a
CLSM.
Penetration of multicellular tumor spheroids measured by z-stack confocal
laser scanning microscopy
MCTS with a diameter of ~700 μm were treated with Comp-NPs (1 μg/mL
BODIPY) by replacing 50% of the media with drug supplemented media in
the dark. The MCTS was incubated with the nanoparticles for 7 h at
37 °C in the dark. After this time, the MCTS was washed three times
with media. Z-stack CLSM images were taken with a microscopy (LSM-800,
ZEISS, Germany). Comp-NPs: λ[ex] = 650 nm, λ[em] = 745 nm.
Light induced cellular singlet oxygen generation in multicellular tumor
spheroids by confocal laser scanning microscopy
MCTS with a diameter of ~700 μm were treated with Comp-NPs (1 μg/mL
BODIPY) by replacing 50% of the media with drug supplemented media in
the dark. The MCTS was incubated with the nanoparticles for 7 h at
37 °C in the dark. The MCTS was further incubated with the ROS specific
probe 2´,7´-dichlorofluorescein diacetate for 30 min. After this time,
the MCTS was washed three times with media. CLSM images were taken with
a microscopy (LSM-800, ZEISS, Germany). Comp-NPs: λ[ex] = 650 nm,
λ[em] = 745 nm, 2´,7´-dichlorofluorescein diacetate: λ[ex] = 488 nm,
λ[em] = 520 nm.
Light induced cellular singlet oxygen generation in 4T1 cells by flow
cytometry
4T1 cells were treated with Comp-NPs (0.5 μg/mL BODIPY) by replacing
50% of the media with drug supplemented media in the dark. The cells
was incubated with the nanoparticles for 7 h at 37 °C in the dark. The
cells were further incubated with the ROS specific probe
2´,7´-dichlorofluorescein diacetate for 30 min. After this time, the
cells were washed three times with media. The ROS production was
measured with a flow cytometry (Cytomics FC500, Beckman). Comp-NPs:
λ[ex] = 650 nm, λ[em] = 745 nm, 2´,7´-dichlorofluorescein diacetate:
λ[ex] = 488 nm, λ[em] = 520 nm.
Cytotoxicity
8 × 10^3 cells were seeded on 96 well plates and allowed to adhere
overnight. The cells were treated with increasing concentrations of the
sample diluted in cell media achieving a total volume of 200 μL. The
cells were incubated with increasing concentrations of NP1, NP2,
Comp-NPs or cisplatin for 24 h and after this time the medium with
serum refreshed. To study the phototoxic effect, the cells were exposed
to irradiation (650 nm, 0.1 W cm^−2, 60 J cm^−2, 10 min). To study the
dark cytotoxicity effect, the cells were not irradiated and after the
incubation time the medium with serum exchanged. The cells were grown
for an additional 48 h at 37 °C. After this time, the medium with serum
was replaced with fresh medium containing
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT,
10 µL of a 5 mg/mL solution in PBS buffer) and the cells further
incubated for 4 h. Acidified SDS solution was then added (100 µL/well)
and the plates kept in the dark for an additional 12 h. Absorption
measurements were performed on a Bio-Rad plate reader at 570 nm (peak
absorbance) and at 650 nm (background absorbance).
Apoptotic cell death
4T1 cells were incubated with the nanoparticles NP1, NP2, or Comp-NPs
(0.5 μg/mL BODIPY) for 12 h at 37 °C and after this time the medium
with serum refreshed. To study the phototoxic effect, the cells were
exposed to irradiation (650 nm, 0.1 W cm^−2, 60 J cm^−2, 10 min). To
study the dark cytotoxicity effect, the cells were not irradiated and
after the incubation time the medium with serum exchanged. The cells
were grown for an additional 12 h at 37 °C. After this time,
FITC-Annexin V/Propidium Iodide as apoptosis markers were incubated for
15 min. The cell death was assessed with a flow cytometer.
