An Oxygen Self-sufficient Fluorinated Nanoplatform for Relieved

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Biological and Medical Applications of Materials and Interfaces

An Oxygen Self-sufficient Fluorinated Nanoplatform for Relieved Tumor Hypoxia and Enhanced Photodynamic Therapy of Cancers Shengnan Ma, Jie Zhou, Yuxin Zhang, Bo Yang, Yiyan He, Chen Tian, Xianghui Xu, and Zhongwei Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19840 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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ACS Applied Materials & Interfaces

An Oxygen Self-sufficient Fluorinated Nanoplatform for Relieved Tumor Hypoxia and Enhanced Photodynamic Therapy of Cancers Shengnan Mab, Jie Zhoub, Yuxin Zhangb, Bo Yanga, Yiyan Hea,b,*, Chen Tiana, Xianghui Xu a,b, and Zhongwei Gua,b,*

a College

of Materials Science and Engineering, Nanjing Tech University, No. 30 Puzhu Road(S),

Nanjing 211816, P. R. China.

b National

Engineering Research Center for Biomaterials, Sichuan University, No. 29 Wangjiang

Road, Chengdu 610064, P. R. China.

* Corresponding

author

E-mail address: [email protected] (Y. He), [email protected] (Z. Gu)

Keywords: oxygen self-sufficient, tumor oxygenation, tumor penetration, photodynamic therapy, orthotopic breast cancer

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Abstract The efficacy of photodynamic therapy (PDT) in the solid tumor is hampered by many challenges, including its oxygen self-consuming nature, insufficient oxygen levels within the hypoxic tumor microenvironment, and limited penetration of photosensitizers within tumors. Herein,

we

develop

the

IR780@O2-SFNs/iRGD

as

an

oxygen

self-sufficient

and

tumor-penetrating nanoplatform, which consists of IR780 loaded pH-sensitive fluorocarbon functionalized nanoparticles (SFNs) and iRGD as a tumor targeting peptide that can penetrate deeper within the tumor. Because of the high oxygen affinity and outstanding permeability of the obtained nanoplatform, oxygen and IR780 which are encapsulated in the same core can play their roles to the utmost, resulting in remarkably accelerated singlet oxygen production, as demonstrated in vitro by the 3D multicellular spheroids and in vivo by tumor tissues. More interestingly, a single-dose intravenous administration of IR780@O2-SFNs/iRGD into mice bearing orthotopic breast cancer could selectively accumulate at the tumor site, highly alleviate the tumor hypoxia, significantly inhibit the primary tumor growth and reduce the lung and liver metastasis, enabling the improved photodynamic therapeutic performance. Thus, this work paves an effective way to improve PDT efficacy through increasing tumor oxygenation and selective delivery of photosensitizers to the deep and hypoxic tumor.


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1.

Introduction

Photodynamic therapy (PDT) is an appealing non-invasive tumor therapy.1 The efficacy of PDT is reliant upon the photosensitization reaction between photosensitizers, oxygen molecules, and light.2 In the PDT process, photosensitizers accumulated in tumor tissue are irradiated by light of a particular wavelength, which interacts with oxygen in the tissue to generate toxic reactive oxygen species (ROS), especially the singlet oxygen (1O2) that plays the important role in driving localized necrotic and apoptotic cell death.3 The exact amount and tissue localization of this PDT-induced 1O2 determine the outcomes of this therapy. The availability of molecular oxygen to the areas of photosensitizers localization is, therefore, a necessary factor to gain the desired PDT effect. However, hypoxia in tumor microenvironment exists initially — the partial pressure of oxygen (pO2) in many hypoxic regions is about two orders of magnitude lower than that in normal tissues.4-5 Moreover, the photosensitization that induces continuous the depletion of tumor oxygen during the PDT process would further potentiate tumor hypoxia.6 Thus, insufficient oxygen supply in tumor regions could cause limited 1O2 production and resistance of solid tumors to PDT. Hence numerous techniques have been proposed to modulate the tumor hypoxia for further potentiation of PDT antitumor efficacy, such as extending irradiation with a low fluence rate 7, fractionating irradiation into light/dark periods,8-9 supplementing hyperbaric oxygen during radiation therapy,10 and inhibiting hypoxia-inducible factor 1 (HIF-1) pathway.11-12 Recently, nanomedicine-based approaches have been developed that offer improved biocompatibility by ACS Paragon Plus Environment


solubilizing the hydrophobic photosensitizers and enhance their tumor-selective delivery. These approaches have been further utilized in efforts to alleviate tumor hypoxia, and have shown great promise for solid tumor PDT.13 Such strategies include the enhancement of oxygen supply using red blood cells as oxygen transporters to facilitate PDT,14-15 and the reoxygenation of hypoxic tumor from the direct splitting of water into H2 and O2 using carbon nitride-based multifunctional nanocomposites.16 Meanwhile, a new technology via nanoparticles compose of catalase, CaO2 or MnO2 could catalyze intracellular hydrogen peroxide to generate O2.17-24 Moreover, metformin, a potent inhibitor of respiration, could also significantly enhance liposomal Ce6-mediated PDT efficiency by reducing tumor oxygen consumption.25 An alternative approach based on oxygen self-enriched perfluorocarbon nanocarriers is emerging to optimize tumor reoxygenation during PDT process.6, 26-28 Oxygen-shuttle nanoperfluorocarbon29 that could adsorb O2 in the lung and release O2 in the tumor would have better effects on reducing intratumoral hypoxia. Although no effective methods could thoroughly reverse the tumor hypoxia, sufficient intratumoral O2 supply is still indispensable to enhance the efficacy of PDT. Thus, optimizing the efficacy of limited O2 supplementation is critically important for the improvement of PDT. Another crucial challenge for efficient PDT is the limited tumor selectivity and penetration of photosensitizers in solid tumors,30 although some novel photosensitizers have been developed for deep permeability into tissues using near-infrared (NIR) excitation. However, the nature of the solid tumors such as the irregular blood vasculature increased interstitial fluid pressure, and high density extracellular matrix31-32 may restrain the penetration of photosensitizers within ACS Paragon Plus Environment

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tumors. To date, many approaches including the use of size- or surface- changeable nanoparticles to maintain or alter their physicochemical properties have been explored for deeper tumor penetration.33-34 Therefore, it would be highly desired to develop multifunctional photodynamic nanoplatforms, which are not only able to relieve the tumor hypoxia, but also can facilitate the penetration and distribution of drugs within the solid tumor, thereby achieving better photodynamic therapeutic performance. IR780 is a hydrophobic cyanine dye with absorption at 780 nm, providing an appropriate penetration depth in tumor tissue. Although it has been reported that IR780 is multifunctional in tumor imaging, PDT and photothermal therapy (PTT) in vivo, the application of IR780 in PDT is limited by its low singlet oxygen quantum yield. Hence, a tactic of utilizing IR780 to up-regulate 1O

2

production is urgently needed. To address all these challenges in PDT, we herein develop an

