A Reactive 1O2 - Responsive Combined Treatment System of Photodynamic and Chemotherapy for Cancer

The development of reactive oxygen species (ROS)-responsive drug delivery and drug release has gradually attracted much attention in recent years as a promising therapeutic strategy. Singlet oxygen (1O2) as the major ROS species is widely used in photodynamic therapy (PDT) of cancer. In the present study, we introduce a combined treatment using ROS-sensitive thioketal (TK) linkage as a linker between upconversion nanoparticles (UNs)-based PDT and doxorubicin (DOX)-based chemotherapy. UNs can not only play a role in PDT, but can also be used as a nanocarrier for drug delivery of DOX. Moreover, the products of 1O2 during PDT are able to cleave TK linker inducing the release of DOX which can further achieve the goal of chemotherapy. By using this 1O2-responsive nanocarrier delivery system, DOX can easily reach the tumor site and be accumulated in the nuclei to effectively kill the cancer cells, and therefore decreasing the side effects of chemotherapy on the body. Thus, PDT also has the function of controlling drug release in this combination treatment strategy. Compared with monotherapy, the combination of PDT with chemotherapy also possesses excellent drug loading capability and anticancer efficiency.


Ability to produce singlet oxygen in solution.
Under 980 nm laser excitation with proper intensity, the emission band at 540 nm was the main emission of upconversion visible fluorescence from UNs, which matched well with the absorption of photosensitizer MC540 (Fig. 2c). The character of MC540 molecular absorbed on the surface of UN allowed for fluorescence transfer from the UN to MC540, thereby the activating MC540 could generate cytotoxic 1 O 2 with surrounding oxygen molecule. We measured the production of 1 O 2 with light in three different concentration systems of oxygen gas, including N 2 , Air and O 2 -saturate aqueous solution containing the dye 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA). ABDA was used as a water-soluble 1 O 2 monitor by its fluorescence decay due to the reaction with 1 O 2 [35][36][37] . These results provided evidence that UN/MC540 could generate 1 O 2 using oxygen molecular existed in surrounding environment. In addition, the yield of 1 O 2 was affected by illumination time at the same concentration (Fig. 2d).

Detection of 1 O 2 in solution and cancer cells. Singlet oxygen in cancer cells was detected using ROS
Kit (Reactive Oxygen Species Assay Kit) by flow cytometry and confocal laser scanning microscopy (CLSM) methods under 980nm laser irradiation (0.5 W/cm 2 ) 38 . As compared to the faint fluorescence signal in the UN group, significant amounts of 1 O 2 were produced in UN/MC540 group. These results further demonstrated that UN/MC540 possessed the ability to generate 1 O 2 efficiently and could be used in PDT for cancer cells (Fig. 3a,b). ROS-responsive drug release. In addition to kill cancer cells, the generated 1 O 2 in PDT could also control the drug release since DOX was covalently conjugated to UN/MC540 by the 1 O 2 -responsive TK linker 39,40 . To certify the ability of 1 O 2 -responsive drug release, aqueous solution of UN/MC540-DOX was treated by 5 min illumination with power density of 0.5 W/cm 2 . After that, nanoparticles were removed by centrifugation (20,000 rpm, 5 min), and supernatant was collected in another tube to measure DOX fluorescence intensity (488 nm excitation and 585 nm emission). The concentration of DOX released from UN/MC540 was determined by its fluorescent intensity. Compared with the untreated group, the light promoted DOX release from nanoparticle. To further demonstrate this was a 1 O 2 -induced DOX release mechanism, vitamin C (VC) as a ROS scavenger was added into the light treated group. The fluorescence from the DOX was significantly inhibited, thus further confirming the generated 1 O 2 under 980 nm laser excitation acted on the TK linker and induced DOX release from nanoparticle (Fig. 4).      In vivo anticancer efficient. DSPE-PEG endowed nanoparticles with stealth behavior to protect them from the RES (reticulo-endothelial system) and easily reached the tumor tissue via EPR effect. Besides, the grafting FA molecules onto the nanoparticles refered to active targeting and aimed to increase specific cancer cells uptake at the tumor site by FA receptor-mediated endocytosis 32,46-49 . The injection of Cy5.5-labeled FA-UN/MC540-DOX was more likely to accumulate at the tumor site than non-target nanoparticle in the process of PEG-induced long blood circulation (Fig. 7a). Having demonstrated the tumor targeting ability of FA, we moved on to explore the antitumor efficacy of different nanoparticles. In comparison with PBS group, all treatment groups showed varying degree of tumor suppression within three weeks. Among them, combination treatment groups exhibited better antitumor effect than free DOX and UN/MC540 group. Notably, targeted modification group showed the greatest anti-cancer effect. Likewise, nuclear apoptosis of cancer cells analysis by TUNEL staining was in agreement with the above results. FA-UN/MC540-DOX induced the greatest cell apoptosis and the tumor was also the smallest, again confirming the success of our combination therapy strategies for cancer cells (Fig. 7b,c).

