Synergizing Upconversion Nanophotosensitizers with Hyperbaric

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Biological and Medical Applications of Materials and Interfaces

Synergizing Upconversion Nanophotosensitizers with Hyperbaric Oxygen to Remodel Extracellular Matrix for Enhanced Photodynamic Cancer Therapy Jingqiu Li, Jinzhao Huang, Yanxiao Ao, Shiyu Li, Yu Miao, Zhongzheng Yu, Lingtao Zhu, Yanhong Zhu, Yan Zhang, and Xiangliang Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07090 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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ACS Applied Materials & Interfaces

Synergizing Upconversion Nanophotosensitizers with Hyperbaric Oxygen to Remodel Extracellular Matrix for Enhanced Photodynamic Cancer Therapy Jingqiu Lia, Jinzhao Huanga, Yanxiao Aoa, Shiyu Lia, Yu Miaoa, Zhongzheng Yub, Lingtao Zhua, Xiaoli Lanc, Yanhong Zhua, Yan Zhanga*, Xiangliang Yanga* a. National Research Centre for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, P. R. China, 430074. b. School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637459. c. Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province Key laboratory of Molecular Imaging, Wuhan, P. R. China, 430022.

*

Corresponding Author: Yan Zhang, Xiangliang Yang

Tel: (+86) 8374 2274; Email: [email protected], [email protected].

KEYWORDS: upconversion nanoparticles, hyperbaric oxygen, photosensitizers, tumor extracellular matrix, cancer therapy

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ABSTRACT Photodynamic therapy (PDT) holds great promise as a non-invasive and selective cancer therapeutic treatment in preclinic research and clinical practise; however, it has limited efficacy in the ablation of deep-seated tumor due to hypoxia-associated circumstance and poor penetration of photosensitizers to cancer cells away from the blood vessels. To tackle the obstacles,

we

propose

a

therapeutic

strategy

that

synergizes

upconversion

nanophotosensitizers (UNPSs) with hyperbaric oxygen (HBO) to remodel the extracellular matrix for enhanced photodynamic cancer therapy. The UNPSs are designed to have a Nd3+sensitized sandwiched structure, wherein the upconversion core serves as light transducers to transfer energy to the neighbouring photosensitizers to produce reactive oxygen species (ROS). With HBO, photodynamic process can generate abundant ROS in the intrinsically hypoxic tumor. It is revealed for the first time that HBO-assist PDT decomposes collagen in the extracellular matrix of tumor and thus facilitates the diffusion of oxygen and penetration of UNPSs into the deeper area of tumor. Such a synergic effect eventually results in a significantly enhanced therapeutic efficacy at a low laser power density as compared with that using UNPSs alone. In view of its good biosafety, the HBO-assisted and UNPSs-mediated photodynamic therapy provides new possibilities for treatment of solid tumors.


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1. INTRODUCTION Photodynamic therapy (PDT) is a therapy that utilizes photosensitizers (PSs) to produce cytotoxic reactive oxygen species (ROS) under laser excitation, it emerges as a non-invasive and highly specific therapeutic approach to treat cancer in preclinical research and clinical practice.1-3 However, PDT has a low efficacy in the treatment of solid tumor, because hypoxic tumor microenvironment and tumor extracellular matrix (ECM) limit the availability of molecular oxygen and the delivery depth of PSs within tumor, respectively. Moreover, the continuous consumption of oxygen and vasoconstriction during PDT further lower the oxygen pressure and aggravate the hypoxic situation of tumor. To overcome the hypoxic issue during PDT, various oxygen-generating nanoparticles such as constructing MnO2,4-7 MnFe2O4,8 or catalase-based nanoparticles have been developed, which often take advantage of oxidative stress in tumor to decompose endogenic H2O2 to supplement oxygen inside the tumor.9 In addition, perfluorocarbon10 and modified homoglobin carriers11 have been used to directly deliver oxygen into tumor. Although all these approaches showed improved PDT efficacy, they still greatly rely on the location where nanoparticles can reach inside the tumor. Thereby, other alternative approaches that simultaneously enhance the availability of molecular oxygen and the delivery depth of PSs within tumor remain to be revealed. Hyperbaric oxygen (HBO) treatment, as an adjunct therapy in clinic, has been employed to cure various diseases, such as carbon monoxide poisoning, wounds healing, soft tissue infections, etc.12-14 It delivers oxygen to tissues without the requirement of hemoglobin, leading to significant enhancement of both diffusion rate and diffusion distance of oxygen in tissues.15 Study uncovered that HBO treatment can efficiently oxygenate or even completely eliminate the hypoxic region of tumors and repeat treatments may lead to a better therapeutic 3 ACS Paragon Plus Environment


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outcome against tumors.16 HBO has also proved to lower tumor interstitial fluid pressure (IFP), which can be beneficial to the penetration of molecules/nanoparticles.17-18 Despite these advantages, HBO has been rarely used to enhance PDT.19-20 In addition, the underlying mechanism on how HBO can affect the diseases microenvironment is largely unknown. In this study, we report an HBO-assisted and upconversion nanophotosensitizers (UNPSs) mediate synergistic photodynamic approach for cancer therapy (Scheme 1). The UNPSs is composed of two key components: upconversion nanoparticles and Rose Bengal (RB) as the light transducers and ROS-generating PSs, respectively. Upconversion nanoparticles have been widely used for molecular imaging and phototherapy.21-23 As upconversion nanoparticles can convert high tissue-penetrating near-infrared (NIR) light into UV or visible light, they can serve as the light transducers to excite RB.24-25 Such a nanoparticle design solves the shallow tissue-penetration issue of RB, making UNPSs effective for deep-tissue PDT.26 Upon light irradiation at 808 nm and HBO administration, effective photodynamic process can be activated even in the hypoxic tumor microenvironment through energy transfer from the upconversion core to RB, followed by sensitization of oxygen supplied by HBO to generate cytotoxic ROS. Detailed mechanistic studies reveal for the first time that HBO-assisted photodynamic process can remodel the tumor microenvironment by decomposing collagen matrix in the ECM, leading to enhanced penetration of the nanophotosensitizers within solid tumor and better tumor re-oxygenation. Such remodelling-enhanced delivery and oxygenation-facilitated photodynamic process synergistically bring in significantly improved in vivo PDT efficacy for cancer therapy.