O2 self-sufficient photodynamic nanoplatform (designated as IR780@O2-SFNs/iRGD) to enhance PDT antitumor efficacy by improving tumor reoxygenation and drug penetration, which consists of pH-sensitive fluorocarbon functionalized nanoparticles (designated as SFNs) loaded with IR780 as a near-infrared photosensitizer, together with iRGD as a tumor targeting and penetrating peptide. As illustrated in Scheme 1, SFNs are fabricated from a super-hydrophobic fluorinated peptide dendron and a hydrophilic dextran that are connected via a hydrazone linkage that can be cleaved within intracellular lysosomes. Very recently, the drug release behavior of SFNs in physiological pH and acidic tumor conditions was evaluated in our previous work, which demonstrated that SFNs exploited as stimuli-responsive doxorubicin delivery platforms, ACS Paragon Plus Environment


allowing drug release inside cancer cells and maintaining stability during circulation.35 Because of the high electronegativity of fluorine and the super-hydrophobicity as well as good membrane permeability of perfluorinated carbon molecules,36-37 the hydrophobic core of SFNs possesses excellent oxygen affinity. Moreover, Anderson group reported that the mammary carcinoma lines including 4T1 cells highly expressed the αvβ3 integrin.38 iRGD can bind to αvβ3 integrin of cell surface and get into cells by neuropilin-1 dependent way, which enables chemically bonded or co-administered cargos to penetrate into the tumor tissue far away from vessels via a tumor selectivity way. Therefore, the co-administration of iRGD with SFNs could further enhance the cell permeability and tumor penetration, leading to a deeper delivery of both oxygen molecules and hydrophobic photosensitizers into extravascular tumor parenchyma, to achieve excellent therapeutic efficiency of PDT. Overall, we provide a new approach to selectively deliver both oxygen molecules and hydrophobic drugs to deep and hypoxic regions of the solid tumor. 2.

Materials and methods

2.1. Chemicals and reagents IR780 was bought from Sigma-Aldrich. Singlet Oxygen Sensor Green (SOSG) was ordered from Molecular Probes®. 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) was obtained from Beyotime. GL Biochem was the source for iRGD. Butyric anhydride was ordered from Aladdin®. Asta Tech Biopharmaceutical was the source of dimethyl sulfoxide (DMSO), anhydrous methanol, and hydrazine hydrate (NH2NH2·H2O). Regenerated Cellulose Dialysis Membranes were purchased from Spectrum/Por. The RPMI-1640 medium, fetal bovine serum ACS Paragon Plus Environment

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(FBS), penicillin, and streptomycin were purchased from Life Technologies Corporation. The Shanghai Institutes for Biological Sciences provided 4T1 cells. All buffers were prepared using Milli-Q® ultrapure water. 2.2. Measurement The structure of compounds was confirmed by 1HNMR. The self-assembly of nanoparticles was conducted using the ultrasonic (SB-5200 DTD). Dynamic light scattering (DLS) was used for the measurement of nanoparticle sizes at 25 °C on Malvern Zetasizer Nano ZS. Then, the morphology was further confirmed using transmission electron microscopy (TEM, Tecnai GF20S-TWIN, FEI, USA). The standard of ultraviolet absorption curve was obtained at a wavelength of 780 nm by nanodrop (Thermo Nanodrop 2000). The photostability of photosensitizer was evaluated by UV-vis spectrum (U-2900 Spectrophotometer). The fluorescence intensity of oxidized SOSG was detected by a microplate reader (Thermo Varioskan Flash), and the spectra were obtained using fluorescence spectrophotometer (Hitachi F-7000). The images for every section of 4T1 tumor spheres were taken using confocal laser scanning microscopy (CLSM, Leica TCP SP5). The distribution of the IR780-loaded nanoparticles in vivo was detected using fluorescence imaging system (CRi, Inc., USA) and quantitative analysis for IR780 in major tissues was measured using the fluorescence spectrophotometer (Hitachi F-7000). Tumor oxygenation of IR780-loaded nanoparticles was determined by using both the photoacoustic imaging (PA) system (MSOT in Vision 128, Germany) and immunofluorescence staining.



2.3. Generation 2 poly(L-lysine) dendron (Compound 1) synthesis H-Lys-OMe·2HCl (2320.7 mg, 10.0 mmol), Boc-Lys(Boc)-OH (8313.6 mg, 24.0 mmol), EDC·HCl (4600.8 mg, 24.0 mmol) and HOBt (3242.4 mg, 24.0 mmol) were dissolved in 80.0 mL CH2Cl2 solvent under N2 atmosphere and an ice-bath. After stirring for half an hour, DIEA (6.6 mL, 40.0 mmol) was added by dropwise. The solution was then steadily mixed at room temperature for 2 additional days. Generation 2 poly(L-lysine) dendron with Boc groups was obtained by purification using column chromatography. Subsequently, Boc groups of generation 2 poly(L-lysine) dendron (1632.0 mg, 2.0 mmol) were removed by 7.0 mL TFA in 6.0 mL CH2Cl2 for 7 hours. Pure generation 2 poly(L-lysine) dendron (Compound 1) was obtained by precipitation using anhydrous diethyl ether. 2.4. Synthesis of fluorocarbon /hydrocarbon functionalized peptide dendron (Compound 2a) 30.0 mL methanol was used to dissolve Compound 1 (832.0 mg, 2.0 mmol) in the presence of N2 atmosphere. Next, we added TEA (14.0 mL, 100.0 mmol) and heptafluorobutyric anhydride (2.5 mL, 10.0 mmol) and stirred for 2 days at 30 ̊C. Fluorocarbon functionalized peptide dendron (Compound 2a) was obtained by evaporation of the reaction solvent. 2.5. Hydrazine modified peptide dendron (Compound 3) synthesis 5.0 mL of methanol solution of Compound 2a (240.0 mg, 0.2 mmol) and 300 μL of hydrazine hydrate (6.0 mmol) were added to a flask in the presence of N2 atmosphere, and this reaction then proceeded for 2 days at 40 °C. Next, the solution was transferred to a dialysis membrane (MWCO 100 to 500 Da) and dialyzed against deionized water to remove the extra


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hydrazine hydrate. Hydrazine modified peptide dendron (Compound 3a) was gained by centrifugation and drying. The synthesis of Compound 2b and Compound 3b were similar to that of Compound 2a and Compound 3a, respectively. 2.6. Synthesis of SFAP and SHAP (Compound 5) Sodium periodate (NaIO4) was used for dextran oxidation, yielding aldehyde-functionalized dextran (Compound 4). When the molar ratio of dextran to NaIO4 was 8:1, the degree of aldehyde substitution of Compound 4 was 15.8%. Compound 3a (120.0 mg, 0.1 mmol) or Compound 3b (69.5 mg, 0.1 mmol) was conjugated to aldehyde groups of dextran (102.5 mg, 0.6 mmol) in equal mole ratio. Under N2 atmosphere, all reactants were dissolved with 25.0 mL DMSO, after which they were allowed to react for 2 days at 40 °C. pH-sensitive fluorocarbon functionalized amphiphilic polymer (SFAP, Compound 5a) or pH-sensitive hydrocarbon functionalized amphiphilic polymer (SHAP, Compound 5b) were gained via dialysis (MWCO = 3500 Da) and lyophilization. 1H NMR and 19F NMR in DMSO-d6 were used to confirm products. 2.7. Preparation and characterization of IR780-SFNs and IR780-SHNs The photosensitizer, IR780, loaded nanoparticles were prepared by dialysis method. 15.0 mg SFAP or SHAP was respectively soluble in 0.5 mL of DMSO, and adequately mixed with 0.5 mL of IR780 DMSO solution (6.0 mg/mL). The mixed solution was slowly dropped into water at pH 7.4 under the ultrasonic (40 KHz, 300 W, 25 °C). After stirring for 1 hour, the solution was dialyzed against distilled water for 24 hours (MWCO 1000 Da) to remove the free IR780 and organic solvent. Subsequently, the solid IR780-SFNs and IR780-SHNs were obtained by ACS Paragon Plus Environment


