Mitochondria-Targeting and Oxygen Self-Supplying Eccentric Hollow Nanoplatform for Enhanced Breast Cancer Photodynamic Therapy

Photodynamic therapy (PDT) has received increasing attention for tumor therapy due to its minimal invasiveness and spatiotemporal selectivity. However, the poor targeting of photosensitizer and hypoxia of the tumor microenvironment limit the PDT efficacy. Herein, eccentric hollow mesoporous organic silica nanoparticles (EHMONs) are prepared by anisotropic encapsulation and hydrothermal etching for constructing PDT nanoplatforms with targeting and hypoxia-alleviating properties. The prepared EHMONs possess a unique eccentric hollow structure, a uniform size (300 nm), a large cavity, and ordered mesoporous channels (2.3 nm). The EHMONs are modified with the mitochondria-targeting molecule triphenylphosphine (CTPP) and photosensitizers chlorin e6 (Ce6). Oxygen-carrying compound perfluorocarbons (PFCs) are further loaded in the internal cavity of EHMONs. Hemolytic assays and in vitro toxicity experiments show that the EHMONs-Ce6-CTPP possesses very good biocompatibility and can target mitochondria of triple-negative breast cancer, thus increasing the accumulation of photosensitizers Ce6 at mitochondria after entering cancer cells. The EHMONs-Ce6-CTPP@PFCs with oxygen-carrying ability can alleviate hypoxia after entering in the cancer cell. Phantom and cellular experiments show that the EHMONs-Ce6-CTPP@PFCs produce more singlet oxygen reactive oxygen species (ROSs). Thus, in vitro and in vivo experiments demonstrated that the EHMONs-Ce6-CTPP@PFCs showed excellent treatment effects for triple-negative breast cancer. This research provides a new method for a targeting and oxygen-carrying nanoplatform for enhancing PDF effectiveness.


Introduction
Photodynamic therapy (PDT) has attracted widespread attention for cancer therapy, in which the photosensitizers (PSs) efectively kill cancer cells via converting O 2 into reactive oxygen species (ROSs) under the irradiation of a laser [1].PDT shows great promise because of its advantages of low trauma, high spatial selectivity, and a short course of treatment [2][3][4][5][6].Two crucial issues should be considered for improving the PDT efectiveness of cancer.First, oxygen plays a signifcant role in the production of ROSs during PDT [7][8][9][10][11].However, aggressive proliferation of solid cancer and the twisted vascular system lead to liquefaction necrosis [12], resulting in a partial pressure of oxygen below 5 mmHg [13].Second, the lifetime and diffusion distance of ROSs are as short as 200 ns and 20 nm, respectively, resulting in the ROSs only destroying the nearby molecules [14][15][16][17].Terefore, the targeting of PSs in organelles is particularly important for improving the therapeutic efcacy of cancer [18][19][20][21].
Mesoporous organosilica nanoparticles (MONs) have been widely used for the diagnosis and treatment of tumor due to their high specifc surface area (>800 m 2 /g), homogeneous mesoporous channels, easy surface modifcation, and good biocompatibility [22][23][24][25][26][27].We speculate that loading oxygen-carrying molecules into mesoporous organosilica is a feasible strategy to increase oxygen content in cancer and alleviate cancer hypoxia [28][29][30].In addition, mitochondria is the main energy production center and the site of aerobic respiration.Mitochondria plays an important role in the pathway of ROS-induced apoptosis [31][32][33].Yue et al.'s group developed various mitochondria-targeting drug delivery systems based on the triphenylphosphonium (CTPP) molecular structure and achieved excellent targeted therapeutic efects for cancer therapy [34][35][36].Terefore, developing a photosensitizer carrier that can both deliver oxygen to relieve hypoxia and modify CTPP molecules to achieve targeted delivery to mitochondria is expected to enhance the therapeutic efectiveness of PDT.

Synthesis.