2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of Lanthanide Upconversion Nanophotosensitizers. A layer-by-layer growth method was used to synthesize the sandwiched-structured NaGdF4:Yb,Er,Ca@NaYbF4:Ca@NaNdF4:Gd,Ca

upconversion


nanoparticles

(UCNPs)

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(Figure

1a).

Core

nanoparticles

NaGdF4:Yb,Er,Ca,

core@shell

nanoparticles

NaGdF4:Yb,Er,Ca@NaYbF4:Ca and the final UCNPs were well monodispersed with the average diameters of 6, 8 and 10 nm, respectively (Figure 1b-1d). X-ray powder diffraction (XRD) analysis proved the hexagonal phase of the as-synthesized multilayer nanoparticles (Figure S1). Energy-dispersive X-ray spectroscopy (EDX) revealed details of element distribution in UCNPs, indicating that the shells were successively grown on the core nanoparticles (Figure S2). Epitaxial growth of mesoporous silica on UCNPs was further proceeded to render the nanoparticles not only aqueous dispersible and stable but also high drug loading efficiency. Upon coating with mesoporous silica, the average size of resultant UCNPs@mSiO2 was found to be 19 ± 3 nm (Figure 1e). The surface area and average pore size of UCNPs@mSiO2 were measured to be 459.1 m2 g-1 and 3.029 nm, respectively (Figure S3). To improve the colloidal stability of the UCNPs@mSiO2, silane PEG was further used to cover the surface of UCNPs@mSiO2 and the hydrodynamic size of modified nanoparticles increased from 32 to 42 nm (Figure S4). Zeta potential and fourier-transform infrared (FTIR) spectroscopy experiments further verified the success of conjugation of PEG onto the surface of UCNPs@mSiO2 (Figures S5 and S6). Dynamic light scattering (DLS) revealed that the UCNPs@mSiO2-PEG remained stable in aqueous solution for 7 days, showing their suitability for long-term studies (Figure S7). At last, RB was loaded into the pore of PEGylated UCNPs@mSiO2 via diffusion to afford UCNPs@mSiO2-PEG-RB. The color change from white to pink manifested the successful loading of the RB into UCNPs@mSiO2PEG (Figure S8). DLS studies revealed that the resultant UCNPs@mSiO2-PEG-RB remained relatively stable in PBS and cell culture medium for 72 h (Figure S9). The luminescence properties of UCNPs and UCNPs@mSiO2-PEG were firstly measured under 808 nm laser excitation (Figures S10 and S11). UCNPs showed characteristic Er3+ emission peaks at 520, 550, and 650 nm that can be assigned to 2H11/2, 4S3/2 and 4F9/2→4I15/2 transitions, respectively. A similar spectral profile with decreased emission intensity was 5 ACS Paragon Plus Environment


shown for UCNPs@mSiO2-PEG, probably due to the OH-vibration of surrounding water molecules. Upon loading with RB, the emissions at 520 nm and 550 nm decreased as RB concentration increased, whereas the emission at 660 nm remained unaffected (Figure 1f). Such a selective quenching of green upconversion emissions by RB was ascribed to fluorescence resonance energy transfer (FRET) between UCNPs@mSiO2-PEG and RB. This was also partially proved by the fact that the strong absorption band of RB from 450 nm to 580 nm overlaps with the green emissions of UCNPs@mSiO2-PEG at 520 and 550 nm (Figure S12). The non-absorbed RB molecules were extracted from the mesoporous silica using centrifugation. The loading efficiency of RB in the UCNPs@mSiO2-PEG was determined to be 4.28% (denoted as upconversion nanophotosensitizers, UNPSs, hereafter) (Figure 1g). As premature leak of RB from nanoparticles will inevitable influence the ROS generation and undermine PDT efficacy, the leakage profile of RB from UNPSs in the dark was assessed using UV-vis spectra. There was no obvious leak within 24 h, which proved the particles were stable in short term (Figure S13). The generation of ROS under irradiation is critical in PDT. As demonstrated in many previous reports, hypoxia contributes to the resistance of PDT. Therefore, we assessed the availability of dissolved oxygen concentration in aqueous solutions under hypoxic (2% O2), normal (21% O2) and HBO (2.5 atm, pure O2) conditions. As expected, the saturated oxygen under the hypoxic condition was as low as 0.78 mg L-1 and it reached to 5.03 mg L-1 under normal oxygen condition (Figure 1h). Strikingly, under HBO administration, the value remarkably increased to 25.73 mg L-1, suggesting that addition of pressure greatly enhances the dissolve oxygen in the solution. We next evaluated whether the increased dissolved oxygen would be beneficial for the generation of 1O2 using 1,3-diphenylisobenzofuran (DPBF) as an indicator. DPBF is capable of irreversibly reacting with 1O2, resulting in a decrease of absorption at 410 nm. Under 808 nm laser irradiation and normal oxygen condition, a decrease of absorption at 410 nm was observed in a time-dependent manner, suggesting that 6 ACS Paragon Plus Environment

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the loaded RB molecules within UNPSs can efficiently absorb the upconverted emission to generate 1O2 (Figure 1i). Under hypoxic condition, the intensity decreased to only around 89%; however, it decreased to about 53.5% and 12.5% in the normoxia and HBO conditions, respectively. The 1O2 generation efficiency was further quantified using RB as reference (1O2 quantum yield, Φ∆RB = 0.68 in ethanol).27 The corresponding Φ∆UNPSs value under normal condition was calculated to be 0.65, which was comparable to that of RB. The values under hypoxic and HBO conditions are 0.32 and 0.90, respectively (Figure S14). This observation is in accordance with the fact that the oxygen concentration is crucial for the generation of 1O2. Control experiments of RB and UCNPs@mSiO2-PEG only under 808 nm laser irradiation were also carried out, showing no change in DPBF absorption. This proved that UNPSs could efficiently generate 1O2 under NIR laser irradiation and HBO can effectively enhance such a NIR photodynamic process. 2.2. In Vitro ROS Generation and Photodynamic Therapy Efficacy Evaluation. Cellular uptake of UNPSs was first studied by confocal laser scanning microscope (CLSM) and flow cytometry against 4T1 cells (Figure 2a and 2b). The red fluorescence from loaded RB in the UNPSs was detected within the cytoplasm within half an hour, and reached to an optimal at 4 h (Figure 2a). However, much weaker fluorescence was shown from cells incubated with RB alone at the same concentration. This confirmed that nanoparticles could indeed facilitate cellular uptake of PSs, and thus underscore the benefits of using the UNPSs instead of traditional PSs (Figure S15). The ability of UNPSs to trigger ROS generation under the three oxygen conditions was examined by using 2’,7’-dichlorofluorescin diacetate (DCFH-DA). DCFH-DA can be activated by ROS in cells, turning on its green emission at 525 nm. Note that UNPSs had good cytocompatibility, showing a viability greater than 90% at the concentration even up to 200 µg mL-1 (Figure S16). After irradiation at 808 nm for 20 min, the cells under the normoxia condition showed green fluorescence, which was 1.74-times higher than those under the 7 ACS Paragon Plus Environment