freeze-drying. Blank SFNs and SHNs nanoparticles were prepared using the same procedures without IR780 addition. Then, the IR780 loading capacity and encapsulation efficiency were conducted as follows: Firstly, to obtain the standard curve of IR780, we prepared different concentration (5 to 40 μg/mL) of IR780 DMSO solution. Then, the IR780 loaded nanoparticles were dissolved in DMSO and the absorptivity of the solution was detected at the wavelength of 780 nm. The IR780 loading capacity and encapsulation efficiency were calculated as below: LC (%) = [weight of loaded IR780/weight of IR780 loaded nanoparticles] ×100% EE (%) = [weight of loaded IR780/weight of IR780 in feeding] ×100% Meanwhile, the size and morphology of bran-new IR780 loaded nanoparticles was respectively measured by DLS at 25 °C and TEM. Another time, IR780-SFNs and IR780-SHNs were prepared as the mentioned above, and the total concentration of SFNs and SHNs was 4 mg/mL with the weight ratio of SFNs or SHNs to IR780 10:1. In the same process, free IR780 (0.4 mg, 50 μL) was dissolved using DMSO and added to 950 μL ultrapure water. Herein, there was no further dialysis for the IR780-SFNs, IR780-SHNs and IR780 solution. These prepared solutions were stored at 4 °C. 2 weeks later, the photos of samples were taken. Meanwhile, IR780, IR780-SHNs and IR780-SFNs were diluted to 20 μg/mL in terms of IR780, UV-vis absorption spectra of which were scanned after storage in dark.


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2.8. Evolution of oxygen loading and release 1 mL of IR780-SFNs solution (1 and 3 mg/mL), IR780-SHNs solution (1 and 3 mg/mL) and PBS in sample tubes were treated with 20 min oxygen flow for saturation (IR780@O2-SFNs, IR780-SHNs, and PBS), respectively. Next, the 1 mL of the oxygenated solution (1 and 3 mg/mL) was added into 4 mL PBS in 25 mL beaker with sealing film, and the oxygen concentration in real-time was monitored by a dissolved oxygen meter (DOG-3082). To measure oxygen loading, the PBS was deoxygenated by nitrogen, and the oxygen release of 1 mL of oxygenated solution (1 mg/mL) was conducted again. 2.9. Detection of singlet oxygen production in vitro Firstly, the fresh SFNs, SHNs, IR780@O2-SFNs or IR780-SHNs solution was made by using the method mentioned above and diluted to the concentration of 20 μg/mL for IR780, where the mass ratio of nanoparticles to IR780 was 10:1. 100 μL of SFNs, SHNs, IR780, IR780@O2-SFNs or IR780-SHNs solution per well was added to dark 96-well plate. PBS was used as a control. Then, 20 μL of 50 μM SOSG methanol solution was added and mixed together in the well. After irradiation for 20 s (808 nm, 2 W/cm2), the fluorescence spectra of oxidized SOSG were recorded via fluorescence spectrophotometer. At the same time, all samples were irradiated for 0 to 60 s in accumulation and the fluorescence intensity of oxidized SOSG was detected at 525 nm by a multifunctional microplate reader with 504 nm excitation wavelength. Moreover, 0.5, 1, 2, and 4 mg SFAP mixed with 0.1 mg IR780 in 50 μL DMSO, respectively. Then it was dispersed into PBS to make IR780 encapsulated at the mass ratio of 5, ACS Paragon Plus Environment


10, 20, or 40, respectively. After dilution and mixture with SOSG in the dark 96-well plate, the concentration of 16.70, 8.35 and 4.18 μg/mL IR780 solution were obtained. When the samples were irradiated 30 s, the fluorescence intensity of oxidized SOSG was measured by using the above procedure. 2.10.

Detection of ROS generation in multicellular 4T1 tumor spheroids 4T1 cells (2 × 105) were dispersed in 1.5 mL media containing 10% FBS and seeded in

6-well plate coated using 1.5 mL 1% (w/v) agarose PBS per well. After 4 days growing in a humidified environment, the 4T1 tumor spheroid diameters were approximate to 100 μm. Spheroids were transferred to a glass plate and respectively incubated 1 hour with groups of IR780@O2-SFNs/iRGD (with 0.5 mM iRGD), IR780@O2-SFNs, IR780-SHNs and IR780, and the IR780 concentration was 10 μg/mL for each group. And the saline group was as a control. Then, the culture medium of all groups containing IR780 was taken out and PBS was used to wash spheroids. Spheroids were then incubated with 25 mM DCFH-DA for 10 min, washed, and irradiated 20 s using 808 nm light. The fluorescence images of DCF for every section of 4T1 tumor spheroids were obtained via confocal laser scanning microscopy with 488 nm excitation wavelength. 2.11.

Detection of singlet oxygen generation in tumor tissues 4-6 week old Balb/c female mice were ordered from Chengdu Dashuo Biological

Technology Company and the following experiment was conducted under the criterion. 100 μL of 4T1 cell suspension (5×105 cells) was injected into dorsal right side subcutaneous of mice. ACS Paragon Plus Environment

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This tumor model was used in the detection of 1O2 generation and the biodistribution study in vivo. When the tumor volume achieved 400-500 mm3, different formulations were intratumorally injected into tumors. The formulations contain 25 μL SOSG (50 μM) and 25 μL IR780@O2-SFNs/iRGD (with 0.5 mM iRGD), IR780@O2-SFNs, IR780-SHNs, IR780 (0.2 mg/mL) or saline, respectively. Then, tumor tissues of all groups were irradiated by 808 nm light for 5 min. Tumors from four groups were collected. The frozen section of tumor tissue was at 7 μm thickness and the green fluorescence of oxidized SOSG was observed by CLSM with 488 nm excitation wavelength. 2.12.

In vitro cytotoxicity The 4T1 cells were plated at 1×104 cells per well on a 96 well plate and rested for 24 hours.

Cells were exposed to nanoparticle solutions of IR780@O2-SFNs/iRGD (with 0.5 mM iRGD), IR780@O2-SFNs, IR780-SHNs and IR780, respectively. The final concentrations of IR780 ranged from 1.0~7.5 μg/mL. To assess cytotoxicity without irradiation, after treated with nanoparticle solutions for 6 hours, the cells were incubated 24 hours with fresh culture medium in the dark. The media were then supplemented with CCK-8 (Dojindo, Japan) solution and a microplate reader measured the absorbance at 450 nm. Meanwhile, to evaluate cytotoxicity upon irradiation, the cells were pretreated with nanoparticle solutions for 4 hours followed by irradiation (808 nm, 2 W/cm2, 20 s). After 2 hours, the nanoparticles containing media were replaced with fresh culture medium. Then incubation for another 24 hours in the dark, the cell viability was evaluated using CCK-8. Cells incubated normally were used as a negative control. ACS Paragon Plus Environment


2.13.