Detection of ICD hallmarks – translocation of CRT by confocal laser scanning
microscopy
The cells were seeded on 24-well plates (Thermo Scientific, USA) at a
density of 1 × 10^4 cells per well. After 24 h, the medium with serum
was removed and the cells were incubated with the nanoparticles
(0.5 μg/mL BODIPY) diluted in cell media for 6 h. The cells were washed
with phosphate-buffered saline and the cells further incubated with
anti-CRT antibody (Abcam, ab92516, 1:50) for 2 h at 4 °C. Subsequently,
the cells were incubated with Alexa Fluor 488-conjugated secondary
antibody (Abcam, ab150077, 1:500) for 1 h. The cells were exposed to
irradiation (650 nm, 0.1 W cm^−2, 2 min). The translocation of CRT
inside the cells was assessed using CLSM (LSM-800, ZEISS, Germany).
Nanoparticles: λ[ex] = 638 nm, λ[em] = 747 nm, DAPI: λ[ex] = 410 nm,
λ[em] = 506 nm, Alexa Fluor 488-conjugated CRT specific antibody:
λ[ex] = 488 nm, λ[em] = 525 nm.
Detection of ICD hallmarks—translocation of CRT by flow cytometry
The cells were seeded on 24-well plates (Thermo Scientific, USA) at a
density of 1 × 10^4 cells per well. After 24 h, the medium with serum
was removed and the cells were incubated with the nanoparticles
(0.5 μg/mL BODIPY) diluted in cell media for 6 h. The cells were washed
with phosphate-buffered saline and the cells further incubated with
anti-CRT antibody (Abcam, ab92516, 1:50) for 2 h at 4 °C. Subsequently,
the cells were incubated with Alexa Fluor 488-conjugated secondary
antibody (Abcam, ab150077, 1:500) for 1 h. The cells were exposed to
irradiation (650 nm, 0.1 W cm^−2, 2 min). The translocation of CRT
inside the cells was assessed by flow cytometry. Nanoparticles:
λ[ex] = 638 nm, λ[em] = 747 nm, DAPI: λ[ex] = 410 nm, λ[em] = 506 nm,
Alexa Fluor 488-conjugated CRT specific antibody: λ[ex] = 488 nm,
λ[em] = 525 nm.
Detection of ICD hallmarks – secretion of adenosine triphosphate
The cells were seeded on 24-well plates (Thermo Scientific, USA) at a
density of 1 × 10^4 cells per well. After 24 h, the medium with serum
was removed and the cells were incubated with the nanoparticles
(0.5 μg/mL BODIPY) diluted in cell media for 6 h. The cells were washed
with phosphate-buffered saline. The cells were exposed to irradiation
(650 nm, 0.1 W cm^−2, 2 min). The release of adenosine triphosphate in
the cell supernatant was detected by an adenosine triphosphate assay
kit following the manufacturer’s protocols.
Detection of ICD hallmarks—secretion of nuclear HMGB1 protein
The cells were seeded on 24-well plates (Thermo Scientific, USA) at a
density of 1 × 10^4 cells per well. After 24 h, the medium with serum
was removed and the cells were incubated with the nanoparticles
(0.5 μg/mL BODIPY) diluted in cell media for 6 h. The cells were washed
with phosphate-buffered saline and the cells further incubated with
anti- nuclear HMGB1protein antibody (Abcam, ab18256, 1:50) for 2 h at
4 °C. Subsequently, the cells were incubated with Alexa Fluor
488-conjugated secondary antibody (Abcam, ab150077, 1:500) for 1 h. The
cells were exposed to irradiation (650 nm, 0.1 W cm^−2, 2 min). The
secretion of nuclear HMGB1protein inside the cells was assessed using
CLSM (LSM-800, ZEISS, Germany). DAPI: λ[ex] = 410 nm, λ[em] = 506 nm,
nuclear HMGB1protein specific antibody: λ[ex] = 488 nm, λ[em] = 525 nm.