hypoxic condition (Figure 2c and 2d). By contrast, 1.35-times stronger green fluorescence for the cells treated with HBO administration relative to those under the normoxia condition was observed, confirming the enhanced generation of ROS. Besides, there was no detectable green fluorescence for the control groups including PBS, UNPSs, and NIR irradiation only as well as HBO only and UNPSs+HBO, confirming that the observed fluorescence was owing to the production of ROS. In vitro PDT efficacy of UNPSs was next evaluated using MTT assay (Figure 2e and 2f). Upon 808 nm laser irradiation and HBO administration, the cell viability dramatically reduced with increasing concentration of the UNPSs, which was 15% when the dose of UNPSs was 50 µg mL-1. In contrast, the cell viability decreased only to 42% and 81% under normoxia and hypoxia conditions, respectively. Furthermore, the cell apoptosis staining assay, in which Calcein-AM and propidium iodide (PI) were applied to mark live and dead cells, severally, proved again that the improved anticancer performance induced by the HBO-assisted PDT using UNPSs (Figure S17). The data thus suggested HBO indeed amplified the generation of 1

O2 from UNPSs and thus enhanced the therapeutic outcome of PDT.

2.3. In Vivo Photodynamic Therapy. The HBO-assisted PDT using UNPSs was then tested on xenografts mouse model. Fluorescence images of living mice were firstly taken to ascertain the optimal treatment time. Due to the background fluorescence from the melanin and hemoglobin at 541 nm, DiR with emission at 780 nm instead of RB was loaded into UCNPs@mSiO2 (UNPS-D). UNPS-D were administrated to 4T1 tumor-bearing mice via intravenous injection. Fluorescence from tumors was detectable in mice at 6 h after administration and nanoparticles continued to retain in the tumors for 48 h, then cleared away from the tumors gradually (Figure 3a and 3b). To image the fluorescence on the tumor side clearly, the hair of the mice around tumor was removed, and thus, the fluorescence of UNPSs from spleen was shown. Actually, the spleen together with the liver is responsible for the clearance of foreign materials by way of macrophage 8 ACS Paragon Plus Environment

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uptake.28 Therefore, the UNPSs are tend to accumulate more in these two organs. The result is consistent with Yu et al studies,28-29 the fluorescence image of excised organs also confirmed that (Figure S18). As the mean tumor fluorescence increases significantly at 24 h and reached maximal at 48 h post-injection, we chosen 24 h post-injection time as the first irradiation time, followed by the second treatment on 48 h. The treatment groups were also divided into 7 groups, group a: PBS, group b: NIR, group c: HBO only, group d: UNPSs, group e: UNPSs+HBO, group f: UNPSs+NIR, and group g: UNPSs+NIR+HBO. During the treatment, a single dosage of 200 µL (5 mg mL-1) UNPSs was injected via mice tail vein. The tumor sites in groups b, f, g were exposed to 808 nm laser (0.75 W cm-2) for 16 min at 24 h and 48 h after administration. The in situ temperature was closely watched with a IR thermal camera. Groups c and e received two times HBO administration, and group g also received two times HBO administration, followed by 808 nm laser irradiation. PDT therapeutic effect was assessed by measuring the changes of relative tumor volumes over time (Figure 3c). Besides, the comparative study with RB and UNPSs was conducted, relative tumor growth curve is shown in Figure S19. Free RB showed no inhibition of tumor growth with a NIR laser irradiation, either with or without HBO treatment. Nonetheless, UNPSs with a NIR laser irradiation inhibited the tumor growth. With the combination of HBO, the tumor showed significant delay growth, with only 0.6-time increase (Figure 3d and 3f). Remarkable tumor cell apoptosis and necrosis were detected in both histology of hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining analysis (Figure 3g). Tumors treated with only UNPSs+NIR showed medium tumor inhibition with more than 5-time increase compared to the UNPSs+NIR+HBO group at day 14. No observable therapeutic effect was presented for PBS, NIR, HBO or UNPSs only groups (Figure 3f). Furthermore, none of the mice showed noticeable loss of body weight in 14 days post-injection, suggesting no apparent toxicity of UNPSs in vivo (Figure 3e). These data



verified that HBO significantly enhanced PDT efficacy for UNPSs, facilitated NIR lasermediated PDT which is an ideal candidate for curing internal tumors. 2.4. Mechanistic Studies of HBO-Assisted PDT. To understand how HBO enhances UNPSs-mediated PDT, the extent of hypoxia in tumors and the nanoparticle penetration for different PDT treatments were studied ex vivo. Pimonidazole was used to detect hypoxic regions in the tumor slices (Figure 4a) and western blot was conducted to detect the expression quantity of hypoxia-inducible factor (HIF)-1α, an important mediator of cell that was regulated by oxygen level (Figure 4b).30-31 Meanwhile, the nanoparticle penetration was directly measured by detection of the location of UNPSs using the tumor blood vessels as the reference stained with green fluorescent FITC-conjugated CD31 antibody (Figure 5a). Among the four tested groups, the intensity of green fluorescent signals corresponding to the hypoxia region and the protein quantity of HIF-1α were the highest for PBS group, which were followed by UNPSs+NIR group, HBO only group and UNPSs+NIR+HBO group, respectively (Figure 4a and 4b). HIF-1α immunofluorescence staining assay provided additional evidence of the above observation (Figure 4c). Similar trend was observed for the nanoparticle penetration: the penetration depth of UNPSs in the UNPSs+NIR+HBO group was 230 µm, which was approximately 120, 100 and 50 µm further than that in the UNPSs, UNPSs+NIR and UNPSs+HBO groups, respectively (Figure 5a and 5b). Moreover, the distribution of UNPSs was more homogeneous in the UNPSs+NIR+HBO group than those in the UNPSs, UNPSs+NIR and UNPSs+HBO groups. In addition, considering that the limited efficacy of conventional PDT in the treatment of solid tumor was partially attributed to the limited delivery depth of PSs within tumor, we carried out the comparative study of the penetration depth of free RB and the UNPSs (Figure S20). It was found that free RB was mainly localized around blood vessel. With the assistance of HBO, the penetration depth was found to be as further as 50 µm, however, it is not comparable with UNPSs (230 µm). Therefore, when irradiated with NIR light, an enhanced tumor inhibition 10 ACS Paragon Plus Environment

