Light-Activatable Red Blood Cell Membrane- Camouflaged Dimeric Prodrug Nanoparticles for. Synergistic Photodynamic/Chemotherapy

Transcription

1 Supporting Information Light-Activatable Red Blood Cell Membrane- Camouflaged Dimeric Prodrug Nanoparticles for Synergistic Photodynamic/Chemotherapy Qing Pei,, Xiuli Hu,*, Xiaohua Zheng,, Shi Liu, Yawei Li, Xiabin Jing, and Zhigang Xie*, State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,5625 Renmin Street, Changchun, Jilin , P. R. China University of Science and Technology of China, Hefei, Anhui, , P. R. China * xiez@ciac.ac.cn; lily@ciac.ac.cn 1

2 Materials. Paclitaxel (PTX) was purchased from Xian Haoxuan Biological Technology Co., Ltd.. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl, GL Biochem) and 4- dimethylaminopyridine (DMAP, Aladdin) were used as received. Chloroform-d (CDCl 3 ) was purchased from Qingdao Tenglong Weibo Technology Co., Ltd.. Annexin V-FITC apoptosis detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.. Cell viability (live dead cell staining) assay kit was purchased from Jiangsu KeyGEN Biotechnology Co., Ltd.. Ultrapure water was prepared from a Milli-Q system (Millipore, USA). Solvents for chemical synthesis were purified by distillation. Methods Synthesis of PTX 2 -TK. RS-cleavable thioketal linker (TK) was first synthesized according to the literature. 1 Then PTX (299.5 mg, 0.35 mmol) was dissolved in dichloromethane (CH 2 Cl 2 ), and the obtained TK (49.5 mg, mmol), EDC. HCl (147.2 mg, 0.77 mmol) and DMAP (5.5 mg, mmol) were added sequentially. After stirring for 1 h at ambient temperature, additional EDC. HCl (71.2 mg, 0.37 mmol) and DMAP (5.8 mg, mmol) were added, and the reaction was continued for another 24 h. The reaction product was purified by silica gel column chromatography using CH 2 Cl 2 and ethyl acetate (v/v, 2/1) as eluent. White solid was obtained with yield of 84%. 1 H NMR (400 MHz, CDCl 3 ) δ (m, 2H), 7.74 (d, J = 7.3 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), (m, 10H), 7.01 (d, J = 9.1 Hz, 1H), (m, 2H), 5.97 (dd, J = 9.2, 3.3 Hz, 1H), 5.68 (d, J = 7.1 Hz, 1H), 5.53 (t, J = 4.9 Hz, 1H), 4.97 (d, J = 7.7 Hz, 1H), 4.44 (dd, J = 10.9, 6.6 Hz, 1H), 4.32 (d, J = 8.4 Hz, 1H), 4.20 (d, J = 8.4 Hz, 1H), 3.80 (d, J = 7.1 Hz, 1H), (m, 4H), (m, 1H), 2.46 (d, J = 5.8 Hz, 3H), (m, 1H), (m, 3H), (m, 1H), (m, 4H), 1.68 (s, 3H), 1.58 (s, 2

3 3H), (m, 3H), 1.13 (s, 3H). MS (LTQ) m/z: calculated for PTX 2 -TK (C 103 H 114 N 2 30 S 2 ) [M+Na] , found Synthesis of TPC. 5,10,15,20-tetraphenylchlorin (TPC) was synthesized from 5,10,15,20- tetraphenylporphyrin (TPP) according to the literature 2 and characterized by 1 H NMR and LTQ. 1 H NMR (400 MHz, CDCl 3 ) δ 8.57 (d, J = 4.9 Hz, 2H), 8.42 (d, J = 4.4 Hz, 2H), 8.18 (d, J = 4.8 Hz, 2H), 8.11 (dd, J = 7.5, 1.6 Hz, 4H), 7.88 (dd, J = 7.5, 1.6 Hz, 4H), (m, 12H), 4.16 (s, 4H), 1.43 (s, 2H). MS (LTQ) m/z: calculated for TPC (C 44 H 32 N 4 ) [M+H] , found Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis assays. The protein concentration of empty RBCs, RBCVs and RBC(M(TPC-PTX)) was quantified according to the BCA protein assay kit. Electrophoresis in a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used to separate a 60 µg protein aliquot of each sample. The separation efficacy was examined after the gel was stained with coomassie brilliant blue. Cell lines and cell culture. HeLa (human cervical carcinoma) and RAW264.7 (mouse macrophage) cell lines were cultured in Dulbecco s modified Eagle s medium (DMEM, GIBC) with 10% (v:v) heat-inactivated fetal bovine serum (FBS, GIBC). Cells were cultured in a humidified incubator at 37 o C with 5% C 2, and the culture medium was replaced once every day. 3