Cell migration wound healing assay
4T1 cells were seeded into 35 mm dishes at a density of 6 × 10^5 cells
per well. After 24 h, the cells were incubated with the nanoparticles
(0.5 μg/mL BODIPY) diluted in cell media with serum for 6 h. To study
the phototoxic effect, the cells were exposed to irradiation (650 nm,
0.1 W cm^−2, 60 J cm^−2, 10 min). After 12 h, the medium was removed,
the cells were washed twice with phosphate-buffered saline, and the
cells were seeded on a new cell culture plate. Upon reaching confluence
a linear wound was generated in the middle of the plate with a 200 μL
micropipette tip. After 12 h, light microscopy images were recorded.
Western blot assay
4T1 cells were incubated with the nanoparticles NP1, NP2, or Comp-NPs
(0.5 μg/mL BODIPY) for 12 h at 37 °C and after this time the medium
with serum refreshed. To study the phototoxic effect, the cells were
exposed to irradiation (650 nm, 0.1 W cm^−2, 60 J cm^−2, 10 min). To
study the dark cytotoxicity effect, the cells were not irradiated and
after the incubation time the medium with serum exchanged. The cells
were grown for an additional 12 h at 37 °C. After this time, The cells
were then lysed in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 nM
NaCl, 1%NP-40, 0.1% SDS) including 1 mM of phenylmethanesulfonyl
fluoride (PMSF). The protein lysate was obtained by centrifugation at
12000 g for 15 min at 4 °C. Then, the lysates were denatured at 100 °C
and resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The
gels were blotted onto PVDF membrane (Merck Millipore) and blocked by
5% BSA buffer for 2 h at room temperature. After incubated with the
indicated primary antibodies (anti-caspase-3 antibody, ab 184787,
1:1000; anti-β-tubulin antibody, ab78078, 1:1000, anti-β-actin
antibody, ab8226, 1:1000; anti-P70 S6 Kinase (phosphor T389) antibody,
ab2571, 1:1000; anti-mTOR antibody, ab32028, 1:1000) at 4 °C overnight,
the membrane was washed several times with TBST buffer, incubated with
corresponding secondary antibody (Peroxidase-Conjugated Goat
Anti-Rabbit IgG (H + L), CAT:33101ES60, 1:5000 and
Peroxidase-Conjugated Goat Anti-Mouse IgG (H + L), CAT:33201ES60,
1:5000) in 5% BSA solution for 1 h at room temperature, washed several
times with TBST buffer, and then imaged withBio-Rad Chemi Doc XRS
imaging system.
Establishment of 4T1 breast mouse model
1 × 10^6 4T1 cells were dispersed in 150 µL of PBS and subcutaneously
implanted into BALB/c female mice (6 weeks) to establish a
tumor-bearing model. After a week, the tumor volumes of the mice
reached approximately 100 mm^3.
Biodistribution in living 4T1 breast mouse model determined by
phosphorescence imaging
The female Balb/c mice mice were intravenously injected in the tail
vein with Comp-NPs (6 mg BODIPY kg^−1). The phosphorescence was
recorded on an In Vivo Imaging System (IVIS, Perkin Elmer). after
0.1 h, 1 h, 12 h, 24 h, 48 h. λ[ex] = 650 nm, λ[em] = 735 nm;
λ[ex] = 808 nm, λ[em] = 950 nm.
Biodistribution in scarified 4T1 breast mouse model determined by
phosphorescence imaging
The female Balb/c nude mice were intravenously injected in the tail
vein with Comp-NPs (6 mg BODIPY kg^−1). 48 h after injection the animal
model were scarified and the biodistribution determined by recording of
the phosphorescence of the respective tissues with an IVIS spectrum
imaging system (Spectrum CT, PerkinElmer). λ[ex] = 650 nm,
λ[em] = 735 nm.
