Biodegradable hollow mesoporous organosilica nanotheranostics  (HMON) for multi-mode imaging and mild photo-therapeutic-induced mitochondrial damage on gastric cancer

doi:10.21203/rs.3.rs-22585/v2

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Biodegradable hollow mesoporous organosilica nanotheranostics (HMON) for multi-mode imaging and mild photo-therapeutic-induced mitochondrial damage on gastric cancer

https://doi.org/10.21203/rs.3.rs-22585/v2

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Journal Publication

published 20 Jul, 2020

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Background: CuS-modified hollow mesoporous organosilica nanoparticles ([email protected]) have been preferred as non-invasive treatment for cancer, as near infrared (NIR)-induced photo-thermal effect (PTT) and/or photo-dynamic effect (PDT) could increase cancer cells’ apoptosis. However, the certain role of [email protected]&PDT inducing gastric cancer (GC) cells’ mitochondrial damage, remained unclear. Moreover, theranostic eﬃciency of [email protected] might be well improved by applying multi-modal imaging, which could offer an optimal therapeutic region and time window. Herein, new nanotheranostics agents were reported by Gd doped HMON decorated by CuS nanocrystals (called [email protected]/Gd).

Results: [email protected]/Gd exhibited appropriate size distribution, good biocompatibility, L-Glutathione (GSH) responsive degradable properties, high photo-thermal conversion eﬃciency (82.4%) and a simultaneous reactive oxygen species (ROS) generation effect. Meanwhile, [email protected]/Gd could efficiently enter GC cells, induce combined mild PTT (43-45 °C) and PDT under mild NIR power density (0.8W/cm²). Surprisingly, it was found that PTT might not be the only factor of cell apoptosis, as ROS induced by PDT also seemed playing an essential role. The NIR-induced ROS could attack mitochondrial transmembrane potentials (MTPs), then promote mitochondrial reactive oxygen species (mitoROS) production. Meanwhile, mitochondrial damage dramatically changed the expression of anti-apoptotic protein (Bcl-2) and pro-apoptotic protein (Bax). Since that, mitochondrial permeability transition pore (mPTP) was opened, followed by inducing more cytochrome c (Cyto C) releasing from mitochondria into cytosol, and finally activated caspase-9/caspase-3-depended cell apoptosis pathway. Our in vivo data also showed that [email protected]/Gd exhibited good fluorescence (FL) imaging (wrapping fluorescent agent), enhanced T1 imaging under magnetic resonance imaging (MRI) and infrared thermal (IRT) imaging capacities. Guided by FL/MRI/ IRT trimodal imaging, [email protected]/Gd could selectively cause mild photo-therapy at cancer region, efficiently inhibit the growth of GC cells without evident systemic toxicity in vivo.

Conclusion: [email protected]/Gd could serve as a promising multifunctional nanotheranostic platform and as a cancer photo-therapy agent through inducing mitochondrial dysfunction on GC.

Nanoscience

Hollow mesoporous organosilica nanoparticles (HMON)

Photo-thermal therapy (PTT)

Photo-dynamic therapy (PDT)

Multi-modal imaging

Mitochondrial damage

Gastric cancer (GC) is one of the most common malignant tumors and its mortality rate ranks third worldwide. Despite of the great progress of traditional chemotherapy and molecular target therapy, the prognosis of GC is still relatively poor, especially in China [1, 2]. Considering the limitations of current diagnosis and therapy methods, there is a pressing need to identify novel potential strategies to oﬀer new, improved diagnosis and therapy methods for GC.

Recently, reactive oxygen species (ROS) have attracted more attention due to its regulation in cancer development. Moderate levels of ROS could promote tumor progression by inducing DNA mutations, genomic instabilities or acting as signaling molecules that accelerate cancer cell proliferation and metastasis [3]. In contrast, excessive levels of ROS might enhance cellular oxidative stress, cause DNA/proteins/lipids damage, and lead to apoptotic cell death [4]. As the main organelle for ROS production, mitochondria are often the victim of exogenous elevated ROS exposure with deadly consequences, such as oxidative damage of mitochondrial DNA (mitoDNA), oxidative respiratory chain and mitochondrial membrane permeability [5, 6]. To our knowledge, mitochondria play a significant role in numerous biological processes, including the initiation of cell death, cellular energy generation and metabolic integration [7, 8]. Accumulating evidence has identified that mitochondrial dysfunction was always accompanied with interfered oxidative respiratory chain, which might reduce intracellular ATP levels and fail to produce enough energy for tumor growth [9, 10]. Meanwhile, mitochondrial damage induces cytochrome C (Cyto C) leakage from mitochondria into cytosol, while high levels of Cyto C subsequently activate caspase-depended apoptosis pathway [11, 12]. Therefore, boosting ROS could be a crucial way for activating mitochondria-depended apoptosis pathway, while precise ROS-generation at GC region is still a big problem.

Near infrared light (NIR)-based photo-therapy (PT) has been preferred for tumors targeting treatment, due to the advantages of minimal harm to normal tissues, non-invasiveness, and efficient therapeutic abilities [13, 14]. Emerging studies have also identified that with the assistance of NIR, photosensitizers could increase the production of ROS to cause mitochondria-depended cancer cell death [15-17]. To some extent, the combination of photo-thermal therapy (PTT) and photo-dynamic therapy (PDT) could achieve much better therapeutic efficiency, as hyperthermia has been reported with the ability to elevate the level of oxygen in the tumor due to the increase of blood flow [18, 19], thus overcoming the hypoxiaassociated resistance for PDT. Several hollow mesoporous organosilica nanotheranostics (HMON) carrying CuS, ICG or 7AAG, had been reported with ROS generation ability and intrinsic photo-thermal property under single laser irradiation, which could be used for PDT&PTT synergistic therapy, utilizing the good dispersion and high drug-loading capability of HMON and the photothermal ability of HMON [20, 21].