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efficacy was shown in UNPSs+NIR+HBO group, indicating that HBO exerts a synergistic effect on nanophotosensitizer mediated photodynamic therapy. To find out the reason why UNPSs+NIR+HBO had the minimal hypoxia and deepest penetration among these treatments, the effect of HBO and UNPSs on the tumor ECM was studied. Because collagens are considered as the major structural component that involves in the formation of fibrillar networks of ECM, basement membranes and has been implicated to alter the behavior of molecules/nanoparticles, anti-collagen I antibody was used to stain the status of collagens in tumor slices for different treatments.32-33 Green fluorescence of anticollagen I antibody was heterogeneously distributed in the tumor slices of the PBS group, while it decreased for other groups, following the order of UNPSs+NIR > HBO > UNPSs+NIR+HBO (Figure 6a). Quantitative analysis of average fluorescence degree of the collagen content in tumor slices revealed that the collagen in UNPSs+NIR+HBO treated group was 14.6, 9.9 and 4.2 times less than PBS, UNPSs+NIR and HBO groups, respectively (Figure 6b). These data confirmed that HBO and UNPSs+NIR had the synergetic effect on breaking down the tumor collagens. This was further validated by in vitro assays using sirius red to measure the [Gly-X-Y] tripeptide helical structure on collagen fibrillin,34 showing UNPSs+NIR+HBO could reduce the collagen amount to 35%, whereas UNPSs+NIR group decreased it to only 83% (Figure 6c). No obvious difference was observed in PBS, NIR light, UNPSs and HBO groups. Such an observation was consistent with the previous studies reporting that ROS could break collagens in tissues.35-36 These ex-vivo studies reveal that HBO-enhanced PDT is closely associated with the ROSinduced breakdown of collages in tumors. HBO administration improves the oxygen concentration within tumors and thus facilitates the production of ROS generated by UNPSs under NIR irradiation in the intrinsically hypoxic tumor environment. The overproduced ROS not only ablates cancer cells, but also cleaves collagens in tumor microenvironment. Such a breakage of collagen fibrillar networks of ECM enhances perfusion and reduces IFP, which 11 ACS Paragon Plus Environment


together promote the diffusion of oxygen and penetration of UNPSs in solid tumor. Thus, although PDT consumes oxygen, the combination of HBO with PDT leads to the reduced hypoxia (Figure 4) and enhanced nanoparticles penetration in tumor (Figure 5). Such a synergetic effect of HBO and PDT eventually amplifies the PDT efficacy (Figure 3). 2.5. In Vivo Toxicity Evaluation of the Upconversion Nanophotosensitizers. In vivo systematic toxicity was studied to evaluate their translational potential. The biodistribution of UNPSs in the mouse major organs at different time interval after i.v. injection was assessed using ICP (Figure 7a). The data implied that UNPSs concentrate primarily in liver and spleen and reached maximal at 12 h post-injection. Although they declined slowly in the liver and spleen, still high level was evident at day 3. The amount in the liver and spleen was greatly reduced at day 14 post-injection. The UNPSs in the lung increased at 12 h and decreased thereafter, whereas the amount in the kidney was low initially, but increased up to 24 h and then declined significantly by 7 days. On the contrary, the amount of UNPSs in the heart was high at the 6 h post-injection, whereas it decreased afterwards. The combination of HBO caused no obvious difference to the biodistribution of UNPSs in the major organs (Figure 7b). Overall, these data indicated that the major organs of UNPSs distribution were liver and spleen, which was consistent with previous reports that primary clearance of upconversion nanoparticles occurs in the liver by Kupffer cells phagocytosis.37-38 Blood biochemical analysis of UNPSs using hepatic indicators, aminotransferase (ALT) and aminotransferase (AST), and kidney index, blood urea nitrogen (BUN) and creatinine (Cr), manifested that liver and kidney function normal during treatments (Figure 7c). H&E staining also confirmed no pathological morphologies in the major organs (liver, lung, spleen, kidney, and heart) (Figure 7d). We therefore believe

negligible toxicity of UNPSs in vivo.

3. CONCLUSIONS 12 ACS Paragon Plus Environment

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In summary, we demonstrate a simple yet effective treatment option by synergizing upconversion nanophotosensitizers and HBO. The HBO administration promotes the generation of ROS by the nanophotosensitizers under NIR irradiation. The detailed mechanistic studies reveal that HBO-assisted PDT can deplete collagen fibers in the extracellular matrix, which not only reduces the tumor hypoxia but also enhances the penetration of nanophotosensitizers into deeper area from the tumor blood vessels. Such a synergistic effect ensures better eradication of cancer cells in solid tumor, leading to much better therapeutic efficacy as compared with the PDT alone. As no observable toxicity for mice after administration of the nanophotosensitizers alone or with HBO administration, the HBO-enhanced PDT using the upconversion nanophotosensitizers represent a promising option to treat tumors.