In vivo biodistribution study The tumor established as the above description grew to approximately 150 mm3. Different

formulations were administered by tail intravenous injection (0.5 mg/kg IR780), with the saline group as a control. Post-injection 1 hour, 3 hours, 6 hours, and 9 hours, the biodistribution of fluorescence were inspected by using in vivo fluorescence imaging system (CRi, Inc., USA). For the quantification of IR780-loaded nanoparticles in tumor and tissues, after 1 hour injection, the BALB/c mice with 4T1 tumor models were dissected, and the tumor, heart, liver, spleen, lung and kidneys were collected. Then the tumor and tissues were washed with 0.9% sodium chloride solution, dried using filter paper, and weighed respectively, the tumor and tissues were ground and suspended in 1.5 mL NaOH (0.1 mol/L) with 1% SDS every 100 mg. The samples were kept 24 hours in the dark and centrifuged 10 min at 3000 r/min. The supernatant and standard samples of IR780 were quantified by using fluorescence spectrophotometer (excitation at 704 nm and emission at 808 nm). Organs collected from saline treated mice were also measured to determine the background levels in different organs. Results are given as ng of IR780 per g of tissue extracts. 2.14.

Photoacoustic imaging and immunofluorescence The orthotopic breast cancer model was formed by injection 5 × 105 4T1 cells into the

mammary gland of female BALB/c nude mice. When tumor reached 150 mm3, the mice were administered with 200 μL of IR780@O2-SFNs/iRGD (with 0.5 mM iRGD), IR780@O2-SFNs and IR780-SHNs by tail intravenous injection (0.5 mg/kg IR780). Post-injection 1 and 2 hours, ACS Paragon Plus Environment

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the signals of IR780 for all groups were collected using photoacoustic imaging (PA) system. Also, the signals of oxygenated hemoglobin and deoxygenated hemoglobin in tumor tissue for different groups were detected. Then, the tumors were excised for histological observation, and their frozen sections were immunofluorescence staining by DAPI and HIF-1α. 2.15.

In vivo antitumor effect of photodynamic therapy When orthotopic breast tumor grew to 100 mm3, the BALB/c nude mice were separated

into 5 groups (6 per group). They were treated with 200 μL saline, IR780, IR780-SHNs, IR780@O2-SFNs, and IR780@O2-SFNs/iRGD by intravenous injection (0.5 mg/kg IR780). Post-injection 1 hour, tumor regions were irradiated for 5 min (808 nm, 2 W/cm2). During the therapy, the weight of the body and the volume of the tumor (1/2 × length × width2) were determined every other day. 2.16.

Histological and immunohistochemical analysis After 12 days mice were sacrificed, and the organs and tumors were taken out for paraffin

section, which was fixed in 4% (v/v) formalin for 24 hours. Samples were paraffin embedded and sectioned at 5 micron thickness. Next, hematoxylin and eosin (H&E) was used to stain sections for histopathological evaluation. Platelet endothelial cell adhesion molecule-1 (CD31) test, monoclonal antibody Ki-67 response for proliferation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assessments were performed to conform the antitumor efficacy of PDT in vivo.



2.17.

Statistical analyses There was a minimum of 6 samples per experiment, and all measurements were conducted

in triplicate. Data are means ± standard deviation. **For Figure 4, 5B, 6B, 6C, 7B, 8B and 9A, a one-way ANOVA followed by Tukey's post-hoc test was used to assess significance. SPSS v19.0 for Windows was used for all testing. P < 0.05 was the chosen significance threshold. 3.

Results and discussion

3.1. SFAP and SHAP synthesis and characterization

Scheme 1. Schematic illustration of an oxygen self-sufficient and tumor-penetrating nanoplatform for enhanced PDT against the orthotopic breast cancer. (A) Structure and oxygen self-sufficient mechanism of IR780@O2-SFNs/iRGD nanoplatform. IR780 and oxygen are loaded in the core of SFNs. The cleavage of hydrazone bond in pH 5.0 results in the disassembly of nanoplatform. Accompanied by self-sufficient oxygen, IR780 induces reactive oxygen species generation under NIR irradiation. (B) The membrane-permeable SFNs co-administrated with iRGD facilitating the selective and effective accumulation in the tumor region after intravenous injection, and enabling tumor penetration and oxygenation in hypoxic tumor microenvironment for further potentiation of PDT efficacy under NIR irradiation.


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In this work, IR780@O2-SFNs/iRGD nanoplatform with oxygen self-sufficient nature and tumor penetrating ability was designed, as illustrated in Scheme 1. This nanoplatform consisted of pH-sensitive fluorocarbon-functionalized nanocarriers (SFNs) and the tumor-targeting and penetrating peptide, iRGD. SFNs were derived from the self-assembly of the pH-sensitive fluorocarbon functionalized amphiphilic polymer (SFAP) in aqueous. In briefly, the synthesized second generation peptide dendron which containing four super-hydrophobic fluorocarbon chains was modified with hydrazine, then the hydrazine group was further conjugated onto the aldehyde-dextran using a hydrazone bond that could be cleaved within intracellular lysosomes to obtain desirable SFAP. The pH-sensitive hydrocarbon-functionalized amphiphilic polymer with dendrons end-capped with hydrocarbon analogues (SHAP) was also synthesized as a negative control. The synthesis of SFAP and SHAP referred to the progress that we have reported previously

35

with a minor modification. Details about the synthetic routes (Figure S1 and S2)

and characterization (Figure S3 and S4) of both SFAP and SHAP could be found in Supplementary data. The 1H-NMR results (Figure S3) confirmed that both SFAP and SHAP were successfully synthesized, and the average number of dendrons attached to per 100 repeating glucose units of the dextran was estimated to be 7.08 (the degree of substitution: 7.08%). The 19F-NMR

spectrum of SFAP respectively presented three peaks at 80.34 ppm, 119.83 ppm and -

126.91 ppm, which revealed three fluorinated species in the fluorocarbon chain (Figure S4).



3.2. Preparation and characterization of IR780-SFNs and IR780-SHNs Both pH-sensitive amphiphilic SFAP and SHAP polymers were able to self-assemble into SFNs and SHNs (pH-sensitive hydrocarbon-functionalized nanocarriers) micelles, respectively. Since the SFNs contained hydrophobic fluorinated cores, we then investigated the ability of SFNs as reservoirs for hydrophobic photosensitizers. IR780, a typical near-infrared (NIR) heptamethine indocyanine dye, was used as a model hydrophobic photosensitizer for PDT. Using a dialysis method, SFAP and SHAP could easily encapsulate IR780 in the hydrophobic core to form IR780-SFNs and IR780-SHNs nano-micelles, respectively. According to the standard curve of IR780 at the wavelength of 780 nm (Figure S5), the IR780 loading content (LC) of SFNs and SHNs was 10.20% and 11.56%, respectively (Table S1). To better investigate how fluorocarbon domains influence the oxygen dissolving capacity and 1O2 generation efficacy, we controlled the mass ratio of amphiphilic polymers and IR780 at 10 to prepare IR780-SFNs and IR780-SHNs for the following study.