Discussion
In conclusion, we have developed an efficient combination control approach for tumor therapy, which consisted of UN-based PDT and chemotherapy. FA-UN/MC540-DOX was applied to an ideal nanoscale drug delivery device with high drug loading for DOX. And the molar percentage of PEG introduced onto the surface of UN/ MC540 endowed the nanocarrier with superior stealth ability to enhance circulation time, and led to tumor accumulation of DOX via EPR effect. Moreover, FR-targeted modification nanoparticle increased cellular uptake and promoted more efficient DOX delivery in vivo. Once FA-UN/MC540-DOX arrived at the tumor site or was uptaken by cancer cells, the produced 1 O 2 in the PDT could quickly trigger the release of DOX from this nanoparticle via cleavage of TK linker. Meanwhile, free DOX was easily able to travel through the nuclear envelope to kill cancer cells efficiently. In addition, UN/MC540-based PDT could also overcome the limitations found in conventional chemotherapy. Thus, our present combination treatment with controlled-release ability exhibits a better anticancer efficacy and paves the way for further research on the clinical application.  Oleic acid (6 mL) and 1-octadecene (ODE) (15 mL) were added to the above solution, and the mixture was stirred at room temperature for 10 minutes. Methanol solution (5 mL) containing NaOH (2.5 mmol) and NH 4 F (4 mmol) were added into this flask. After that, methanol was evaporated by heating at 100 °C, and the mixture was maintained at 300 °C for 1 h under argon protection. After cooling the reaction mixture to room temperature, medium solution (10 mL) was collected as the core for the later fabrication of core/ shell NaYF 4 :Yb,Er/NaYF 4 UNs. YCl 3 (1 mmol), NaOH (2.5 mmol) and NH 4 F (4 mmol) dissolved in methanol (5 mL) were added into the collected core solution to prepare core/shell UNs. The solution was stirred for 30 min and heated to remove methanol. After being degassed and maintained under argon protection, core/shell UNs were obtained by centrifugation and washed for three times 27 . TK and DSPE-PEG-DOX were synthesized according to the method reported previously (Fig. 8a) 18,20 . DOX showed the retention at 8.7 min and DSPE-PEG-DOX showed retention at about 7.1 min with major single peak (Fig. 8b). To prepare UN/MC540-DOX, 10 mg of DSPE-PEG-DOX and 5 mg of MC540 were added into a 25 mL flask containing 10 mg of UNs and 5 mL of CHCl 3 . This mixture was stirred overnight at room temperature. In this reaction, 5 mg of DSPE-PEG 2000 -Folate was added to prepare targeted modification of PEG-TK-DOX nanoparticles.

Reagents and materials.
Loading percentage of MC540 and DOX in UNs. MC540 and DOX encapsulated within the UNs were determined in triplicate by HPLC with UV detection at 227 nm and 290 nm (LC-20AT, Shimadzu), respectively. The loading percentage was calculated according to the following formula: Cytotoxicity evaluation. Cytotoxicity of UN nanoparticle was evaluated by CCK-8 kit assay. B16 cells were seeded in 96-well plates at the density of 5 × 10 5 cells/well and cultured for 24 h at 37 °C. Original medium in each well was refreshed with 200 μ L medium containing serial dilutions of UNs (corresponding DOX concentration was 0-10 μ g/mL). After incubation for 48 h, 20 μ L of CCK-8 kit was added to all wells, and then cells were continued to be incubated at 37 °C with 5% CO 2 for 4 h. Infinite M200 microplate spectrophotometer (Tecan, Mannedorf, Switzerland) was used to detect the absorbance at 540 nm. Percent viability was normalized to cell viability in the absence of the samples.
In vivo anticancer efficient. To demonstrate the targeting ability of FA-UN/MC540-DOX in vivo, Cy5.5 dye was loaded into this nanoparticle according to the method described in the preparation of MC540-loaded nanoparticles. B16-bearing mice were injected intravenously with 5 μ g of FA-UN/MC540-DOX and UN/MC540-DOX in 100 μ L of PBS (pH 7.2), respectively. Eight hours after the intravenous administration, mice were anesthetized and scanned using in-vivo imaging system with an excitation at 670 nm and an emission filter at 700 nm.
In the subcutaneous B16 melanoma model, treatments were started when the average tumor volume reached about 100 mm 3 , and mice were randomly divided into five groups. Five groups of eight mice each were intravenously administered PBS (100 μ L, pH 7.2), DOX, UN/MC540, UN/MC540-DOX and FA-UN/MC540-DOX (1 mg/kg of DOX) once every two days, and then tumor sites were irradiated with a 980 nm laser. Tumor sizes were measured with a digital caliper every other day. And tumor volume was calculated by the formula (LxW 2 )/2, where L was the long and W was the short tumor diameter (mm). Until tumor volume reached 5000 mm 3 in PBS group, mice were sacrificed for humane reasons. In other groups, one mouse of each group was sacrificed and tumor tissue was removed for apoptotic analysis. Detection of apoptotic cells by TUNEL (terminal transferase-mediated dUTP nick end labeling) staining accorded to the protocol from Thermo Fisher Scientific.