However, the lasers employed for the PTT&PDT treatments are usually different, thus the time interval between different modes will affect the synergistic efficacy [22]. Meanwhile, since PTT-treated cells readily acquire tolerance to heat stress, a relatively high temperature (>50 ºC) was required to achieve the desired therapeutic effect, which would inevitably cause damage to normal organs near the tumor [23, 24]. Therefore, there is still a great need to provide the synchronous implementation of PDT&PTT utilizing a single laser irradiation at mild-temperatures (43-45 ºC), while targeted delivery of a photosensitizers to the intended area holds potential to pinpoint the site of therapeutic efficacy. Furthermore, the certain role of PTT&PDT influencing GC cells’ apoptosis at mild temperature also needs further exploration.

Herein, HMON nanotheranostics were decorated with CuS nanocrystals ([email protected] NPs) via in situ growth, following the protocol of our previous work where CuS nanocrystals were in situ grown onto the surface of hollow mesoporous nanospheres [19]. The designed nanoplatform performed well PDT and PTT conversion efficiency and better biocompatibility. Furthermore, Gd was doped into the into the hollow structure, while good fluorescence (FL) imaging (wrapping fluorescent agent), enhanced T1 imaging under magnetic resonance imaging (MRI) and infrared thermal (IRT) trimodal imaging could therefore be achieved to guide photo-therapy [25]. After treated with [email protected]/Gd under NIR irradiation, in vitro and in vivo GC proliferation abilities were evaluated. Moreover, to fully elucidate whether ROS-mediated mitochondrial apoptosis pathway contributed to the anti-tumor effects induced by [email protected]/Gd, we detected the changes of ROS levels and the biological function of mitochondria, including mitochondrial transmembrane potentials (MTPs), mitochondrial ROS (mitoROS) and mitochondrial permeability transition pore (mPTP) produced levels, along with the expression levels of Cyto C and apoptosis-related proteins. Above all, our well designed [email protected]/Gd might present a promising approach for the imaging-guided treatment of GC ( See Scheme 1 in the Supplementary Files.)).

Scheme 1. The synthesis process, FL / MRI / IRT trimodal imaging abilities and anti-tumor mechanism of [email protected]/Gd in GC cells

2.1 Materials

Cetyltrimethylammonium chloride solution (CTAC), triethanolamine (TEA), tetraethoxysilane (TEOS), sodium citrate (3-mercaptopropyl)-trimethoxysilane (MPTES), concentrated HCl (37%), ammonia aqueous solution (NH₃·H₂O, 25wt%), bis[3-(triethoxysilyl)propyl]tetrasulfide (BTES), sodium sulfide nonahydrate (Na₂S·9H₂O), copper chloridedihydrate (CuCl₂·2H₂O), Gadolinium chloride hexahydrate (GdCl₃·6H₂O) were purchased from Sigma-Aldrich (MO, USA). The C18PMH-mPEG was purchased frommLaysan Bio Inc. (AL, USA). The PBS, DMEN medium, Fetal bovine serum (FBS) and 0.05% trypsin-EDTA were obtained from Gibco Laboratories (NY, USA). Human stomach normal epithelial cells (GES-1), human stomach cancer cells (HGC-27), human lung normal epithelial cells (BEAS-2B), primary spleen cells of mice (spleen cells) and human liver normal epithelial cells (LO2) were acquired from American Type Culture Collection (ATCC).

2.2 Synthesis of HMON

In this study, we applied an ammonia-assisted selective etching strategy to construct the hollow structure of HMON nanocarriers. Briefly, 2.1 mL of CTAC solution and 50 ul TEA solution were added into 20 mL deionized water (ddH₂O), followed by stirred at 95 ℃, and then 1 mL of TEOS were dropwised into the mix solution. 1 hour later, a mixture of BTES (1 mL) and TEOS (1 mL) were dropwised and reacted for another 4 hours to form [email protected] products. The [email protected] products were washed with ethanol for three times. Subsequently, the [email protected] products were suspended with 30 mL ddH₂O containing 8.4 mL HCl solution (37%) and stirred at 80 ℃ for 12h to remove the unreacted CTAC, then washed with ethanol and repeated the reaction once again. After that, a mixture of 20 mL of ddH₂O and 13.5 mL ammonia solution were added and reacted at 60℃ for another 3 hours. Finally, the HMON products were obtained after centrifugation and washing with ddH₂O for several times.

2.3 Synthesis of [email protected]/Gd

Firstly, 30 mg of HMON products were suspended with 40 mL ethanol solution. Subsequently, 15 mg of CuCl₂·6H₂O were added and reacted for another 6 hours. After washing with ethanol for several times, 30 mg of Na₂S were added and stirred overnight at room temperature. Following that, 15 mg of GdCl₃·6H₂O were added into the obtained solution for 12 hours, washed with ethanol for several times, and finally [email protected]/Gd were collected after centrifugation. Furthermore, in order to the water-solubility and biocompatibilities, [email protected]/Gd were stirred with 15 mg of C18PMH-mPEG [26] and kept stirring for 1 hour before blow-drying of the chloroform. Finally, the PEG-modified [email protected]/Gd were collected by centrifugation and washed with ddH₂O for several times.

2.4 Characterization of [email protected]/Gd

The surface morphology and elemental mapping images of [email protected]/Gd were captured by JEOL JEM-2100F TEM. In addition, zeta potentials and hydrodynamic diameters of various samples were detected using a Zetasizer Nano ZS (Malvern). UV-visible absorption spectrum was measured using shimadzu UV-2600 UV-vis spectrophotometer. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded on a Nicolet 7000-C spectrometer with KBr pellets. The powder X-ray diffraction (XRD) patterns were acquired on a PANalytical X'Pert PRO X-ray diffractometer, while X-ray photoelectron spectroscopy (XPS) measurements were conducted on a PHI 5000 VersaProbe spectrometer using a monochromatic Al Kα radiation source. In order to detect the photothermal effect, different concentrations of [email protected], [email protected] or [email protected]/Gd in PBS solutions (200 μL) were irradiated with an 808 nm NIR laser (0.8 W/cm², 5 mins). The real-time temperatures of the solutions were recorded using a FLIR E50 camera system.