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Figure 1. (A) Size distributions and TEM images (inset) of IR780-SFNs and IR780-SHNs. (B) The optical images of IR780-SFNs, IR780-SHNs and IR780 before and after two weeks of storage at 4 °C. The free IR780 was dissolved in DMSO firstly, then that was dispersed in water solution. IR780-SFNs and IR780-SHNs solutions were freshly prepared. UV-vis absorption spectra of (C) IR780, (D) IR780-SHNs and (E) IR780-SFNs (20 μg/mL in terms of IR780) after storage in dark. (F) The relative absorption of IR780, IR780-SHNs and IR780-SFNs at 780 nm after storage in dark. Dynamic light scattering (DLS) established the size of IR780-SFNs and IR780-SHNs severally were 75 nm and 80 nm (Figure 1A). According to transmission electron microscopy (TEM), these nanoparticles were well dispersed and possessed spherical morphology and the size (about 50 nm) was smaller than the result from DLS. The difference in DLS and TEM outcomes is likely related to the fact that DLS determined the hydrodynamic size of particles and not their true physical size. The images in Figure 1B exhibited that IR780-SFNs solution (4.0 mg/mL) could still be homogenously dispersed and remain stable for more than two weeks. However, obvious precipitation and aggregation could be observed at the bottom of both free IR780 and IR780-SHNs groups. These phenomena demonstrated that the peptide dendrons capped using four super-hydrophobic fluorocarbon chains had a branching structure that allowed SFNs to better harbor hydrophobic photosensitizers and maintain the long-term stability. Meanwhile, the photostability of IR780 loaded nanoparticles was explored using UV-vis spectrum. After storage in dark, the absorption maximum of IR780 and IR780-SHNs declined observably every day (Figure 1C and 1D). IR780-SFNs could keep the relatively high absorption at 780 nm in Figure 1E and 1F. Thus, the IR780-SFNs not only have the thermodynamic stability but also keep the ACS Paragon Plus Environment


photostability. The size and morphology of IR780-SFNs in pH 5.0 buffer were also detected by DLS and TEM. The results (Figure S6) showed that IR780-SFNs in acid condition had a large size and irregular morphology, which was contributed to the rapid disassembly process through cleavage of hydrazone linkages between the dextran and fluorinated dendrimer. 3.3. Oxygen loading and release behavior

Figure 2. Oxygen loading and release behavior. (A) O2 concentration changes after addition of IR780@O2-SFNs, IR780-SHNs and PBS into PBS buffer without deoxygenating. The samples were added into PBS at the 60 seconds. And the oxygen concentration was monitored all the time by a dissolved oxygen meter (DOG-3082). The process was conducted at 1 mg/mL and 3 mg/mL of the nanocarriers. (B) The oxygen release behavior of IR780@O2-SFNs, IR780-SHNs and PBS in deoxygenated PBS buffer. The deoxygenated PBS was processed by nitrogen, which was also monitored by a dissolved oxygen meter (DOG-3082). 1 mg/mL of nanoplatforms was adopted to calculate the oxygen loading capability. Because of the high electronegativity of fluorine and the super-hydrophobicity of perfluorinated carbon molecules,36-37 the hydrophobic core of SFNs generated from fluorinated peptide dendrons should possess excellent oxygen affinity. Therefore, not only hydrophobic ACS Paragon Plus Environment

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photosensitizers but also oxygen molecules could be loaded in the core of SFNs. We thus investigated the potential application of SFNs as oxygen reservoirs, allowing oxygen loading, and gradual release in a hypoxic environment. The oxygen loading and release behaviors were studied using a dissolved oxygen meter (DOG-3082) to test dissolved oxygen concentrations in PBS buffer in real time. Firstly, the as-prepared IR780-SFNs, IR780-SHNs and PBS were thoroughly saturated with oxygen (IR780@O2-SFNs, IR780-SHNs and PBS), and then the oxygenated solution was added into PBS buffer, respectively. The dissolved oxygen concentrations of all groups increased immediately, then saturated followed by decreasing over time due to the overflow of oxygen (Figure 2A). Interestingly, the dissolved oxygen concentration of IR780@O2-SFNs group was markedly greater than that of IR780-SHNs group at an equivalent solution concentration. Moreover, the dissolved oxygen concentration notably enhanced with the increasing of IR780@O2-SFNs solution concentration. There was almost no difference between the IR780-SHNs and PBS groups in dissolved oxygen concentration despite the IR780-SHNs concentration increased. These findings demonstrate the excellent oxygen loading and release capacities of SFNs. To further study the oxygen loading content and release behavior of IR780@O2-SFNs, 1 mL of the oxygen saturated IR780@O2-SFNs solution (1 mg/mL), IR780-SHNs solution (1 mg/mL) and PBS were added into the deoxygenated PBS (prepared by boiling and cooling to 25 °C and pretreated with nitrogen), respectively. Meanwhile, a polytetrafluoroethylene sealing film was used to protect deoxygenated PBS from the air. It was found that IR780@O2-SFNs was distinct ACS Paragon Plus Environment


from IR780-SHNs and PBS groups, and the oxygen saturated IR780@O2-SFNs could obviously enhance the dissolved oxygen concentration in deoxygenated PBS, indicating the efficient oxygen loading and gradual release of IR780@O2-SFNs in a hypoxic condition (Figure 2B). Calculated from the oxygen release curve, the oxygen loading capability of IR780@O2-SFNs at 25 °C was measured to be 9.6 mg oxygen per 1 g SFNs. Hence, the self-assembly SFNs from fluorocarbon functionalized nanoparticles could act as efficient oxygen reservoirs and effectively release oxygen in a hypoxic environment. 3.4. Singlet oxygen production in vitro

Figure 3. 1O2 generation of different groups in vivo as determined based on oxidized SOSG fluorescence. (A) The fluorescence spectra of oxidized SOSG for different groups under 20 s of irradiation by 808 nm laser. (B) 1O2 generation of IR780@O2-SFNs (w/w, 10), IR780-SHNs (w/w, 10), IR780 and PBS groups under different irradiation times. (C) The 1O2 generation of


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IR780@O2-SFNs (16.70, 8.35 and 4.18 μg/mL IR780) with different mass ratios of SFNs and IR780. Once the superiority of encapsulation hydrophobic photosensitizers and oxygen molecules into SFNs was confirmed, we immediately turned to compare the