4. EXPERIMENTAL SECTION 4.1. Materials and reagents. 2-[Methoxy(polyethyleneoxy)9-12propyl] trimethoxysilane (PEG-silane) was obtained from J&K Scientific Ltd. Cyclohexane, ethanol, ethyl acetate and acetone were purchased from Sinopharm Chemical Reagent Co., Ltd. 4,6-diamino-2-phenyl indole (DAPI), RIPA lysis buffer and phenylmethanesulfonyl fluoride (PMSF) were from Beyotime. 1-dioctadecyl-3 ， 3 ， 3 ， 3-tetramethylindotricarbocyanine iodide (DiR iodide) was purchased from AAT Bioquest Inc. Reagents for cell culture and FITC-conjugated secondary antibody were purchased from Thermo Fisher Scientific. Calcein-AM/PI Double Stain Kit was purchased from Yeasen. Hypoxyprobe™-1 Kit was purchased from Hypoxyprobe. Antibodies for collagen I, HIF-1α， CD-31 were purchased from Abcam. Sirius Red Total Collagen Detection Kit was purchased from Chondrex. All of other chemicals were from Sigma-Aldrich and used as received. Millipore water (18.2 MΩ cm at 25℃) was used throughout all experiments. 13 ACS Paragon Plus Environment


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4.2. Synthesis of Nanoparticles. UCNPs were synthesized by co-precipitation method which was established in previous work.39 Typically, an aqueous lanthanide acetates solution (2 mL) was added to a 50 mL flask with 4 mL oleic acid and 10 mL 1-octadecene. The solution was heated at 150 ℃ for 1 h to form the lanthanide-oleate solution. After cooling down to room temperature, a mixture of NH4F (1.6 mmol) and NaOH (1 mmol) in methanol solution (10 mL) was added and stirred at 50 ℃ for 30 min. The reaction was then heated to 100 ℃ for 10 min to remove the remaining methanol in the mixture. The resultant solution was heated to 300 ℃ under the protection of argon gas for 30 min to form the nanoparticles. After cooling down to room temperature by itself, ethanol was added to precipitate the nanoparticles, followed by centrifugation, the nanoparticles were collected and finally re-dispersed in 2 mL of cyclohexane.

The

core-shell

NaGdF4:Yb,Er,Ca@NaYbF4:Ca

nanoparticles

and

the

NaGdF4:Yb,Er,Ca@NaYbF4:Ca@NaNdF4:Gd,Ca nanoparticles (UCNPs) were synthesized following the same protocol except the core and core-shell nanoparticles were added in the precursor solution, respectively. 4.3. Synthesis of UCNPs@mSiO2. UCNPs@mSiO2 were synthesised according to former work39. 2 mL cyclohexane solution containing 10 mg UCNPs was added into 20 mL of CTAB aqueous solution (CTAB, 0.1 g). After stirring for 1 h, the mixture was heated up to 80 ℃ for 30 min to evaporate cyclohexane. The solution was cooled to room temperature, a mixture of 25 mL water and NaOH aqueous solution (1.8 mL, 2 M) was then added. The produced mixture was heated up to 70 ℃ with the addition of TEOS (70 µL), APTES (20 µL) and ethyl acetate (1 mL). After 6 h reaction, the solution was naturally cooled down to room temperature. Nanoparticles were separated via centrifugation and stored in 20 mL ethanol. Then, 40 µL HCl was added to adjust the pH of the dispersion to ~ 1.4. After stirring for 3 h at 60 ℃, the nanoparticles were washed using ethanol and centrifuged. The resultant UCNP@mSiO2 nanoparticles were stored in ethanol.


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4.4. Surface modification of UCNPs@mSiO2 with PEG. 8 µL of TEOS and 50 µL PEGsilane were added into UCNP@mSiO2 solution and the reaction was kept at 70 ℃ for 3 h. The obtained UCNP@mSiO2-PEG were obtained by centrifuge at 12000 rpm for 20 min and redispersed in 0.7 mL DI water and stored at 4 ℃. 4.5. Loading rose bengal into UCNPs@mSiO2-PEG. An aqueous solution of RB was prepared by dissolving 1 mg RB in 25 mL water, then UCNP@mSiO2-PEG (~ 25 mg) were added. The mixture was stirred at ambient temperature overnight and protected from light. The UCNP@mSiO2-RB was obtained by centrifugation at 12000 rpm for 15 min and it was rinsed few times until the supernatant solution clear. 4.6. Detection of singlet oxygen in solution and 1O2 Generation Efficiency. The bleaching of the DPBF absorbance at 410 nm was recorded to measure the production of 1O2. In brief, 400 µL UNPSs (5 mg mL-1) and 250 µL DPBF solution (dissolved in ethanol) were added to 6 mL ethanol, the absorbance at wavelength of 410 nm was recorded using the UV-vis spectrometer after irradiation by 808 nm laser at different time interval. DPBF in solution was used as control. For hypoxia group, argon gas was steadily purged into the solution during irradiation. For HBO group, pure oxygen was steadily purged into the solution during irradiation. Quantification of 1O2 generation efficiency (Φ∆) of UNPSs was determined by using Rose Bengal as a reference compound (Φ∆ = 0.68).27 Φ∆ was calculated using the equation of Φ∆ =Φ∆RB · (WUNPSs · IRB) / (WRB · IUNPSs), Where WUNPSs and WRB are the reaction rate of the DPBF with 1O2 in the presence of UNPSs and RB, which can be obtained from the photobleaching curves while UNPSs and RB were added (Figure S14) ; IUNPSs and IRB are the rates of light absorption of UNPSs and RB individually.40 4.7. Cell culture. The 4T1 cell line were purchased from Shanghai Institutes for Biological Sciences. Cells were cultured following the provider’s direction. 4.8. Cellular uptake images using a confocal laser scanning microscopy. 4T1 cells were seeded in confocal dishes and cultured 24 h before use. Then the culture medium was 15 ACS Paragon Plus Environment