2.5 Calculation of Photo-thermal Conversion Efficiency of [email protected]/Gd at 808 nm.

The photothermal conversion efficiency of [email protected]/Gd was calculated by the following equation: (see Equation 1 in the Supplementary Files)

Where h is the heat transfer coefficient, S is the surface area of the container, T_max is the maximum equilibrium temperature for [email protected]/Gd NPs solution, while T_max,wateris the maximum equilibrium temperature for water. In addition, I refer to the laser power (0.8 W/cm²), and A₈₀₈is the absorbance of the [email protected]/Gd NPs at 808 nm.(see Equation 2 in the Supplementary Files)

Where m_Dand C_D refers to the weight of water and the heat capacity of water respectively. In addition, τ_S is time constant of the sample. (See Equation 3 in the Supplementary Files)

Where T_ambis surrounding ambient temperature. Therefore, the τ_S can be calculated using the linear regression curve between cooling stage and negative natural logarithm of driving force temperature of [email protected]/Gd.

2.6 Detection of singlet oxygen on solutions

After treated with or without NIR laser irradiation, the Singlet Oxygen Sensor Green reagent (Life Technologies, Carlsbad, CA, USA) was used to detect the production of singlet oxygen on the solutions contained [email protected] or [email protected]/Gd using a Jasco (Easton, MD, USA) FP-6200 spectrofluorometer.

2.7 Biocompatibilities of [email protected]/Gd

To address biocompatibility issues, normal GES-1, BEAS-2B, LO2 and spleen cells were treated with PEG-modified or non-PEG-modified [email protected]/Gd solutions for 24h. Then, cell viability was quantified with Cell Counting Kit-8 (CCK-8) assay (Dojindo, Tokyo, Japan) and a microplate reader. In order to evaluate immunotoxicity, the mouse macrophage (RAW264.7) cells were seeded into confocal laser scanning microscope (CLSM) dishes and treated with [email protected]/Gd for 24h. Subsequently, treated cells were washed, fixed with 4% paraformaldehyde, treated with 1% bovine serum albumin (BSA), incubated with 0.1% Triton X-100(Abcam, Cambridge, UK), and finally stained with rhodamine-phalloidin (Abcam) and with 4’,6-diamidino-2-phenylindole (DAPI; Abcam). Finally, CLSM (Olympus, Tokyo, Japan) was used to observe and photograph stain cells.

2.8 Cell uptake of [email protected]/Gd

In order to access the intracellular uptake efficiency, HGC-27 cells were planted in CLSM dishes, and then treated with fluorescein isothiocyanate (FITC)-labeled [email protected]/Gd, which were constructed as the followings: Briefly, 20 mg of [email protected]/Gd products were suspended with 20 mL ddH₂O, and then 1 mg FITC were added and kept stirring for 8 hours. After concentration and washing with ddH₂O, the FITC-labeled [email protected]/Gd were constructed. After incubation for 1, 2 and 4 hours, the cells were washed with PBS and collected. Following that, cells were directly analyzed by flow cytometry assay. For immunofluorescence imaging, treated cells were ﬁxed with 4% paraformaldehyde, and stained with DAPI. Finally, The CLSM dishes were observed and imaged with CLSM (Olympus).

2.9 In vitro photo-therapeutic effect of [email protected]/Gd

HGC-27 cells were pretreated with [email protected]/Gd, with or without an 808 nm laser at a power density of 0.8 W/cm² for 5 mins respectively. 24 hours later, the treated cells were collected and incubated with CCK-8 kit for viabilities detection, with Annexin V-FITC/PI Kit (KeyGEN, Nanjing, China) for apoptosis detection and EDU testing kit (Ruibo, Guangzhou, China) for cell proliferation ability detection. Furthermore, in order to evaluate mitochondrial function, the treated cells were also harvested and detected with JC-1 Kit (Keygen), mitoROS kit (AAT Bioquest, Wuhan, China), ROS Kit (Keygen), mPTP kit (BestBio, Shanghai, China) follow the manual instructions.

2.10 In vivo multi-modal imaging behaviors of [email protected]/Gd

In order to evaluate the multi-modal imaging behaviors of [email protected]/Gd, HGC-27 cells were harvested and suspended with appropriate amount of PBS, and then 50 μL of suspension was injected into the right flank of mice to construct tumor-bearing nude mice, which were randomly divided into different groups. For fluorescence images, the tumor-bearing mice were injected with DIR labeled [email protected]/Gd via tail vein injections, while DIR-labeled [email protected]/Gd were firstly constructed using similar methods shown in 2.8. At the time point of 0, 3, 6, 12, 18 and 24 h after injection, FL images of whole body and their major organs were all detected by FL imaging system (Digital Fprcision Medicine Company, Beijing, China). For IRT images, the tumor-bearing mice were anesthetized before and after injecting with [email protected]/Gd, then thermographic images were captured by a FLIR E50 camera at the present of NIR laser irradiation. For MRI images, single [email protected]/Gd solutions, and tumor-bearing mice were anesthetized before and after injecting with [email protected]/Gd, then MRI images were captured through a 3.0 T MAGNETOM Skyra MRI scanner (Phihips, Amsterdam, Netherlands) using T1-weighted sequence (TR=450.0 ms, TE =15.3 ms, thickness =2 mm).

All animal procedures were approved by Nanfang Hospital of Southern Medical University (Certiﬁcation No. L2017243). All procedures of the investigation were carried out following the rules of the Declaration of Helsinki of 2008 (https://www. wma.net/what-we-do/medical-ethics/declarationofhelsinki/), revised in 2008.

2.11 In vivo photo-therapeutic effect of [email protected]/Gd

In order to clarify the photo-therapeutic effect of [email protected]/Gd, tumor-bearing mice were randomly divided into four groups, which were treated with saline and [email protected]/Gd, with or without irradiation of NIR (0.8 W/cm², 8 mins). The body weight and tumor volume were recorded every three days for 14 days. At the 14th day, the mice were euthanized, then their tumor tissues were excised and fixed in 4% formalin for immunohistochemistry (IHC; Tunel, Abcam, USA) and H&E staining.

2.12 Statistical analysis

Data are showed as mean ± standard deviation (SD). Quantitative analysis of the immunofluorescence intensity, western-blotting bands and IHC stained area by aniline was conducted using the Image J program (National Institutes of Health, Bethesda, MD). The differences between the mean values were analyzed using SPSS 21.0 (International Business Machines Corporation, Armonk, NY) and one-way ANOVO statistical approach. Results were considered statistically signiﬁcant when P<0.05. All experiments were repeated at least 3 times.