Tumor growth inhibition in 4T1 breast mouse model
Twenty tumor-bearing female Balb/c mice (6 weeks) were randomly
separated into 4 groups, resulting in five mice for each group. The
mice were intravenously injected in the tail vein with Comp-NPs (6 mg
BODIPY kg^−1). After 24 h, the mice were anaesthetized and fixed in a
warm three-axes holder. Before irradiation, the tumor site was
disinfected with ethanol. The tumor was exposed to irradiation (650 nm,
0.1 W cm^−2, 60 J cm^−2, 10 min). Following irradiation, the operative
area was disinfection by iodophor. The tumor volume and body weight
were measured and recorded every two days. Tumor volume was calculated
by the following formula: Volume = (Length * Width^2)/2.
Quantification of biochemical markers upon treatment of 4T1 breast mouse
model
Following the previously described treatment, the levels of the
biochemical markers ALT, AST, BUN and CR inside the animal models were
analyzed.
Histological examination of 4T1 breast mouse model
The major organs and tumors were collected and a slice of each one was
fixated with 4% paraformaldehyde. The obtained slices were stained with
hematoxylin and eosin. The histological images were taken using a CLSM
(LSM-800, ZEISS, Germany).
Detection of ICD hallmarks in tumor slices—translocation of calreticulin
(CRT)
Following the previously described treatment, tumor slices from the
respective animals were collected. The tissue was incubated with
anti-CRT antibody (Abcam, ab92516, 1:50) for 2 h. Subsequently, the
tissue was incubated with Alexa Fluor 594-conjugated secondary antibody
(Abcam, ab150080, 1:500) for 1 h. The translocation of CRT inside the
cells was assessed using CLSM (LSM-800, ZEISS, Germany). DAPI:
λ[ex] = 410 nm, λ[em] = 506 nm, Alexa Fluor 488-conjugated CRT specific
antibody: λ[ex] = 590 nm, λ[em] = 617 nm.
Detection of ICD hallmarks in tumor slices – secretion of nuclear
high-mobility group box 1 (HMGB1) protein
Following the previously described treatment, tumor slices from the
respective animals were collected. The tissue was incubated with
anti-nuclear HMGB1 protein antibody (Abcam, ab18256, 1:50) for 2 h.
Subsequently, the tissue was incubated with Alexa Fluor 594-conjugated
secondary antibody (Abcam, ab150080, 1:500) for 1 h. The secretion of
nuclear HMGB1 protein inside the cells was assessed using CLSM
(LSM-800, ZEISS, Germany). DAPI: λ[ex] = 410 nm, λ[em] = 506 nm,
nuclear HMGB1 protein specific antibody: λ[ex] = 590 nm,
λ[em] = 617 nm.
Detection of ICD hallmarks in tumor slices—detection of CD8^+ T-cells
Following the previously described treatment, tumor slices from the
respective animals were collected. The tissue was incubated with CD8^+
T cells specific fluorescent probe. The presence of CD8^+ T cells were
assessed using CLSM (LSM-800, ZEISS, Germany). DAPI: λ[ex] = 410 nm,
λ[em] = 506 nm, CD8^+ T-cells fluorescent probe: λ[ex] = 488 nm,
λ[em] = 525 nm.
Detection CD and DC cells in tumor slices
Fresh tumors and draining lymph node tissue were collected for
antitumor immune response analysis via FACS. Briefly, samples were
dissociated into single-cell suspensions, and then red blood cells were
removed with red blood cell lysing buffer (Beyotime). After that,
samples were blocked with 0.1% BSA in PBS followed by incubation with
relevant antibodies for 1 h at room temperature. For characterizing T
cells and DC cells in tumor, cells were stained by anti-CD3-PE
(elabscience, E-AB-F1013D, 1:100), anti-CD4-PC5.5 (elabscience,
E-AB-F1097J, 1:100), anti-CD8-FITC (elabscience, E-AB-F1104C, 1:100),
anti-mouse IFN-γ (biolegend, 505810.1:00), anti-CD11c-PE (elabscience,
E-AB-F0991D, 1:100), anti-CD80-FITC (elabscience, E-AB-F0992C, 1:100)
and anti-CD86-APC (elabscience, E-AB-F0994C, 1:100). For analyzing DCs
in tumor and lymph nodes, cells were stained by anti-CD11c-PE,
anti-CD80-FITC, and anti-CD86-APC. All the antibodies used above were
all purchased from elabscience. Flow cytometric data acquisition was
performed with CytExpert software, and the data were processed using
FlowJo software. Data were expressed as mean ± SD (n = 3).