1O

2

generation of

IR780@O2-SFNs with that of IR780-SHNs in vitro and in vivo, as determined using the fluorescence intensity of oxidized SOSG. SOSG reagent was employed as an indicator of 1O2 production. The fluorescence of SOSG is quenched in its initial state and generates strong fluorescence upon oxidation by 1O2,26, 29 The evaluation of 1O2 production from IR780@O2-SFNs, IR780-SHNs, IR780, SFNs and SHNs as well as PBS after different irradiation time was performed by measuring the oxidized SOSG fluorescence. After 20 s of irradiation, the oxidized SOSG fluorescence from IR780@O2-SFNs was much higher than that from IR780-SHNs or IR780 group (p < 0.05, Figure 3A). With increasing irradiation time, the fluorescence intensity of oxidized SOSG for IR780@O2-SFNs increased obviously. Whereas there was a negligible increasement in fluorescence signal from IR780-SHNs or IR780 groups (Figure 3B). Overall, compared with IR780-SHNs and IR780, IR780@O2-SFNs group obtained a significantly higher level of 1O2 production (p < 0.05), suggesting that SFNs serving as oxygen molecules and photosensitizers carriers could enhance the level of 1O2 generation, by virtue of the high electronegativity of fluorine from fluorocarbon chains functionalized peptide dendrons.36-37 To further validate the influence of the fluorocarbon functionalized nanoparticles on 1O2 production, we prepared a series of fresh IR780@O2-SFNs solutions with the mass ratios of



SFNs vector to IR780 ranging from 0 to 40 (Figure 3C). Following irradiation, the oxidized SOSG fluorescence from all IR780@O2-SFNs groups was stronger than that from IR780 group (SFNs to IR780 ratio is 0). When the concentration of IR780 was diluted from 16.70 to 4.18 μg/mL, it caused a remarkable relative reduction in fluorescence intensity between IR780@O2-SFNs (SFNs to IR780 ratio is 10) and IR780 (SFNs to IR780 ratio is 0) groups, from 3.63-fold to 1.52-fold. Additionally, at the same concentration of IR780, the fluorescence intensity of oxidized SOSG depended on the mass of SFNs vector. It illustrates that SFNs play a key role in 1O2 production in photodynamic therapy. The nature of this phenomenon is that the fluorinated inner core of SFNs has a positive effect on the oxygen loading capacity. 3.5. ROS generation in multicellular 4T1 tumor spheroids


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Figure 4. (A) ROS generation in 4T1 tumor spheroids as detected with DCFH-DA (scale bar = 25 μm). The spheroids were incubated with IR780@O2-SFNs/iRGD, IR780@O2-SFNs, IR780-SHNs, IR780 and saline for 1 hour. DCFH-DA was a ROS indicator. (B) 1O2 production in tumor tissues determined using oxidized SOSG in frozen sections (scale bar = 25 μm). Mice bearing tumors were administered different formulations containing SOSG through intratumoral injection followed by 5 min of irradiation. Green fluorescence indicates the generation of 1O2. Encouraged by the considerable enhancement in 1O2 generation, we then inspected the influence of SFNs on the generation of ROS including

1O

2

in three-dimensional (3D)

multicellular 4T1 tumor spheroids, with the use of DCFH-DA as a fluorometric assay for intracellular

ROS

detection.19,

24

3D

multicellular

spheroids

were

incubated

with

IR780@O2-SFNs/iRGD, IR780@O2-SFNs, IR780-SHNs, IR780 and saline for 1 hour respectively, then ROS production was detected using DCFH-DA following irradiation. The images of different sections for 3D multicellular spheroids were obtained and overlaid by using confocal laser scanning microscopy (Figure 4A). Both IR780-SHNs and IR780-treated 3D multicellular spheroids showed similar green fluorescence intensity of DCF, which were weaker than that of the IR780@O2-SFNs group. Meanwhile, IR780@O2-SFNs/iRGD group showed a slight advantage over IR780@O2-SFNs. More interestingly, most of the fluorescence pixels of IR780-SHNs and IR780 groups located on the periphery of tumor spheroids. However, the green pixels of IR780@O2-SFNs and IR780@O2-SFNs/iRGD groups could be observed inside the tumor spheroids, which were far from the periphery. Collectively, the phenomena described above suggested that IR780@O2-SFNs and IR780@O2-SFNs/iRGD groups could dramatically



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improve ROS generation in 3D multicellular 4T1 spheroids, revealing the enhanced 1O2 generation.

Moreover,

accompanied

by

the

tumor-penetrating

iRGD

peptide,

the

IR780@O2-SFNs could achieve deep penetration and uniform distribution of ROS. These results could indeed be ascribed to the excellent oxygen affinity, good membrane permeability of fluorocarbon

segments

36-37

and

prominent

stability

of

IR780@O2-SFNs

and

IR780@O2-SFNs/iRGD as compared with hydrocarbon analogues. 3.6. Singlet oxygen generation in tumor tissues In view of the excellent achievements of IR780@O2-SFNs and IR780@O2-SFNs/iRGD in 3D multicellular spheroids, it was possible to produce the same performance in vivo. Mice bearing

tumors

were

administered

different

formulations

(IR780@O2-SFNs/iRGD,

IR780@O2-SFNs, IR780-SHNs, IR780 and saline) containing SOSG through intratumoral injection followed by irradiation (808 nm, 2 W/cm2, 5 min). By comparing fluorescence images of tumor frozen sections among all groups (Figure 4B), it was found that the tumors treated with IR780@O2-SFNs/iRGD or IR780@O2-SFNs showed the marked fluorescence, whereas negligible fluorescence signals could be detected in IR780-SHNs, IR780 and saline groups. Although faced with the complex environment in tumor tissue, higher generation and deeper penetration of 1O2 by IR780@O2-SFNs/iRGD and IR780@O2-SFNs could also be successfully achieved in vivo. It would not be ignored that the signal of 1O2 generation in tumor tissues was enhanced by doping iRGD peptide. By the above results, it is meaningful to the further research in anticancer effect.


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3.7. Cytotoxicity with or without irradiation

Figure 5. (A) Cytotoxicity of different formulations against 4T1 cells without irradiation. After incubated with 4T1 cells 6 hours, the formulations were replaced with the free medium. 4T1 cells were incubated another 24 hours and viability was determined via CCK-8. (B) Phototoxicity of different groups against 4T1 cells upon laser irradiation (808 nm, 2 W/cm2, 20 s). After thoroughly investigating the influence of SFNs as oxygen and photosensitizers vectors on 1O2 generation in vitro and in vivo, we performed in vitro photodynamic therapy against 4T1 cells. Moreover, iRGD was employed to further improve the tumor selectivity39 and penetration ability.40 Because iRGD contains a tissue penetration element called CendR, it is able to be actively transported through tumor vessels and through the tumor stromal environment in a manner dependent on αvβ3 integrin and neuropilin-1.41 As shown in Figure 5A, all formulations never displayed any measurable toxic effect in cells without irradiation in the selected IR780 concentration ranges from 1.0 to 7.5 μg/mL. Notably, a striking decline in the cell viability was detected in both IR780@O2-SFNs/iRGD and IR780@O2-SFNs groups after irradiation. Additionally, the cell viability of IR780@O2-SFNs/iRGD and IR780@O2-SFNs groups treated 4T1 cells was less than 20% when IR780 concentration increased to 5.0 μg/mL (Figure 5B).