3.1 Preparation, characterization and in vitro photo-thermal effect of [email protected]/Gd

HMON were synthesized via a mild selective etching progress. In brief, mesoporous organosilica nanospheres with large pore were firstly synthesized by the dual hydrolysis and condensation of BTES and TEOS under the catalyzation of TEA, while CTAC was used for pore forming. As the dominant -Si-O- bonds were weaker than the –Si-C- bonds within the outer sites, the inner part of the mesoporous organosilica nanospheres were then etched using diluted ammonia to form the hollow mesoporous structure, bridged by –S-S- bonds [21]. Though it has been reported that CuS could be conjugated onto the surface of HMON through thiol groups, it required respective synthesis of HMON and CuS following the conjugation progress. The multi-steps protocol might be simplified as it has also been reported that Si-O- groups on the surface of the HMON have the potential to bond with metal ions (such as Fe³⁺, Au⁺, etc.) without the need of additional molecular chelator [27, 28]. In our previous work, CuS nanocrystals were in situ grown onto the surface of hollow mesoporous TaOx nanospheres without the help of thiol groups [19]. Herein, CuS nanocrystals were in situ grown onto the surface of the HMON similarly. Briefly, Cu²⁺ ions were added directly into the HMON solution and adsorbed onto the HMON, following the addition of Na₂S to achieve the in situ nucleation and growth of CuS nanocrystals. Finally, the [email protected]/Gd NPs were constructed with the addition of GdCl₃·6H₂O. Then, the element mapping of [email protected]/Gd showed that the Cu²⁺ and Gd³⁺ions were successfully jointed together onto the surface of HMON (Figure 1A). It is noticeable that the etching degree by diluted ammonia has great influence on the ability of the HMON to absorb Cu²⁺ ions. HMON with excessive etching leading to the poor conjugation with Cu²⁺ and Gd³⁺ions due to the depletion of –Si-O- bonds. Eventually, C18PMH-mPEG was applied to improve the stability and dispersity of the [email protected]/Gd nanospheres.

The as-prepared HMON showed specific strong and broad peaks at 2927.32 cm^-1 and 2855.28 cm^-1 (attributed to -CH₂) [29], which represented the covalent grafting of C18PMH-mPEG chains onto the surface of HMON, compared to non-PEGylation HMON. Meanwhile, new peak at 627.03 cm^-1 (attributed to Cu-S) was also observed in [email protected] and [email protected]/Gd [30]. Therefore, the FI-TR results consistently confirmed the successful synthesis of [email protected]/Gd (Supplementary files, Figure S1). UV-vis spectra results revealed that [email protected] and [email protected]/Gd both have strong absorption in NIR region (Supplementary files, Figure S2), which is mainly contributed to the effective adherence of CuS nanocrystals on the surface of HMON [21]. XPS results demonstrated the presence of C (C 1s peaks at 284.95 eV), O (O 1s peaks at 531.99 eV), Si (Si 2p peaks at 102.53 eV), Cu (Cu 2p peaks at 933.27 eV), S (S 2p peaks at 163.36 eV) and Gd (Gd 4d peaks at 142.72 eV) (Supplementary files, Figure S3) [31]. From XRD results, [email protected] and [email protected]/Gd both showed specific characteristic diffraction peaks [30], which is indexed as a typical covellite crystalline phase of CuS (Supplementary files, Figure S4). It is noteworthy that CuS crystal was found in [email protected] and [email protected]/Gd, while no Gd-related crystal was observed in [email protected]/Gd, indicating that Gd was doped in HMON, but not as a crystal form. Taken together, the above results consistently confirmed the successful synthesis of [email protected]/Gd. In addition, the diameters of HMON was 115.5±1.46 nm, while CuS or/and Gd³⁺ loading led to a slight increase in the size of the [email protected] (116.7±1.51 nm) and [email protected]/Gd (117.1±1.71 nm), and all of them exhibited unimodal size distribution. Meanwhile, the data also indicated that both of [email protected] and [email protected]/Gd possessed negative net charge, which could help to improve their circulation stability (Supplementary files, Figure S5). Compared to these PEG modified HMON NPs, HMON-based materials without PEG modification also showed negative net charge, and slight minor diameters. However, the diameters’ SD value in HMON (108.2±4.54 nm), [email protected] (109.4±3.41 nm) and [email protected]/Gd (110.1±4.15 nm) NPs were larger than those in PEG-modified HMON NPs, indicating that C18PMH-mPEG could improve their stability (Supplementary files, Figure S6). Consequently, PEG modified HMON NPs were chosen for further experiments in our research. Due to the presence of the disulfide bonds in the framework of HMON, [email protected]/Gd exhibited time-dependent biodegradable behavior in the glutathione (GSH) solutions (Figure 1B), indicating that [email protected]/Gd NPs could be bio-degradable and eliminated through feces and urine, thus the drug accumulation leaded toxicity could be avoided.