Typically, 75 mL of ethanol was added to 170 mL of CTAB (6 mM) in water.Ten, 0.1 mL of ammonia was added to the mixed solution.Te solution was stirred at 35 °C and 500 rpm.After 5 min, 0.2 mL of tetraethyl orthosilicate (TEOS) was slowly added, and the temperature of the reaction system was raised to 60 °C.After 24 h, mesoporous silica nanoparticles (MSNs) were obtained and washed three times with ethanol.Next, 3.5 mg of MSNs was dispersed in 0.5 mL of ethanol, 8.5 mL of deionized water, 15 mg of CTAB, and 0.7 mL of ammonia-mixed solution, and the solution was stirred evenly at 35 °C and 500 rpm for 30 min.Ten, 0.1 mL of 1,2-bis (triethoxysilane) ethane (BTSE) was slowly added to the mixed solution, and the solution was stirred for 3 h.Te white focculent precipitate was washed with ethanol three times and water once.Te precipitate was dispersed in 35 mL of water for 12 h at 80 °C.Te EHMONs were collected via centrifugation and redispersed in an ethanol/HCl solution (Vethanol � 10 mL, VHCl � 20 μL) to remove CTAB.
One milliliter of CTPP (20 mg/mL) and 1 mL of photosensitizer Ce6 (20 mg/mL) were mixed with 0.5 mL of EDC (20 mg/mL, dissolved in DMF) and 0.5 mL of NHS (20 mg/mL, dissolved in DMF), respectively.Te mixture was shaken at room temperature for 3 h to activate the carboxyl groups.EHMONs were dispersed in 42 mL of ethanol, and 0.2 mL of ammonia and 0.3 mL of APTES were added to the solution.Te mixed solution was shaken for 12 h.Te precipitate was collected by centrifugation.30 mg of the precipitation was dispersed in the mixed solution of activated CTPP and Ce6.After 12 h, the surfacefunctionalized EHMONs-Ce6-CTPP were collected by centrifugation.

Loading Oxygen.
At normal temperature and pressure, a large amount of oxygen can be directly dissolved, and its oxygen solubility is three times that of blood (35-70 ml/dl at 25 degrees Celsius).Terefore, 1 mg of EHMONs-Ce6-CTPP was centrifuged and washed twice.Under vacuum, 0.1 mL of perfuoropentane solution was quickly added to EHMONs-Ce6-CTPP, and ultrasound was performed in an ice bath with a temperature below 4 °C for 2 min to obtain EHMONs-Ce6-CTPP@PFC.

Hemolytic Assay and In Vitro Toxicity.
In order to explore the hemolysis performance of EHMONs, one milliliter of blood obtained from Jinling Hospital was centrifuged at 2000 rpm for 5 min to collect the red blood cells (RBCs).Te obtained RBCs were diluted with physiological saline to 2 ml.Te RBCs suspension (0.2 ml) was incubated with the EHMONs-Ce6-CTPP in physiological saline (0.8 ml) at 35 °C 2 Bioinorganic Chemistry and Applications for 2 h.RBCs incubated with water and physiological saline were set as the positive and negative control, respectively.Finally, the supernatants were collected to measure the absorbance at 630 nm.Te hemolysis percentage of RBCs (%) was calculated by the following formula: Hemolysis rate � absorbance of sample -absorbance of negative control absorbance of positive control?absorbance of negative control × 100%. ( To determine the in vitro cytotoxicity, 4T1 human breast cancer cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 5% CO 2 concentration and 95% relative humidity, then seeded in 96-well plates (1 × 10 4 cells/per well) for 24 h.Afterwards, diferent concentrations of EHMONs-Ce6-CTPP were dispersed in RPMI 1640 medium (100 μL) and added to each well.After 24 h or 48 h, the medium from each well was replaced with 100 μL complete medium containing 10 μL CCK-8.After 1 h, the absorbance of the medium in each well was measured at 490 nm with a microplate reader.Cell activity was calculated by the following formula: Cell viability � the absorbance at 490 nm of experimental group the absorbance at 490 nm of control group × 100%. (2)

Targeted Mitochondria.