discarded and 500 µL UNPSs (50 µg mL-1) or comparable RB in fresh medium was added with different incubation time. Then cells were washed with PBS for three times and incubated with 4% paraformaldehyde for 20 min, followed by another three times PBS washing. Then, cells were marked by DAPI for another 10 min and cellular uptake was studied by confocal laser scanning microscopy (CLSM, Olympus FV1000, Japan). The emission of RB was used to analyze the localization of the UNPSs. 4.9. Cellular uptake by flow cytometry. 4T1 cells were seeded in 24 well-plate overnight before use. 50 µg mL-1 UNPSs or comparable RB was added into the medium and incubated for different time. After removing supernatant, PBS was used to wash cells for three times in case of residual UNPSs or RB, then cells were trypsinzed and centrifuged for collection, finally resuspended for flow cytometry analysis (Beckman). 4.10. In vitro cytotoxicity evaluation of UNPSs. 4T1 cells were inoculated in 96-well plate overnight. After incubation with of UNPSs at different concentrations and time in the darkness, the MTT assay was then performed to report the cell viabilities according to standard procedure. 4.11. HBO administration. The hyperbaric chamber (Weifang Huaxin, China) was supplied with pure oxygen. For HBO administration, the chamber was first perfused with oxygen and the pressure arise to 2.5 atm in 15 min at a constant velocity. The pressure was maintained for 120 min, and then, it was reduced to 1 atm in 15 min at a constant velocity. 4.12. In vitro ROS detection. 4T1 cells were inoculated in 24 well-plate overnight. Cells were then incubated with 50 µg mL-1 UNPSs for 4 h and protected from light. Afterwards, cells were incubated with 2’,7’-DCFH-DA in FBS free RPMI 1640 medium in darkness for 20 min. The irradiation was performed using 808 nm laser, the power density was 1.0 W cm-2 and the time was 20 min. For hypoxia groups, cells were cultured in a tri-gas incubator (Hua Xi Electronics Technetronic Co., Ltd, China) with a 2% oxygen concentration. Afterwards, the cells were putted into an anaerobic bag while incubation with DCFH-DA before 16 ACS Paragon Plus Environment

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irradiation. For HBO groups, cells were placed in HBO chamber for 2 h, then replaced to a sealable bag full of oxygen while incubation with DCFH-DA before irradiation. After irradiation, PBS was used to wash the cells three times and the production of ROS was visualized with a fluorescence microscopy (Olympus FV1000, Japan). The intracellular ROS generation was then determined by the fluorescent intensity from DCF. 4.13. In vitro PDT evaluation. 4T1 cells were seeded and incubated with gradient concentration of UNPSs for 4 h at 37 ℃ in the darkness. The medium was discarded to remove the non-internalized UNPSs and PBS was used to cleanout the residual UNPSs. The irradiation condition was the same as that of in vitro ROS detection. For hypoxia groups, cells were cultured in a tri-gas incubator with a 2% oxygen concentration and then been putted in an anaerobic bag during irradiation. For HBO groups, cells were placed in HBO chamber for 2 h and then put into a sealable bag full of oxygen before irradiation. After irradiation, cells were replaced with fresh medium with further culture for 24 h. The MTT assay was then performed conducted to standard procedure. 4.14. In vitro live and dead assay. 4T1 cells were seeded and received treatments as the former section mentioned. Live and dead assay was carried out by Calcein-AM/PI Double Stain Kit following the manufacturer's instructions. After trypsinization and centrifugation, cells were stained with Calcein-AM and PI following the instruction of Kit. After 30 min incubation at room temperature, the prepared cells were imaged with a confocal laser scanning microscopy (CLSM, Olympus FV1000, Japan). 4.15. Tumor models. Female Balb/c mice (4 ~ 5 weeks old, 18 ~ 20 g) were obtained from the Hubei Research Centre of Experimental Animals. All experiments of mice were implemented in accordance with Institutional Animal Care and Use Committee Guidelines of Huazhong University of Science and Technology, China. To establish the tumor model, suspension of 4T1 cells in PBS was prepared in advance, then approximately 1.5×106 cells



were injected into the right flank of mice subcutaneously. The mice received treatments when the tumor size grew up to ~100 mm3. 4.16. In vivo fluorescence imaging of DiR loading UNPSs. Balb/c mice with 4T1 tumor received intravenously injection of 100 µL of UCNPs@mSiO2@DiR (5 mg mL-1 in PBS). At different time interval, tumor fluorescence of DiR was measured by an IVIS Lumina XR in vivo imaging system (Perkin Elmer, USA) at the indicated time after injection, the excitation and emission wavelength were 748 nm and 780 nm, respectively. 4.17. In vivo PDT evaluation. Divided 4T1 tumor-transplanted mice into 11 groups (n = 5): PBS, NIR, HBO, UNPSs, UNPSs+HBO, UNPSs+NIR, UNPSs+NIR+HBO, RB, RB+HBO, RB+NIR, RB+NIR+HBO. Mice received intravenously injection of 200 µL of UNPSs (5 mg mL-1) or comparable RB (30 µg mL-1) or 200 µL of PBS. HBO administration was performed by transferring mice into HBO chamber for 2 h and it was performed at 24 h and 48 h after administration, respectively. At 24 h after injection, 808 nm laser irradiation was conducted, the power density was 0.75 W cm-2 and the irradiation process maintained for 16 min. Mice in the UNPSs+NIR+HBO group were irradiated soonest as HBO administration finished. Since the treatment initiated, the width and length were monitored with caliper every other day. The volume of tumor was figured out as length×width2/2. Body weight of each mouse was weighted every 2 days. Mice were sacrificed at 14 days after treatments. Tumors were excised, weighed and fixed with 4% paraformaldehyde. H&E staining and TUNEL staining were performed according to the standard protocol, respectively. 4.18. Immunofluorescence staining of hypoxic area. For detecting the hypoxic area, mice were arranged into 4 groups: PBS, HBO, UNPSs+NIR and UNPSs+NIR+HBO. After predetermined treatments, mice were i.v. injected with Hypoxyprobe™-1 (60 mg kg-1). An hour and a half later, tumors were collected and slides were made as mentioned before, then the staining was conducted according to the instruction of Hypoxyprobe™-1 Kit (Hypoxyprobe, USA). After incubation with primary antibody, slices were rinsed with PBS18 ACS Paragon Plus Environment