It could also be expected that the as-prepared NPs possess the potential to be applied as MRI imaging contrast agent due to the addition of Gd [32, 33]. In vitro experiment demonstrated that T1 signal intensity was enhanced with the increasing concentration of [email protected]/Gd solutions. As calculated, significant increase of SNR (signal to noise ratio) could also be observed, indicating the outstanding potential for MRI imaging (Figure 1C&D). Besides the MRI imaging properties, we further investigate the photo-thermal conversion efficiency of [email protected]/Gd by monitoring the temperature changes under NIR laser irradiation in vitro using an infrared thermal imaging camera. After NIR laser irradiation (0.8 W/cm²) for 5 mins, dramatical temperature increase was observed in [email protected]/Gd and [email protected] groups, while no obvious temperature change was shown in PBS or [email protected] groups with the same laser irradiation. The maximum increased temperature (ΔT_max) of [email protected]/Gd and [email protected] groups (1000µg/mL) ∼38 °C, whereas the ΔT_max of other [email protected]/Gd and [email protected] groups (100µg/mL, 50µg/mL and 12.5µg/mL) increased to ∼16.0 °C, ∼8.0 °C and ∼6.0 °C respectively (Figure 1E&F; Supplementary files, Figure S7). It is worth pointing that [email protected]/Gd and [email protected] showed good temperature response under the NIR laser irradiation conditions, which demonstrating that the photo-thermal conversion efficiency was contributed to the presence of CuS. Moreover, [email protected]/Gd NPs showed an excellently stable photo-thermal performance, as the heat-cool curve of [email protected]/Gd NPs had no significant difference within five cycles of NIR laser irradiation (Supplementary files, Figure S8). According to the linear regression curve between the cooling stage and negative natural logarithm of driving force temperature of [email protected]/Gd, the photo-thermal conversion efficiency of [email protected]/Gd NPs was calculated to be 82.4% (Figure 1G). In addition, to determine whether [email protected]/Gd NPs could produce ROS under NIR laser irradiation, a singlet oxygen sensor was applied to detect ROS levels. As shown in Figure 1H, [email protected]/Gd NPs exhibited concentration-dependent production of ROS in deionized water, indicating that [email protected]/Gd NPs possessed excellent PDT ability. Moreover, [email protected] showed similar ROS generation ability in the presence of NIR, while no excessive singlet oxygen was observed in [email protected] NPs, with or without NIR treatment (Supplementary files, Figure S9), which demonstrating that the ROS generation capacity of [email protected]/Gd NPs were contributed to the presence of CuS. Therefore, [email protected]/Gd would be promising PTT, PDT and functional imaging NPs for the treatment of GC

3.2 In vitro photo-therapeutic effect of [email protected]/Gd nanoparticles in GC cells

Firstly, we tested the in vitro photo-therapeutic effects of [email protected]/Gd on HGC-27 cells. Briefly, HGC-27 cells were first incubated with [email protected]/Gd at different concentrations for 4 h and then were irradiated with an 808 nm laser at a power density of 0.8 W/cm² for 5 mins. After 24 h, CCK-8 assay was applied to evaluate the antitumor therapeutic efficacies of [email protected]/Gd. As shown in Figure 2A, free [email protected]/Gd and single NIR laser treatment did not induce significant changes in cell death, since the half maximal inhibitory concentration (IC50) of [email protected]/Gd was as high as 570.01µg/mL. Furthermore, dramatical decreased cell viabilities were observed in [email protected]/Gd plus NIR group (IC50=50.51µg/mL), indicating that [email protected]/Gd exhibited an effective photo-therapy effect with NIR irradiation. In the other hand, [email protected] exhibited similar anti-tumor effect with NIR irradiation, compared to [email protected]/Gd plus NIR irradiation group, indicating that photo-therapy effect was contributed to the addition of CuS, while Gd showed negligible cell toxicity (Supplementary files, Figure S10). Consistently, LDH assay revealed that [email protected]/Gd plus NIR induced higher levels of LDH leakage than blank group, while [email protected]/Gd only and single NIR laser did not induce any LDH changes (Figure 2B). These results suggested that [email protected]/Gd could provide a promising killing effect to GC cells, with the irradiation of NIR.

As it has been reported, several CuS-based NPs had been applied for PDT&PTT synergistic therapy [34, 35]. However, the therapeutic effect of [email protected] PTT and PDT at mild temperature, has not been fully discussed. It is still wondered whether the therapeutic effect was achieved by PTT ablation alone, or PDT also played some certain role in the therapeutic progress. In order to evaluate the PDT effect, HGC-27 cells were placed on an ice box while being irradiated with NIR light and the temperature was controlled below 10°C to minimize the effect of NIR-induced PTT ablation. To our surprise, significant cell death was still observed in this condition, though not as obvious as [email protected]/Gd plus NIR treatment, as IC50 raised to 122.4 µg/mL (Figure 2C). Furthermore, evident LDH leakage was also found, shown in Figure 2D.

When referring to PTT effect, clinicians have pointed out that tumor cells might occur apoptosis under 43-45°C, while normal cells are generally tolerant to this temperature condition [36]. In another word, PTT effect refers to generating external 45 °C heating at cancer region, which is supposed to have a direct cytotoxic effect on tumor cells, and enhance the efficacy of chemotherapy and radiotherapy, improve the body's immunity, and thus suppress tumor progression [18, 37]. Coincidentally, it could be observed by the IR camera that when HGC-27 cells were treated with [email protected]/Gd (50 µg/mL) plus NIR irradiation (0.8 W/cm², 5 mins), the temperature could raise up to around 45 °C at 37 °C temperature condition. Consequently, [email protected]/Gd with 50 µg/mL concentration were chosen for the following cellular experiments. Simulated 45 °C external temperature condition using a cell culture incubator was set as control. It could be seen that slight cell death and LDH leakage were observed in the external heating group, comparing with NIR irradiation group, indicating hyperthermia might not be the only factor (Figure 3C&D). Taken together, it could be concluded that [email protected]/Gd could induce combined PDT and PTT effect, while this obvious anti-tumor cell death was contributed to their PDT&PTT synergetic effect.

To address biocompatibility issues, in vitro experiments were performed by incubating [email protected]/Gd NPs with normal GES-1 cells. CCK-8 assay revealed above 85% cell viability rates for GES-1 as the concentration ranging from 0-512 µg/mL, while the IC50 of [email protected]/Gd was as high as 957 µg/mL to GES-1 cells (Figure 2E). However, when treated with non-PEG modified [email protected]/Gd NPs, cell viability rates for GES-1 cells showed significant decrease (IC50=312 µg/mL; Supplementary files, Figure S11), indicating the addition of PEG could improve their bio-safety, and PEG modified [email protected]/Gd were consequently chosen for further biological experiments in our research. Surprisingly it could be observed that IC50 to GES-1 cells was much higher than HGC-27 GC cells, indicating that the as-prepared [email protected]/Gd showed specific toxicity to GC cell. Furthermore, [email protected]/Gd (50 µg/mL) were also co-incubated with RAW264.7 murine macrophage-like cells. This immunotoxicity experiment revealed that [email protected]/Gd treatment did not elicit any inflammatory response at the cellular level (Figure 2F). These results strongly indicated that [email protected]/Gd NPs have good in vitro biocompatibilities, highlighting their value for clinical translation as drug carriers.