Obviously, the photodynamic therapy effect in vitro could be improved because the 1O2 production was increased by using oxygen self-sufficient SFNs. 3.8. In vivo biodistribution

Figure 6. In vivo biodistribution. (A) NIRF images of 4T1 tumor-bearing nude mice following systemic administration of IR780 formulations. Different formulations were administered by tail intravenous injection (0.5 mg/kg IR780). White dashed lines indicate the tumor locations. Arrows mark the location of the urinary bladder. (B) The fluorescence intensity of IR780 formulations for tumor tissue at different time points. (C) The quantification of IR780-loaded nanoparticles in tumor and organs by fluorescence spectrophotometer. Taken together, the significant enhancement of 1O2 generation in vitro and in vivo, as well as the preliminary in vitro phototoxicity data encouraged us to investigate the PDT effect of


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IR780@O2-SFNs/iRGD in vivo. In general, the biodistribution of drug loaded nanoparticles in tumor tissue determines the antitumor efficacy. To further confirm the targeting ability of IR780@O2-SFNs/iRGD mediated by passive tumor targeting and active tumor homing of the iRGD peptide, a 4T1 tumor-bearing mice model was established to evaluate in vivo biodistribution

using

in

vivo

fluorescence

imaging

system.

A

single

dose

of

IR780@O2-SFNs/iRGD, IR780@O2-SFNs, IR780-SHNs and IR780 at 0.5 mg/kg IR780, as well as saline was intravenously injected into 4T1 tumor-bearing mice. Figure 6A showed that intense signal could be clearly visualized and exhibited prominent accumulation in tumor area of the mice at 3 hours after IR780 treatment. This phenomenon could be explained by the preferential in vivo tumor accumulation property of IR780 alone42. However, IR780 was cleared from the circulation and accumulated in the urinary bladder and urinary meatus of the mice by 6 h post-injection. The observed fluorescence signals of IR780 treated mice enabled us to conceive that some intravenously injected IR780 was rapidly excreted through the renal route43-45. More importantly, the fluorescence signals accumulated in tumors of both IR780@O2-SFNs treated mice and IR780@O2-SFNs/iRGD treated mice were much higher than that of IR780-SHNs treated mice, demonstrating passive tumor targeting of SFNs due to high stability and the enhanced permeability and retention (EPR) effects. Fluorescence signals in tumors achieved their maximum levels at 1 hour after treatment (Figure 6B), which might result from the rapid tumor accumulation and disassembly of nanoparticles. As the released IR780 could be cleared by free diffusion, the fluorescence in tumor areas of IR780-SHNs and IR780-treated mice gradually



decreased to near background levels, whereas the IR780@O2-SFNs/iRGD treated mice still showed strong signals in tumor, suggesting the efficient tumor accumulation and retention of IR780@O2-SFNs/iRGD, which would be of great benefit in increasing the therapeutic index of PDT without any side effects upon systemic administration. After the validation of biodistribution by using in vivo real-time imaging, we further conducted a quantitative investigation to more accurately assess the relative levels of IR780 in the tumors and vital organs (heart, liver, spleen, lung and kidneys). Those mice were sacrificed and organs were excised at 1 hour after intravenous injection for biodistribution study. After dissolution and centrifugation, the collected supernatant and standard samples of IR780 were detected using fluorescence spectrophotometer to determine IR780 levels in those samples. Organs collected from saline-treated mice were also measured to determine the background levels in different organs. It could be clearly observed that the liver and the tumor showed strong IR780 accumulation 1 hour after intravenous injection of different IR780 formulations (Figure 6C), suggesting that foreign entities were primarily retained in the reticuloendothelial system (RES) organs, such as the liver via blood circulation.46 Nevertheless, compared with free IR780 treated mice, IR780 tumor distribution was increased to 2.2-fold when delivered with IR780@O2-SFNs. Notably, the IR780 distribution of IR780@O2-SFNs/iRGD treated group in the liver was reduced by almost half and increased to 3.5-fold in the tumor as compared with free IR780 treated group. These findings might be attributable to the hydrophilic dextran and stability of IR780@O2-SFNs that allowed IR780 to escape from the capture of RES and thereafter ACS Paragon Plus Environment

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undergo preferential accumulation within the tumor via EPR. Additionally, a more desirable biodistribution was observed in the IR780@O2-SFNs/iRGD treated group, with higher tumor and lower liver accumulation, emphasizing the enhanced accumulation in tumors via iRGD. The IR780 concentration in the other organs was quite low and inadequate to induce adverse effects. Thus, this strategy could enhance the specificity of photosensitizers delivery, reduce accumulation in healthy tissues, and maximize photosensitizers concentration at the tumor site, resulting in a greater potentiation of photodynamic therapy. 3.9. Relieving Tumor Hypoxia in vivo

Figure 7. (A) Photoacoustic imaging of nude mice bearing the orthotopic breast cancer before and after 1 hour systemic administration of IR780@O2-SFNs/iRGD, IR780@O2-SFNs and IR780-SHNs (0.5 mg/kg IR780). The signals of Hb (blue) and HbO2 (red) were collected using photoacoustic imaging (PA) system. The white circled line represents the position of tumor.


(B)


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The improved percent of HbO2 in tumors by treatment of IR780@O2-SFNs/iRGD, IR780@O2-SFNs and IR780-SHNs. The mean intensity of HbO2 was improved by 50.1%, 45.2% and 8.6%, respectively. Next,

we

investigated

whether

the

nanoplatforms

(IR780@O2-SFNs/iRGD,

IR780@O2-SFNs and IR780-SHNs) could relieve tumor hypoxia upon systematic administration. Therefore, the in vivo photoacoustic imaging system (MSOT in Vision 128) and the HIF-1α staining assay were used to estimate the tumor hypoxia status in a 4T1 orthotopic breast tumor model before and after treatment. Meanwhile, photoacoustic images (Figure S7) showed the IR780 signals in tumor tissues of IR780@O2-SFNs/iRGD, IR780@O2-SFNs and IR780-SHNs treated mice, respectively, which were consistent with NIRF images (Figure 6A). Photoacoustic imaging could also detect the signals of oxygenated hemoglobin (HbO2, λ = 850 nm) and deoxygenated hemoglobin (Hb, λ = 750 nm) in the blood, reflecting the status of blood oxygenation inside the tumors.28-29,

47

As collected by the photoacoustic imaging system and

shown in Figure 7, the red and blue signals represented HbO2 and Hb, respectively. 1 hour after intravenous injection of IR780@O2-SFNs/iRGD and IR780@O2-SFNs, the red HbO2 signals over the entire tumor area were stronger than that of pre-administration, while the signal of HbO2 was still very slight after treatment of IR780-SHNs group (Figure 7A). The mean intensity of HbO2 was improved by50.1%, 45.2% and 8.6% for IR780@O2-SFNs/iRGD, IR780@O2-SFNs and IR780-SHNs group, respectively (Figure 7B). These result above demonstrated that the SFNs acting as an oxygen self-sufficient nanoplatform play a key role in relieving tumor hypoxia. Meanwhile, iRGD as a tumor-penetrating peptide could enhance the photosensitizers ACS Paragon Plus Environment

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concentrated in tumor tissue (Figure 6A and S7) and tumor oxygenation. Hence, it could be concluded that IR780@O2-SFNs/iRGD could deliver both oxygen and photosensitizers in the hydrophobic core to the deep and hypoxic regions of solid tumors, and significantly improve the tumor oxygenation.