Establishment of a patient-derived mouse xenograft model
For patient-derived xenograft models, fresh carcinoma blocks about 2
mm^3 were subcutaneously transplanted into the right flank of the
BALB/c nude mice. The mice were used for experiments when the tumor
volume reached approximately 100 mm^3.
Tumor growth inhibition in patient-derived mouse xenograft model
20 nude tumor-bearing nude mice were randomly separated into 4 groups,
resulting in five mice for each group. The mice were intravenously
injected in the tail vein with Comp-NPs (6 mg BODIPY kg^−1). After
24 h, the mice were anaesthetized and fixed in a warm three-axes
holder. Before irradiation, the tumor site was disinfected with
ethanol. The tumor was exposed to irradiation (650 nm, 0.1 W cm^−2,
60 J cm^−2, 10 min). Following irradiation, the operative area was
disinfection by iodophor. The tumor volume and body weight were
measured and recorded every two days. Tumor volume was calculated by
the following formula: Volume = (Length * Width^2)/2.
Histological examination of patient-derived mouse xenograft model
The major organs and tumors were collected and a slice of each one was
fixated with 4% paraformaldehyde. The obtained slices were stained with
hematoxylin and eosin stain or as terminal deoxynucleotidyl transferase
dUTP nick end labeling stain. The histological images were taken using
a CLSM (LSM-800, ZEISS, Germany).
Proteomics analysis
Nine nude hepatocellular carcinoma patient-derived tumor-bearing mice
were randomly separated into 3 groups, resulting in 3 mice for each
group. The mice were intravenously injected in the tail vein with
Comp-NPs (6 mg BODIPY kg^−1). After 24 h, the tumor was exposed to
irradiation (650 nm, 0.1 W cm^−2, 60 J cm^−2, 10 min). The tumors were
collected and incubated in the absence and the presence of 10 μM PDS,
respectively, at 37 °C for 24 h. The cells were then individually
harvested, lysed on ice and extracted whole cell proteins by total
protein extraction kit (BestBio). The concentration of raw protein
extracts was measured by BCA Kit (Beyotime). The extracted proteins
were digested and stable isotopic labeled. The labeled peptide mixtures
were pre-fractionated by HPLC (Agilent Technologies 1260 infinity) with
Agilent ZORBAX 300 Extend-C18 column. Mass spectrometric quantification
was performed on an Orbitrap Fusion Lumos mass spectrometer coupled
with an EASY-nLC 1200 nanoUPLC system equipped with an Acclaim™ PepMap™
100 pre-column (20 mm × 75 μm, 3 μm) and an Acclaim™ PepMap™ RSLC C18
analytical column (150 mm × 75 μm, 2 μm). Raw MS/MS data were searched
in Proteome Discoverer (Thermo Scientific, version 2.3) database for
peptide and protein identification.
Statistics and reproducibility
All statistical analyses were performed using GraphPad Prism. Data were
analyzed using unpaired two-tailed t test, one-way ANOVA with Tukey’s
multiple comparisons test and two-way ANOVA with Šídák’s multiple
comparisons test for the calculation of P values. The number of
replicates performed is indicated in each figure legend, where
applicable.
Reporting summary
Further information on research design is available in the [219]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[220]Supplementary Information^ (4.8MB, pdf)
[221]Reporting Summary^ (317.6KB, pdf)
Source data
[222]Source Data^ (1.5MB, xlsx)
Acknowledgements