To detect performance of EHMONs in mitochondrial targeting, 4T1 human breast cancer cells were incubated in the culture plate at an inoculation density of 1 × 10 3 .100 μL Ce6, EHMONs-Ce6-CTPP, and EHMONs-Ce6-CTPP@PFC medium suspension (with Ce6 equivalent concentration of 1 μM) were then added to the cells, respectively.After 2 h, the culture medium was sucked out, and the cells were washed three times with PBS to remove the residual culture solution.Te cells were stained with mitochondrial stain for 20 min.Ten, excess stain was sucked out, and the cells were washed with PBS three times.DAPI dye was added to the cells to stain the nuclei.After 10 min, the staining solution was sucked out, and 100 μL PBS was added to the cells.Te cells were observed using laser confocal microscope (CLSM).Te fuorescence localization results were statistically analyzed by ImageJ software.

In Vitro Singlet Oxygen Detection.
To detect the singlet oxygen generation ability of EHMONs in vitro, 100 μL Ce6, EHMONs-Ce6-CTPP, and EHMONs-Ce6-CTPP@PFC medium suspension with equivalent Ce6 concentration (1 μM) were mixed with 10 μL SOSG (50 μM) and added to the cells, respectively.Te cells were irradiated by a 660 nm laser with a power of 1 W/cm 2 for 0, 1, 3, 5, and 10 min, respectively.Each experimental group was set up in three wells.Te fuorescence emission intensity of the sample well was measured using a microplate reader at the wavelength of 530 nm.
To further detect the generation of singlet oxygen at the cellular level, 4T1 human breast cancer cells were incubated for 24 h, 100 μL of Ce6, EHMONs-Ce6-CTPP, and EHMONs-Ce6-CTPP@PFC medium suspension with equivalent Ce6 concentration (1 μM) were added to the cells, respectively.Ten, the cells were incubated in the dark for 12 h, the medium was removed from the well, and the cells were washed three times with PBS.Ten, 100 μL of DCFH-DA (20 μM) was added to the cells.After 4 h, the residual solution was sucked out, and the cells were washed once.Ten, 100 μL of 1640 medium was added to the cells, and each well was irradiated with a 660 nm laser with a power of 1 W/cm 2 for 5 min.Te fuorescence emission intensity of each well was recorded with a microplate reader at 485 nm.

In Vitro Photodynamic Terapy.
To evaluate the efect of photodynamic therapy on EHMONs, 4T1 human breast cancer cells were incubated for 24 h, and then 100 μL Ce6, EHMONs-Ce6-CTPP, and EHMONs-Ce6-CTPP@PFC medium suspension with equivalent Ce6 concentration (1 μM) were added to the cells, respectively.After 12 h, the cells were washed three times with PBS and 100 μL of fresh medium was added to the cells, and they were irradiated with a 660 nm laser for 5 min (1 W/cm 2 ).Te medium in each well was replaced with 100 μL medium containing 10 μL CCK-8.After 1 h, the absorbance value of each well at 490 nm was detected by using a microplate reader, and 5 multiple wells were set for each experimental group.4T1 breast cancer cell activity was calculated by the following formula: Cell viability(100%) � the absorbance at 490 nm of experimental group the absorbance at 490 nm of control group × 100%.
Bioinorganic Chemistry and Applications 2.8.In Vivo Antitumor Efect.4T1 breast tumor model was constructed by injected into 1 × 10 6 cells into BALB/c mice on day 0. When the tumors reached to ∼130 mm 3 , the mice were randomly divided into 4 groups and injected with PBS, Ce6, EHMONs-Ce6@PFC, and EHMONs-Ce6-CTPP@PFC at an equivalent Ce6 dose of 20 μg per mouse at days 7, followed by 635 nm irradiation with a 660 nm laser for 5 min (1 W/cm 2 ) on day 8. Tumor volumes and body weights were recorded every two days during the two weeks.Te tumor volume was calculated according to the formula: tumor volume � length × width 2 × 0.5 mm 3 .
2.9.Characterization.S4800 scanning electron microscope (SEM, Hitachi, Tokyo), HT7700 transmission electron microscope (TEM, Hitachi, Tokyo, Japan), and FEI Talos F200x electron microscope were employed to capture SEM, TEM, and elemental mapping images, respectively.Nitrogen adsorption-desorption isotherms were measured using a Micromeritics ASAP 2020 analyzer at −196 °C.Before the measurements, the samples were degassed under vacuum at 150 °C for at least 10 h.Te specifc surface area (S BET ) was calculated with the Brunauer-Emmett-Teller (BET) method using the adsorption data in a relative pressure (P/P 0 ) range from 0.14 to 0.25.Te pore size distribution was obtained by applying proper nonlocal density functional theory (NLDFT) methods from the adsorption branch of isotherms.