3.3 In vitro cellular uptake and anti-GC effect of [email protected]/Gd nanoparticles

Cellular uptake of [email protected]/Gd were then investigated by flow cytometry and CLSM assays, while FITC were firstly encapsulated into [email protected]/Gd NPs. After incubating with [email protected]/Gd (50 µg/mL) for 1-4 h, the FITC fluorescence intensities increased significantly as time passed by (Figure 3A&B), indicating the excellent cellular uptake efficiency of [email protected]/Gd. Since [email protected]/Gd could efficiently enter GC cells, we further detected the apoptosis rates determined by flow cytometry, which demonstrated that [email protected]/Gd plus NIR laser irradiation induced 2-fold higher levels of total apoptosis (14%) than blank group (5%), while free [email protected]/Gd and single NIR laser did not induce any apoptotic change (Figure 3C). EdU dye is a kind of thymidine nucleoside analogues, which could specifically insert into DNA molecules of rapid proliferation cells, and higher EdU-positive cell rates usually demonstrate better cell growth abilities [38]. In our research, after conjugated reactions with EdU, we found that [email protected]/Gd plus NIR laser irradiation induced 2-fold lower levels of EdU-positive rates (15%) than blank group (40%), while free [email protected]/Gd NPs and single NIR laser did not induce any EdU-positive rates change (Figure 3D). These results suggested that [email protected]/Gd provided a promising anti-proliferation and promoting apoptotic effects to GC cells, with the irradiation of NIR.

3.4 Anti-tumor mechanism of photo-therapeutic effect induced by [email protected]/Gd

Though excellent anti-tumor effect has been proved, their relevant photo-therapeutic mechanism remained unknown. To examine the cancer-killing mechanism of [email protected]/Gd, we incubated HGC-27 cells with [email protected]/Gd (50 µg/mL) for 4 h, irradiated with an 808 nm laser (0.8 W/cm², 5 mins), then observed by TEM. Surprisingly, dozens of mitochondria were shown in non-treated HGC-27 cells, while no mitochondria were observed in treated HGC-27 cells (Figure 4A). Meanwhile, it could be proved that MTPs of HGC-27 cells was reduced by [email protected]/Gd NPs with the assistance of NIR irradiation, further indicating the damage of mitochondria (Figure 4B). Consequently, we assumed that [email protected]/Gd induced PTT&PDT probably resulted in mitochondrial damage to exert its function.

Mitochondria are central organelles for the regulation of cancer cell life and death, to which the damage can directly activate the intrinsic apoptosis pathway. When cells receive certain external stimuli, the mitochondrial electron transport is blocked, MTPs changes, the maintain of mPTP is disrupted and Cyto c release, followed by activates caspase-depended apoptosis pathway [39]. Actually, the mitochondrial-dependent damage could be triggered via a range of exogenous and endogenous stimuli, such as oxidative stress, ischemia and DNA damage [40, 41]. As it is well-known, ROS are inevitable products of cell metabolism, while high levels of intracellular ROS could attack mitochondria and cause mitochondrial-damage-dependent apoptosis [42]. Accumulating studies had proved that NIR-mediated PTT&PDT could induce the outbreak of ROS, following by activating oxidative stress and mediating mitochondrial damage in cancer cells [15, 16, 43]. As it has been demonstrated in the CCK-8 results, [email protected]/Gd did induce obvious anti-tumor cell death, partly contributed by their PDT effect. In order to clarify the ROS generation, DCFH-DA was applied as biological probe to monitor the intracellular level of ROS [44]. Increasing ROS generation was found to be significantly increased in [email protected]/Gd plus NIR treated HGC-27 cells (Figure 4C). To further identify the PDT effect, HGC-27 cells were placed on an ice box while being irradiated with NIR light, and the temperature was controlled below 10°C to minimize the effect of NIR-induced PTT ablation. Interestingly, dramatical increase of ROS was still observed in this condition, though not as obvious as [email protected]/Gd plus NIR treatment. Meanwhile, our results showed that slight ROS was produced in the external heating group (Supplementary files, Figure S12). Consequently, it could be concluded that [email protected]/Gd could induce ROS generation, was also mainly contributed to their PDT effect.

Meanwhile, in order to clarify whether the generated ROS could cause mitochondrial dysfunction, MitoSOX Red was used as an indicator to discern the superoxide in mitochondria through flow cytometry analysis (FACS) in this study [45]. Notably, a sharp increase in red fluorescence (refer to mitochondrial ROS, also called mitoROS) was detected in [email protected]/Gd plus NIR group (Figure 4D). Taken together, we assumed that photo-therapeutic effect induced by [email protected]/Gd NPs might promote intracellular ROS level and induce mitochondrial dysfunction to some extent. The formation of much more amount of mitoROS species was also discovered, which in turn aggravate the damage of mitochondria and might activate the Caspase-depended apoptosis pathway. To verify whether the Caspase-depended apoptosis pathway was activated, the expression of pro-apoptotic protein (Bax) and anti-apoptotic protein (Bcl-2) was detected. It could be seen that pro-apoptotic protein (Bax) dramatically elevated and anti-apoptotic protein (Bcl-2) decreased (Figure 4E; Supplementary files, Figure S13). As Bax was a pro-apoptotic protein with multiple Bcl-2 homology domains, this alteration could alter mPTP [46] (Figure 4F; Supplementary files, Figure S14). MPTP opening is the primary event of the mitochondrial intrinsic apoptosis pathway, could be referred as sudden mitochondrial permeability transition and loss of inner mitochondrial potential, leading to the increase of cytochrome c (Cyto C) (Figure 4E; Supplementary files, Figure S13). Considering Cyto c release from mitochondria to the cytoplasm has been verified as the most important event in caspase depended mitochondrial mediated apoptosis signal transduction pathway, the expression of apoptotic proteins in treated cells were further detected. From the western blot results, the scale of the expressed well-defined apoptosis protein markers (cleaved caspase-9/caspase-9, cleaved caspase-3/caspase-3) was markedly increased in [email protected]/Gd plus NIR group, compared with other three groups (Figure 4G; Supplementary files, Figure S15). Through the involvement of caspase-9, Caspase-3 is subsequently cleaved, which could activate DNA fragmentation, thereby inducing cell apoptosis [47, 48]. Taken together, our research demonstrates that [email protected]/Gd plus NIR treatment did induce mitochondrial damage to trigger GC cells’ caspase-dependent apoptosis pathway.