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Tween 20, incubated with FITC-conjugated secondary antibody. After staining with DAPI, slices were imaged by confocal microscopy. 4.19. Western blotting analysis of HIF-1α. After the same treatments in the immunofluorescence staining section, tumors were collected and grinded in RIPA lysis buffer that containing PMSF. Then, lysate was sonicated with an ultrasonic cell disruptor (Scientz, China) and centrifuged. The supernatant was mixed with loading buffer and kept in 100 ℃ for 10 min. Western blotting was performed according to the standard protocol. 4.20. Immunofluorescence staining of HIF-1α. Divided Balb/c mice into 4 groups: PBS, HBO, UNPSs+NIR and UNPSs+NIR+HBO. After predetermined treatments, tumors were collected and slices were prepared as mentioned before. Then, slices were blocked with serum and was incubated with anti-HIF-1α antibody, followed by further incubation with FITCconjugated secondary antibody. After staining with DAPI, slices were imaged by confocal microscopy. 4.21. Penetration staining of UNPSs. Balb/c mice were divided to 6 groups: UNPSs, UNPSs+HBO, UNPSs+NIR, UNPSs+NIR+HBO, RB+NIR and RB+NIR+HBO. 200 µL of UNPSs (5 mg mL-1) or comparable RB (30 µg mL-1) were i.v. injected, HBO administration and NIR irradiation were performed the same as in vivo PDT. After collection of tumors, the slices were made as described before in immunofluorescence staining section. After serum blocking, the blood vessels of tumor were marked with anti-CD31 antibody and FITCconjugated secondary antibody. The prepared slices were photographed with confocal microscope (Olympus FV1000, Japan) and analysed with Image J software. 4.22. Immunofluorescence staining of collagen. Balb/c mice were divided into 4 groups: PBS, HBO, UNPSs+NIR and UNPSs+NIR+HBO. After predetermined treatments, tumors were collected and tumor slices were prepared as mentioned before. After blocking with serum and anti-collagen I antibody incubation, tumor slices were then incubated with FITC-



conjugated secondary antibody. After staining DAPI, slices were imaged by confocal microscopy. 4.23. In vitro collagen depletion evaluation. 50 µg mL-1 collagen from rat tail was dissolved with 0.05 mol L-1 acetic acid. UNPSs (5 mg mL-1) was mixed with collagen solution with a ratio of 1:1 and indicated treatments were conducted. Then, 808 nm laser irradiation was performed for 30 min and the power density was adjusted to 5 W cm-2. Hypoxia treatment was performed the same as MTT assay did. HBO treatment was performed by keeping the mixed solution in HBO chamber for 2 h. The temperature was closely monitored to be under 37 ℃ under treatments. The content of collagen was then detected by Sirius Red Total Collagen Detection Kit (Chondrex, USA). 4.24. In vivo biodistribution of UNPSs. Divided female healthy Balb/c mice into 2 groups (n = 3): UNPSs and UNPSs+HBO. Mice received intravenously injection with 200 µL of UNPSs (5 mg kg-1). After gradient time, the main organs (heart, liver, spleen, lung and kidney) were collected and weighted, and then incubated with 3 mL HNO3 and 1 mL HClO4 and maintained at 320 ℃ until the organs digested completely. The digestive solution was cooled to ambient temperature. Deionized water was added to each of the solutions to 2 mL.

Neodymium was chosen as component on behalf of elements of UNPSs, and ICP (inductive coupled plasma) was used to determine the amount of Nd element in organs. 4.25. Blood analysis. Balb/c mice were divided to 3 groups (n = 3): PBS, UNPSs, UNPSs+HBO. 14 days after injection of 200 µL UNPSs (5 mg mL-1) or PBS, blood samples were collected and centrifuged for the supernatant, then kept in water bath of 37 ℃ for 2 h. After dilution with PBS, samples were used to measure the level of ALT, AST, BUN and Cr. 4.26. H&E staining. Divide balb/c mice into three groups (n = 3): PBS, UNPSs and UNPSs+HBO. The mice were injected with 200 µL PBS or UNPSs (5 mg mL-1) via tail vein. After 2 weeks, the mice were executed and main organs (heart, liver, spleen, lung and kidney) were gathered for H&E staining and imaged by an inverted microscope (DMI3000, Leica). 20 ACS Paragon Plus Environment

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4.27. Statistical analysis. All data was analyzed by Student’s test and shown as Mean± SD if not specially emphasized. Statistical significance is measured with P < 0.05. 4.28. Instrumentation. Morphological features were obtained from Transmission electron microscopy (TEM) measurements carried on a JEOL JEM-2100F microscope operating at an acceleration voltage of 200 kV, and element composition from Energy-dispersive X-ray (EDX) spectroscopy with a JEOL FESEM-7600F field emission scanning electron microscope. Powder X-ray diffraction analysis (XRD) delivered details about crystal structure recorded on a D8 Advance Bruker powder X-ray diffractometer with Cu Kα radiation (λ = 1.5406 A) from 10° to 80°. Malven Nano Zetasizer system provided comprehensive information of hydrodynamic size distribution, zeta potential and stability of the nanoparticles. Ultraviolet-visible absorption spectra was recorded on a Shimadzu UV-3600 spectrophotomer, while upconversion fluorescence emission spectra on a Fluoromax-4, Horiba Jobin Yvon spectrofluorometer equipped with 808 nm NIR laser. Fourier transform infrared spectroscopy (FTIR) was performed on a Digilab FTS 3100. Brunauer-Emmett-Teller (BET) was assessed in micromeritics ASAP 2020 V3.01H. As for in vitro tests, Olympus FV1000 was utilized for confocal laser scanning microscopy (CLSM) imaging, and FC500, Beckman for Flow cytometric analysis.



Figure 1.

Synthesis and characterization of UNPSs. (a) Schematic illustration for the

synthesis of NaGdF4:Yb,Er,Ca@NaYbF4:Ca@NaNdF4:Gd,Ca@mSiO2 nanoparticles. TEM images of (b) core NaGdF4:Yb,Er,Ca; (c) core/shell NaGdF4:Yb,Er,Ca@NaYbF4:Ca; (d) core/shell/shell NaGdF4:Yb,Er,Ca@NaYbF4:Ca@NaNdF4:Gd,Ca. Scale bar: 50 nm. (e) TEM image of NaGdF4:Yb,Er,Ca@NaYbF4:Ca@NaNdF4:Gd,Ca@mSiO2 nanoparticles. Scale bar: 20

nm.