A Brookhaven Zeta-PLAS analyzer (USA) was employed to characterize the hydrodynamic size and surface electronegativity of the nanoparticles.An Infnite M200 PRO microplate reader (Tecan, Switzerland) was employed to measure the absorbance.Material characterization was repeated at least three times.

Results and Discussion
Eccentric hollow mesoporous organosilica nanoparticles with organic-inorganic hybrid frameworks were prepared by anisotropic encapsulation and hydrothermal etching.In particular, MSNs with a uniform particle size were used as a seed.Mesoporous organosilica was anisotropically encapsulated on the MSNs to form the eccentric mesoporous organosilica nanoparticles by using 1,2-bis (triethoxysilyl) ethane (BTSE) as an organosilica precursor and the hexadecyltrimethylammonium bromide (CTAB) as a structural template.After hydrothermal treatment, the MSNs were etched away, and eccentric hollow mesoporous organosilica nanoparticles (EHMONs) were formed.TEM images showed that the obtained EHMONs had very good dispersion and uniform size (300 nm) (Figure 1(a)).Highmagnifcation TEM images showed clearly that the EHMONs possessed a large internal cavity (about 140 nm) which was conducive to the storage of guest molecules (Figure 1(b)).SEM and high-angle annular dark-feld scanning TEM (STEM-HAADF) images revealed that the EHMONs have ultrathin shells near the cavity side with a size of about 16 nm (Figures 1(c) and 1(d)).Te ultrathin shell was extremely favorable for the difusion of drug molecules.Elemental mapping images clearly show that C, Si, and O elements were evenly distributed in the EHMONs (Figure 1(e)), indicating their organic-inorganic hybrid frameworks.High-magnifcation TEM images showed that the surface of EHMONs possesses a highly ordered mesoporous structure (Figure 1(f )), which extremely facilitates the loading and release of drug molecule.We further investigated the physicochemical properties of the EHMONs.Te Fourier transform infrared spectrum (FT-IR) of the EHMONs showed two characteristic peaks at 2980 cm −1 and 1414 cm −1 (Figure 2(a)), which were assigned to the C-H stretching vibration.In contrast, the peaks were absent in MSN, indicating that EHMONs possessed the organic-inorganic hybrid framework.Nitrogen adsorption-desorption isotherms of the EHMONs showed a type IV curve (Figure 2(b)), indicating a typical mesoporous structure.In addition, the pore size of the mesoporous shell was calculated to be 2.3 nm by nonlocal density functional theory (Figure 2(c)).UV-visible spectra of the EHMONs-Ce6-CTPP showed two absorption peaks at 400 nm and 660 nm (Figure 2(d)), which coincided with the characteristic absorption peak of Ce6, indicating successful modifcation of Ce6.Te loading content of Ce6 in the EHMONs-Ce6-CTPP was determined to be up to 30 μg/mg.Te zeta potential of the EHMONs, EHMONs-Ce6, and EHMONs-Ce6-CTPP was measured to be −21 mV, −17.5 mV, and −5 mV (Figure 2(e)), respectively, indicating the successful modifcation of functional molecules.Dynamic light scattering (DLS) showed the hydrodynamic diameters of EHMONs and EHMONs-Ce6-CTPP were 370 nm and 390 nm, respectively.Te polydispersibility index (PDI) values of the EHMONs and EHMONs-Ce6-CTPP were 0.118 and 0.123, respectively, demonstrating excellent uniformity (Figure 2(f )).STEM-HAADF image of the modifed nanoparticles clearly showed no change in the morphology (Figure 2(g)).Elemental mapping images showed that C, O, Si, and P elements were distributed in the organic-inorganic hybrid framework of the EHMONs-Ce6-CTPP (Figure 2(h)), indicating the successful modifcation of CTPP on the nanoparticles.