In order to further confirm whether the mitochondrial-damage-dependent apoptosis pathway contributed by [email protected]/Gd induced PDT effect, N-Acetyl-L-cysteine (NAC, an anti-oxidant containing sulfhydryl group) was applied for the rescue experiments [49]. With the addition of NAC, intracellular ROS level in [email protected]/Gd plus NIR treated HGC-27 cells was dramatically decreased (Figure 5A). Interestingly, MTPs, mitoROS and mPTP in treated HGC-27 cells was consistently reversed (Figure 5B&C&E; Supplementary files, Figure S16). Furthermore, we also found the expression of apoptosis protein markers (Bax) and Cyto C was significantly decreased, while the anti-apoptotic protein (Bcl-2) increased (Figure 5D; Supplementary files, Figure S17), which both indicated that mitochondrial damage was partly rescued. Since Cyto C has been reported to be one of the main activators during the caspase-dependent cell death, caspase-pathway would be inhibited if Cyto C’s release was decreased. As expected, the expression of apoptosis protein markers (cleaved caspase-9/caspase-9, cleaved caspase-3/caspase-3) were significantly decreased (Figure 5F; Supplementary files, Figure S18). Furthermore, cell viabilities, LDH leakage, cell proliferation and apoptotic levels were consistently reversed, when ROS was suppressed by NAC (Figure 5G&H&I&J). In all, these results indicated that [email protected]/Gd plus NIR could exert anti-proliferation effect through activating the ROS/mitochondria-damage/caspase pathway.

3.6 In vivo multi-mode imaging behaviors of [email protected]/Gd nanoparticles

Before moving forward to study in vivo tumor photo-therapy, in vivo biodistribution behaviors of such agent in HGC-27 tumor-bearing mice were studied. DIR, as a near-infrared lipophilic carbocyanine dye, has been used for many cell biology applications [50, 51]. DIR were first encapsulated into [email protected]/Gd NPs ([email protected]/Gd-DIR). Following that, tumor-bearing mice were treated with [email protected]/Gd-DIR via tail vein injections. After 0, 3, 6, 12, 18 and 24 hours, the mice were imaged using a small animal in vivo fluorescence imaging system (DIGITAL FPRCISION MEDICINE Company, Beijing, China). The in vivo fluorescence images indicate that [email protected]/Gd could access the cancer region in 3 hours, then gradually enriched in the tumor site from 3h to 24h (Figure 6A; Supplementary files, Figure S19). As shown in Figure 6A, a signiﬁcant ﬂuorescence signal accumulated in the tumor in the [email protected]/Gd-treated mice and peaked at 24h, which provided an optimal therapeutic time window for subsequent therapy in vivo. Meanwhile, tumor-bearing mice were sacriﬁced 24h post injection, and major organs were collected for ﬂuorescence imaging. Ex vivo imaging also showed that [email protected]/Gd NPs were mainly accumulated in the tumor region (Figure 6B), which further conﬁrming that their passive tumor-targeting abilities through enhanced permeability and retention (ERP) effect [52, 53]. Since [email protected]/Gd NPs were accumulated in lung, spleen and liver, we have already carried out CCK-8 assays to further test the biocompatibility of [email protected]/Gd NPs, by incubating [email protected]/Gd with lung cells (BEAS-2B), primary spleen cells of mice (spleen cells) and liver cells (LO2). From the in vitro results, all the cells showed above 85% cell viabilities rates, which indeed confirm the biosafety of [email protected]/Gd NPs (Supplementary files, Figure S20). Moreover, to further demonstrate the enhancement of MRI, HGC-27 tumor-bearing mice were also detected using a 3.0 T MAGNETOM Skyra MRI scanner, as it has been verified that [email protected]/Gd could be used as MRI T1 contrast agent in vitro. [email protected]/Gd solution (6 mg kg⁻¹) was tail-intravenous injected, and then MRI images of the tumor were obtained after 24h. Figure 6C&D displayed that [email protected]/Gd could significantly enhanced T1-weighted signal after tail-intravenous administered, and these results not only highlighted the potential of [email protected]/Gd for MRI, but also again confirmed that [email protected]/Gd could gradually access the cancer region within 24 hours, and 24h could serve as an optimal therapeutic time window for subsequent therapy in vivo. Subsequently, the IRT imaging abilities of [email protected]/Gd in vivo were further investigated. HGC-27 tumors on mice that were exposed to NIR irradiation (0.8 W/cm², 8 mins), after tail-intravenous injected with [email protected]/Gd for 24 hours. As shown in Figure 6E&F, the tumor temperature of tumors increased from ∼36 °C to ∼44 °C within 3 mins, while maintained at 43°C∼45 °C in the next 5 mins. This temperature was much greater than that (36 °C) experienced by mice administered saline plus NIR irradiation (0.8 W/cm², 8 mins), indicating that [email protected]/Gd did exhibit mild PTT effect (43-45 °C) under NIR irradiation (under 0.8 W/cm²) , which would be much more attractive for clinical photo-therapeutic treatment.

Development of multifunctional nanoplatforms integrating both diagnostics and treatment functions for cancer nano-theranostics have attracted widespread research interest in nanobiotechnology [54, 55]. However, it is still worth to construct theranostic nano-systems with multi-modal imaging to guide therapy. Our results revealed that [email protected]/Gd did exhibit good FL, MRI and IRT imaging capacities. Furthermore, [email protected]/Gd NPs could selectively cause PT at cancer region, guided by FL/MRI/ IRT imaging. Taken together, [email protected]/Gd NPs might be a promising treatment for solid tumors with precise photo-therapeutic eﬃciency.