(f)

Upconversion

spectrum

of

NaGdF4:Yb,Er,Ca@NaYbF4:Ca

@NaNdF4:Gd,Ca@mSiO2-PEG with different RB loading efficiency. (g) UV-vis spectrum of NaGdF4:Yb,Er,Ca@NaYbF4:Ca@NaNdF4:Gd,Ca@mSiO2-PEG with diverse RB loading efficiency. (h) The dissolved oxygen in the cell medium under hypoxia, normoxia and HBO conditions. (i) The generation of 1O2 determined by the decreased absorption of DPBF with RB, UCNPs@mSiO2-PEG and UNPSs under hypoxia, normoxia and HBO conditions.


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Figure 2. In vitro cellular uptake, ROS generation and PDT evaluation. (a) Confocal fluorescence imaging and (b) flow cytometry analysis of 4T1 cells that have been incubated with 50 µg mL-1 UNPSs in time gradient. Red and blue fluorescence represent for RB and DAPI, individually. Scale bar: 50 µm. (c) Quantitative study of the mean fluorescence intensity of DCF based on the images shown in (d) (1.0 W cm-2, 20 min). (d) Intracellular ROS detection via DCFH-DA staining after irradiation with an 808 nm laser (1.0 W cm-2, 20 min) at different conditions. The fluorescence of DCF was imaged. Scale bar: 100 µm. (e) Viability of 4T1 cells after various treatments and UNPSs (50 µg mL-1) under hypoxia, normoxia and HBO conditions. (f) Viability of 4T1 cells after treatment with different UNPSs concentration under hypoxia, normoxia and HBO conditions. Data are shown as Mean ± SD.


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Figure 3. In vivo fluorescent images and PDT evaluation via intravenous injection. (a) In vivo fluorescence photographs of tumor-transplanted mice, pictures were taken at different time after injection of DiR loaded UNPSs. (b) Quantification of relative mean fluorescence intensities of tumor based on images shown in (a). The data were shown as Mean ± SD. (c) Changes of relative tumor growth curves upon different treatments. *P < 0.05. Error bars were based on standard deviations (n=5). (d) Average weight of excised tumor after different 25 ACS Paragon Plus Environment


treatments. **P < 0.01. Error bars were based on standard deviations (n=5). (e) Changing curves of mice body weight upon different treatments. The data was shown as Mean ± SD (n=5). (f) Picture of tumors obtained from each group of mice two weeks after treatments started. (g) TUNEL staining and H&E staining of tumors from mice in each group. 1, PBS; 2, NIR; 3, HBO; 4, UNPSs; 5, UNPSs+HBO; 6, UNPSs+NIR; 7, UNPSs+NIR+HBO.


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Figure 4. Hypoxic microenvironment evaluation. (a) Immunofluorescence staining of hypoxic regions (green) and nuclei (blue) with pimonidazole and DAPI upon various treatments. (b) Expression of HIF-1α by western blotting. (c) Immunofluorescence staining of 27 ACS Paragon Plus Environment


HIF-1α (green) and nuclei (blue) with HIF-1α and DAPI from 4T1 tumor-bearing mice upon various treatments. UNPSs concentration is 5 µg mL-1. Scale bar: 200 µm.


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Figure 5. In vivo penetration studies of UNPSs into tumors upon various treatments. (a) Confocal imaging of tumor vessels stained with anti-CD31 antibody (green) after intravenous injection of UNPSs upon different treatments. Red fluorescence was from the loaded RB in the UNPSs. Scale bar: 50 µm. (b) Quantification of the mean fluorescence strength of RB in a random line showed in (a) from loaded RB in the UNPSs to away from tumor vessels. Line length = 230 µm. Fluorescence intensities were analysed with Image J.



Figure

6.

Determination

of

collagen

content

upon

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various

treatments.

(a)

Immunofluorescence staining of collagen-I (green) and nuclei (blue) with collagen-I antibody and DAPI, individually. Scale bar: 50 µm. (b) Quantification of the mean fluorescence intensity of collagen-℃

based on fluorescence shown in (a). Error bars were based on SD

(n=3). (c) In vitro quantitative studies of collagen-I (50 µg mL-1) reduction by staining 30 ACS Paragon Plus Environment

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collagen with sirius red after incubation with UNPSs or PBS upon various treatments. The remaining collagen content was analyzed by Sirius Red Total Collagen Detection Kit. The relative content of collagen shown in PBS group was used as control. *P < 0.05, **P < 0.01. Error bars were based on SD (n=3).



Figure 7. In vivo biosafety evaluation of UNPSs. Biodistribution of UNPSs at different time interval (a) without or (b) with HBO administration after i.v. injection of UNPSs into normal Balb/c mice. The amount of neodymium in major organs (heart, liver, spleen, lung and kidney) 32 ACS Paragon Plus Environment

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was measured by ICP. Error bars were based on SD (n=3). (c) Blood biochemistry analysis of ALT, AST, BUN and Cr collected from PBS, UNPSs and UNPSs+HBO treated normal Balb/c mice. PBS or UNPSs was injected into mice via tail vein. Data were shown as Mean ± SD (d) H&E staining of major organs obtained from PBS, UNPSs and UNPSs+HBO treated normal Balb/c mice. Scale bar: 50 µm.

Scheme 1. Schematic illustration of synergizing upconversion nanophotosensitizers (UNPSs) with or without HBO about remodelling collagen in extracellular matrix to better oxygenation and deeper penetration of nanophotosensitizers for enhanced photodynamic cancer therapy.



ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figures of XRD pattern and EDX of UCNPs. BET of UCNPs@mSiO2. DLS, zeta potential, FTIR and colloidal stability of UNPSs. Upconversion spectra of UCNPs and UCNPs@mSiO2. The leak study of RB from UNPSs. DPBF photobleaching curves of RB and UNPSs. Cellular uptake of RB, in vitro cytotoxicity of UNPSs and cell apoptosis studies. Calcein-AM/PI double stain. Fluorescence image of excised organs. Tumor inhibiting curves and penetration study of RB related treatments.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected] (Y.Z.). *Email: [email protected] (X.Y.). ORCID Jingqiu Li: 0000-0002-8422-1390 Yanhong Zhu: 0000-0002-6258-6065 Yan Zhang: 0000-0002-8811-5552 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work is funded by National Basic Research Program of China (2015CB931802), National Natural Science Foundation of China (81703031, 81573013, 81627901) and Huazhong University of Science and Technology (2017KFYXJJ162).


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Synergizing Upconversion Nanophotosensitizers with Hyperbaric

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