Te PFC possessed the ability to carry oxygen to relieve the hypoxic microenvironments, so, the oxygen concentrations in H 2 O, EHMONs-Ce6-CTPP, EHMONs-Ce6-CTPP@PFC, or Ce6 solution was detected.Te result showed that the oxygen concentrations in the EHMONs-Ce6-CTPP@PFC solution increased to 13.4 mg/L within 2 min and remained constant for over 5 min, which is 1.9, 1.7, and 1.8-fold higher than that in H 2 O, EHMONs-Ce6-CTPP, and Ce6 groups, respectively (Figure 3(a)).Furthermore, the singlet oxygen generation capacity of EHMONs-Ce6-CTPP@PFC was also studied.SOSG was used to detect the generation of singlet oxygen.As shown in Figure 3(b), the fuorescence signal intensity of SOSG in the Ce6 group did not change signifcantly with the increase in laser irradiation time.In contrast, the fuorescence signal intensity of the EHMONs-Ce6-CTPP@PFC and EHMONs-Ce6-CTPP groups increased rapidly within 1 min, and tended to be stable after 5 min.Notably, the fuorescence signal intensity was much higher in the EHMONs-Ce6-Bioinorganic Chemistry and Applications  Bioinorganic Chemistry and Applications CTPP@PFC group compared to other groups, indicating that the loading oxygen-carrying PFCs in EHMONs-Ce6-CTPP@PFC can signifcantly enhance the generation rate and amount of the singlet oxygen under laser irradiation.Furthermore, the ROSs generated cellular level was detected using DCFH-DA.Te results showed that the fuorescence signal intensities of the EHMONs-Ce6-CTPP and EHMONs-Ce6-CTPP@PFC groups were signifcantly higher than those of other groups after laser irradiation (Figure 3(c)).It was noteworthy that the EHMONs-Ce6-CTPP@PFC + laser showed the strongest fuorescent signal in cancer cells, suggesting an increase in ROS content.
Te mitochondrial targeting performance of the EHMONs-Ce6-CTPP was further investigated.Compared with the other two groups, the Ce6 group showed the weakest fuorescence signal (Figure 4(a)).Te fuorescence intensity of the EHMONs-Ce6 group was slightly stronger than that of the free Ce6 group.In contrast, the EHMONs-Ce6-CTPP group shows the strongest fuorescence intensity, indicating that the mitochondria-targeting molecular CTPP contributed to improving the uptake efciency and quantity of particles by cells.Te ImageJ software results showed that the position of the green fuorescence of Ce6 in the EHMONs-Ce6-CTPP group was overlapping with that of the red fuorescence of mitochondria (Figure 4(b)), and the green fuorescence intensity was signifcantly higher than that of the EHMONs-Ce6 group (green area).Tese results indicated that EHMONs-Ce6-CTPP can target mitochondria and increase aggregation in mitochondria after entering cells.
Te hemolysis and cytotoxicity of the EHMONs were further investigated to demonstrate their potential for biomedical applications.After mixing red blood cells (RBCs) with the EHMONs-Ce6-CTPP at concentrations ranging from 25 to 800 μg/mL, only slight red color was observed in the supernatants (inset in Figure 5(a)).Quantitative analysis   6 Bioinorganic Chemistry and Applications indicated that the hemolytic activity of the EHMONs-Ce6-CTPP was 1.75% at the concentration up to 800 μg/mL (Figure 5(a)), suggesting that the EHMONs-Ce6-CTPP induced very light hemolysis toward RBCs.Te survival rate of cells remained above 80% after the cells were incubated with diferent concentrations of EHMONs-Ce6-CTPP (less than 200 μg/mL) for 24 h (Figure 5(b)).Upon further incubation to 48 h, the survival rate of 4T1 cells remained above 80%, indicating that EHMONs-Ce6-CTPP nanoparticles had good in vitro biocompatibility.Finally, we evaluated the photodynamic therapy efect of EHMONs-Ce6-CTPP@PFC on tumor cells.Te results showed that the cell survival rate of Ce6 + laser, EHMONs-Ce6 + laser, EHMONs-Ce6-CTPP + laser, and EHMONs-Ce6-CTPP@ PFC + laser groups were 90%, 79%, 53%, and 35% at a concentration of 200 μg/mL, respectively, indicating that the modifed targeting molecule CTPP signifcantly improves the efectiveness of photodynamic therapy.Notably, the EHMONs-Ce6-CTPP@PFC with oxygen-carrying PFC molecules killed about 65% of cancer cells, suggesting that the strategy of alleviating hypoxia and mitochondriatargeting efectively enhances the efect of PDT (Figure 5(c)).