3.7 In vivo photo-therapeutic treatments of [email protected]/Gd nanoparticles in GC

To further analyze the antitumor effects of [email protected]/Gd NPs in vivo, we constructed tumor-bearing mice. Then, we randomly divided these mice into four groups, treating them with saline (negative control), saline plus NIR irradiation, [email protected]/Gd and [email protected]/Gd plus NIR irradiation. Fourteen-days’ post-treatment, we found that the relative tumor volume showed no significant difference between the saline group and saline plus NIR group, which meant that NIR itself could not inhibit tumor growth (Figure 7A&B&C&D). Furthermore, inhibited tumor growth rate in the [email protected]/Gd plus NIR irradiation group was approximately 80% compared to control group, while free [email protected]/Gd induced no significant changes (Figure 7A&B&C&D). We also observed the body-weight-change in these four groups, while no body-weight-loss was found (Figure 7E). The blood serum samples were sent for the biochemical analysis of clinically relevant indicators (aspartate aminotransferase and alanine aminotransferase) for the liver and (blood urea nitrogen and creatinine) for the kidneys [47]. No significant differences were observed in free NIR, free [email protected]/Gd and [email protected]/Gd plus NIR groups, compared to saline control groups, indicating that no damage happened in livers or kidneys (Figure 7F). Therefore, these results indicated that the photo-thermal therapy induced by [email protected]/Gd NPs could efficiently kill tumor cells with less side effect, indicating [email protected]/Gd NPs have a promising application for GC treatment.

The isolated issues were further sent for histological detection, while H&E staining (Figure 7G) further confirmed the collected specimen were tumors. TUNEL termed as “Terminal deoxynucleotidyl transferase dUTP nick end labeling”, which is a classic marker to detect DNA fragmentation in apoptotic tumor cells [56]. Interestingly, we found that expression of TUNEL was upregulated in [email protected]/Gd plus NIR group, while no evident changes were found in other groups, compared with saline group (Figure 7G; Supplementary files, Figure S21). Consequently, the results of IHC consistently highlight the superiority of [email protected]/Gd plus NIR irradiation on anti-tumor proliferation, since this group showed highest expression of TUNEL.

In this work, we successfully constructed biocompatible and biodegradable Gd doped HMON decorated by CuS NPs (called [email protected]/Gd). The as-prepared NPs exhibited high photo-thermal conversion eﬃciency (82.4 %) and ROS generation ability. Differently from the reported [email protected] NPs, it was found that hyperthermia might not be the only factor of the cell apoptosis, while ROS induced by PDT also plays an important role. PDT induced ROS would attack MTPs, promote mitoROS production and finally induced mitochondrial damage. Meanwhile, mitochondrial damage again changed anti/pro-apoptotic proteins, opened mPTP, released Cyto C into cytosol, and finally activated caspase-9/caspase-3-depended cell apoptosis pathway. In vivo data showed that [email protected]/Gd could serve as a nanoplatform for fluorescence/ MRI/IRT triple modal imaging guided photo-therapy at cancer region, inhibit the growth of GC cells without evident systemic toxicity. Taken together, [email protected]/Gd could serve as a promising multifunctional nanotheranostic platform through inducing mitochondrial damage on GC.

HMON: Hollow mesoporous organosilica nanotheranostics; [email protected]: CuS-modified hollow mesoporous organosilica nanoparticles; NIR: Near infrared; PTT: Photo-thermal therapy; PDT: Photo-dynamic therapy; GC: Gastric cancer; [email protected]/Gd: Gd doped HMON decorated by CuS nanocrystals; GSH: L-Glutathione; ROS: Reactive oxygen species; MTPs: Mitochondrial transmembrane potentials; MitoROS: Mitochondrial reactive oxygen species; mPTP: mitochondrial permeability transition pore; FL: Fluorescence; MRI: Magnetic resonance imaging; IRT: Infrared thermal; mitoDNA: Mitochondrial DNA; Cyto C: Cytochrome C; PT: Photo-therapy; CTAC: Cetyltrimethylammonium chloride solution; TEA: Triethanolamine; TEOS: Tetraethoxysilane; MPTES: Sodium citrate (3-mercaptopropyl)-trimethoxysilane; BTES: Bis[3-(triethoxysilyl)propyl]tetrasulfide; CuCl₂: Copper chloridedihydrate; Na₂S·9H₂O: sodium sulfide nonahydrate; GdCl₃·6H₂O: Gadolinium chloride hexahydrate; FBS: Fetal bovine serum; ATCC: American Type Culture Collection; ddH₂O: Deionized water; FT-IR: Fourier transform infrared spectroscopy; XRD: X-ray diffraction; XPS: X-ray photoelectron spectroscopy; CLSM: Confocal laser scanning microscope; BSA: Bovine serum albumin; FITC: Fluorescein isothiocyanate; IHC: Immunohistochemistry; SD: Standard deviation; IC50: Half maximal inhibitory concentration; FACS: Flow cytometry analysis; NAC: N-Acetyl-L-cysteine; EPR: Enhanced permeability and retention.

Ethics approval and consent to participate

All the animal experiments were conducted in accordance with the guidelines and the ethical standards of the Institutional Animal Care and Use Committee of the Nanfang Hospital of Southern Medical University.

Consent for publication

All authors agree to be published.

Competing interests

The authors declare no competing financial interest.

Funding

This work was supported by grants from National Natural Science Foundation of China (81601550; 81971746), China Postdoctoral Science Foundation (2019M662977; 2019M662986), Special Funds for the Cultivation of Guangdong College Students' Scientific and Technological Innovation (pdjh2020a0106, pdjh2020a0109, pdjh2020a0108), Guangzhou City Science and Technology Project-Zhujiang Technology New Star Project (20160521606488), Natural Science Foundation of Guangdong Province (2017A030306023), Science and Technology Project of Guangdong Province (2018B090944002), National Key R&D Program of China (2017YFC1103400) and Outstanding Youths Development Scheme of Nanfang Hospital, Southern Medical University (2017J006)

Authors' contributions

WG, ZC, JC, BZ and YH designed the study. XF, YY, HH, YL (Yanrui Liang) and WH collated the data, carried out data analyses and produced the initial draft of the manuscript. GS, YL (Yu Liang), CP and YL (Yanbing Li) revised the figures and tables. XF, YH, and GL contributed to drafting the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors want to thank PD Dr. Miaomiao Yuan from Sun Yat-sen University for technical support

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

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Biodegradable hollow mesoporous organosilica nanotheranostics (HMON) for multi-mode imaging and mild photo-therapeutic-induced mitochondrial damage on gastric cancer

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