Figure 8. (A) The immunofluorescence imaging of tumor slices images. The hypoxia area and nucleus was indicated by HIF-1α (green) and DAPI (blue). (B) The quantification of tumor hypoxia for different groups. Meanwhile, to further confirm the tumor hypoxia relieving by IR780@O2-SFNs/iRGD in vivo, we performed the hypoxia-inducible factor (HIF)-1α staining assay in 4T1 orthotopic breast tumor model after intravenous injection of different formulations. HIF-α is a hypoxia-associated protein that can drive vascularization, and this protein breaks down in normoxic environments.16, 18, 48-49

Thus, the signal of HIF-α can reflect the level of tumor hypoxia. It was notable that the

HIF-1α signals obviously decreased in both IR780@O2-SFNs and IR780@O2-SFNs/iRGD treated groups (Figure 8A), which were consistent with photoacoustic imaging results, revealing



the effective improvement of tumor oxygenation. The green HIF-α positive signal of IR780@O2-SFNs/iRGD treated tumor tissue was slightly higher than that of IR780@O2-SFNs treated tumor tissue, with values of 12.20 ± 0.03% and 8.29 ± 0.03%, respectively (Figure 8B). Such an enhancement in tumor oxygenation of IR780@O2-SFNs/iRGD could be attributed to the efficient active tumor homing of the iRGD peptide. Interestingly, as compared with the hypoxic status of SFNs treated groups, the tumors treated with IR780-SHNs and saline showed much stronger HIF-1α positive signals and more hypoxic areas. And there was no difference between IR780-SHNs and saline groups, indicating severe hypoxic conditions of tumors. Thus, both in vivo photoacoustic imaging results and ex vivo immunofluorescence staining data demonstrated that the tumor hypoxia was significantly relieved by the oxygen self-sufficient SFNs. 3.10.

In vivo antitumor effect of photodynamic therapy To investigate the PDT-induced antitumor effect of IR780@O2-SFNs/iRGD in vivo, we

intravenously injected IR780@O2-SFNs/iRGD, IR780@O2-SFNs, IR780-SHNs, IR780 and saline into orthotopic tumor models and irradiated (808 nm, 2 W/cm2, 5 min) the tumors 1 hour post-injection. Because of the maximum tumor accumulation, 1 hour post-injection was chosen for laser irradiation. The changes of relative tumor volumes (V/V0) were monitored as a function of time (Figure 9A). After PDT, The IR780-SHNs and IR780-mediated PDT showed low tumor inhibition efficiency which could be explained by insufficient 1O2 production and the lack of tumor targeting. However, an obvious decline in the tumor growth rate of IR780@O2-SFNs treated mice was found (P < 0.05), and IR780@O2-SFNs/iRGD worked even better than


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IR780@O2-SFNs in inhibiting tumor growth, which was revealed that oxygen self-sufficient photosensitizers vectors could enhance the phototoxicity in the hypoxic tumor and iRGD improved the PDT efficacy given its tumor penetrating capability. Accompanying by PDT effects, the PTT of IR780 in the irradiation was not evitable avoidable but parallel for all IR780-invovled groups. Thus, the enhanced antitumor effect was mainly attributed to 1O2 production in PDT. Meanwhile, the body weights (Figure S7A) and survival rates (Figure S7B) of the mice were not significantly affected by PDT, implying no severe side effects during the therapy.

Figure 9. In vivo PDT efficacy against orthotopic breast cancer of nude mice. The nude mice were treated with 200 μL IR780, IR780-SHNs, IR780@O2-SFNs, IR780@O2-SFNs/iRGD, and saline by intravenous injection (0.5 mg/kg IR780). (A) The tumor volume changed every two days during the treatment. (B) Immunohistochemical analysis (CD31 and TUNEL) of tumor tissues after PDT. (C) Histological analysis of tumor, liver and lung for different groups after PDT. The blue arrows and oval indicated the diseased regions. The antitumor efficacy was also confirmed using the histological and immunohistochemical analysis of tumor tissues (Figure 9B, Figure S8 and Figure S9). Stained by haematoxylin and ACS Paragon Plus Environment


Page 36 of 42

eosin (H&E), remarkable necrosis was observed in the tumor section from the IR780@O2-SFNs/iRGD

treated

mice.

Meanwhile,

substantially

more

apoptotic

cells

(TUNEL-positive cells) were present in tumor tissues following IR780@O2-SFNs/iRGD mediated PDT therapy. Compared with other treatment groups, the tumor vessels and the cell proliferation of IR780@O2-SFNs/iRGD treated tumor was significantly inhibited as determined by

the

use

of

CD31

test

and

Ki-67

assay,

respectively.

The

histological

and

immunohistochemical analyses for tumors were consistent with the retarded growth of tumors. The histopathology of organs was further investigated using H&E staining (Figure 9C). There was almost no abnormal morphology in heart, spleen or kidney for all treatment groups. Notably, distinct tumor metastasis occurred in livers and lungs for saline, IR780 and IR780-SHNs treated mice.

However,

no

visible

metastasis

was

found

in

IR780@O2-SFNs

and

IR780@O2-SFNs/iRGD treated mice. These phenomena confirmed that a single-dose intravenous injection of IR780@O2-SFNs/iRGD into orthotopic breast cancer model could remarkably inhibit the primary tumor growth and reduce the lung and liver metastasis.

4.

Conclusion

In conclusion, we demonstrated that SFNs combined with iRGD could act as an oxygen self-sufficient and tumor-penetrating nanoplaform (IR780@O2-SFNs/iRGD) for overcoming insufficient oxygen supply and limited tumor penetration in PDT. SFNs as photosensitizers


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vectors could improve the solubility of hydrophobic IR780 in aqueous solution and remain stable in storage. Meanwhile, oxygen self-sufficient SFNs as IR780 vectors could remarkably enhance the tumor oxygenation and thus increase the generation of 1O2 in vitro and in vivo. The tumor accumulation and penetration ability of IR780@O2-SFNs could be further improved by employing iRGD. Moreover, a single-dose intravenous injection of IR780@O2-SFNs/iRGD into orthotopic breast cancer model could remarkably inhibit the primary tumor growth and reduce the lung and liver metastasis. Thus, this work provides an effective means of improving PDT efficacy through increasing the oxygen solubility and selective delivery of photosensitizers to deep and hypoxic regions of the solid tumor.

Acknowledgements The National Natural Science Foundation of China (31871000, 31500810, 81621003 and 31771067), the National Key Research and Development Program of China (2017YFC1103501), and the Scientific Research Foundation for Talent Introduction of Nanjing Tech University (39803130 and 39803132) all provided financial support for this work. This project was also funded by the China Postdoctoral Science Foundation (2017M610602).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. The synthesis of compound 3 and 5, 1H NMR spectra and 19F NMR spectra, the standard curve of IR780 by ultraviolet absorption, IR780 loading content (LC) and



encapsulation efficiency, size distribution and TEM image at pH 5.0, the PA images of IR780 signal in vivo, histological and immunohistochemical analyses.

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An Oxygen Self-sufficient Fluorinated Nanoplatform for Relieved

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