To evaluate the safety of HMONs-Ce6-CTPP@PFCs in vivo, we investigated the blood physiological and biochemical parameters in mice treated with HMONs-Ce6-CTPP@PFCs and PBS.Te results showed no signifcant diferences in alanine aminotransferase (ALT), creatine kinase (CK), aspartate aminotransferase (AST), total bilirubin (TBIL), or direct bilirubin (DBIL) between the various groups of mice for diferent treatments (Figures 6(a)-6(f )).Moreover, we further examined the major organs of the mice using hematoxylin and eosin (H&E) staining.Te results Bioinorganic Chemistry and Applications revealed no signifcant morphological changes in the major tissues and organs of mice treated with HMONs-Ce6-CTPP@PFCs compared with those treated with PBS (Figure 6(g)).Tese fndings indicate that EHMONs-Ce6-CTPP@PFCs are safe for use in vivo during cancer We further evaluated the in vivo anticancer efcacy of the mitochondria-targeting and oxygen-self-supplying nanomedicines for the breast cancer.To this end, the 4T1 tumor model was constructed by inoculation of 4T1 cells into BALB/c mice.On day 7, the 24 mice were randomly divided into 4 groups, and intratumorally administered with PBS, Ce6, EHMONs-Ce6@PFC, and EHMONs-Ce6-CTPP@PFC, followed by NIR irradiation at 24 h postadministration.Te tumor volume of the mice was measured over 14 days (Figure 7(a)).Te tumor volume of the PBS group with laser irradiation presented a remarkable increase and reached 1980 mm 3 day 14 (Figure 7(b)).By contrast, the free Ce6 exhibited a slight inhibition of tumor growth (1504 mm 3 ) (Figure 7(b)).Moreover, the EHMONs-Ce6@PFC treatments showed moderate delay growth, while EHMONs-Ce6-CTPP@PFC treatment exhibits the distinct inhibition of tumor progression (392 mm 3 ).Tis result indicated that the tumor growth could be efectively 8 Bioinorganic Chemistry and Applications suppressed by combination of PFC and the mitochondriatargeting molecular TCPP.In addition, the administration of EHMONs-Ce6@PFC and EHMONs-Ce6-CTPP@PFC did not result in any signifcant reduction in body weight compared with the control groups (Figure 7(c)), indicating a lack of acute toxicity of the mitochondria-targeting and oxygen nanomedicine.After the treatments, it was observed that the photographs and weights of tumor on the tumor-bearing mice exhibited the same results (Figures 7(d) and 7(e)), suggesting that the mitochondriatargeting and oxygen self-supplying EHMONs are a promising nanomedicine for the photodynamic therapy of tumors.

Conclusion
In summary, we construct an eccentric hollow nanoplatform with hypoxia-alleviating and mitochondrial-targeting ability for enhancing photodynamic efcacy.Te prepared EHMONs possess a unique eccentric hollow structure, a large cavity, and ordered mesoporous channels.Tanks to their active surface properties and large cavity, the EHMONs are modifed with the mitochondria-targeting molecule CTPP and photosensitizer Ce6 and loaded with carrying compound PFCs.Hemolytic assays and in vitro toxicity experiments show that the EHMONs-Ce6-CTPP possesses good biocompatibility and mitochondrial targeting property, which increase the enrichment of photosensitizer Ce6 in mitochondria of tumor cells.Te EHMONs-Ce6-CTPP@PFCs increase the content of singlet oxygen in 4T1 triple-negative breast cancer cells.Tus, the EHMONs-Ce6-CTPP@PFCs show a signifcantly improved killing efect for triple-negative breast cancer cells, which also verifed in 4T1 tumor-bearing mouse models.Tis work provides a new route for photodynamic therapy of cancer by alleviating hypoxia and targeting mitochondrial strategies.