4 Cellular uptake measured by CLSM. The cellular uptake of M(TPC-PTX) and RBC(M(TPC- PTX)) were examined by using a confocal laser scanning microscope (CLSM). HeLa cells were seeded in 6-well plates (a clean cover slip was put in each well) at the density of cells per well and allowed to adhere for 24 h. And then the medium was replaced with M(TPC-PTX) or RBC(M(TPC-PTX)) diluted with fresh culture medium to a final TPC concentration of µm. Thereafter cells were incubated for additional 2h, 4h, and 6h at 37 o C. Subsequently, the supernatant was removed and the cells were washed gentlely three times with PBS (ph 7.4), fixed with 4% paraformaldehyde (1 ml/ each well) for 15 min and washed thrice with cold PBS. Hoechst was employed to stain the cell nuclei. For lysosome colocalization, the cells were first incubated with RBC(M(TPC-PTX)) for 6 h, and then treated with Lyso-Tracker Red and Hoechst for 30 min and 4 min at 37 C to stain lysosomes and cell nuclei, respectively, and observed by CLSM. The influence of light irradiation on endosomal escape was investigated by culturing HeLa cells with RBC(M(TPC-PTX)) for 6 h, and subsequent irradiation with 638 nm laser lamp (power density of 100 mw/cm 2 ) for 20 min. And then the cells were stained by Lyso- Tracker and observed by CLSM. RAW264.7 cellular uptake of RBC(M(TPC-PTX)) measured by CLSM. RAW264.7 cells ( cells/per well) were seeded in the 6-well plates and cultured for 24 h. Afterwards, the cells was added with M(TPC-PTX) and RBC(M(TPC-PTX)) and incubated for 6 h. Then the cells were washed with PBS and fixed with 4% paraformaldehyde. After the cell nuclei was stained with Hoechst, cells were subjected to CLSM observation. RAW264.7 cellular uptake of RBC(M(TPC-PTX)) measured by FCM. 4

5 RAW264.7 cells ( cells/per well) were seeded in the 6-well plates and cultured for 24 h. Afterwards, the cells was added with M(TPC-PTX) and RBC(M(TPC-PTX)) and incubated for 6 h. And then the supernatant was removed and the cells were washed gently three times with cold PBS (ph 7.4), subsequently treated with trypsin. The harvested cells were suspended in PBS and centrifuged at 1000 r/min for 5 min. The rinse and centrifugation was repeated for three times. Then the supernatants were discarded and the cell pellets were re-suspended in 0.5 ml PBS before analysis by Guava easycyte 6-2L Base System (Merck Millipore, USA). Cellular uptake measured by FCM. The HeLa cells were seeded in 6-well plates at about a density of cells per well in 1.5 ml of DMEM medium and allowed to adhere for 24 h. And then the medium was replaced with M(TPC-PTX) or RBC(M(TPC-PTX)) diluted with fresh culture medium to a final TPC concentration of µm. After incubation for 2 h, 4 h and 6 h at 37 C, the supernatant was removed and the cells were washed gently three times with cold PBS (ph 7.4), subsequently treated with trypsin. The harvested cells were suspended in PBS and centrifuged at 1000 r/min for 5 min. The rinse and centrifugation was repeated for three times. Then the supernatants were discarded and the cell pellets were re-suspended in 0.5 ml PBS before analysis by Guava easycyte 6-2L Base System (Merck Millipore, USA). Intracellular RS detection. RS generation was detected by a cell-permeable RS-sensitive fluorescent probe, 2,7 -dichlorofluorescin diacetate (DCFH-DA). DCFH-DA is nonfluorescent, but it could be rapidly oxidized to a fluorescent molecule (dichlorofluorescein, DCF) by RS. 3 Hela cells were incubated with RBC(M(TPC-PTX)) with TPC concentration of µm for 6 h, and then illuminated by 638 nm laser lamp (power density of 100 mw/cm 2 ) for 20 min. Hela 5

6 cells without illumination treatment were used as negative control. After the irradiation, the medium was replaced with culture medium. Then DCFH-DA (10 µm) was added and the cells were incubated for 20 min. After removing the DCFH-DA-containing medium and washing three times, Cells was subjected to observation fluorescence by CLSM. VC was used to reduce the fluorescence of DCF by quenching the generated RS. Cell viability assays. The cytotoxicity of M(TPC), M(TPC-PTX), RBC(M(TPC)) and RBC(M(TPC-PTX)) with or without laser irradiation was examined via MTT protocol. Briefly, HeLa cells harvested in a logarithmic growth phase were seeded in 96-well plates at an initial density of cells/well and incubated in 100 µl DMEM at 37 o C in 5% C 2 atmosphere for overnight. After removing incubation medium, M(TPC), M(TPC-PTX) and RBC(M(TPC-PTX)) (100 µl) dispersions diluted with cell culture media to the desired concentration were added to cell wells. After 6 h incubation, cells were illuminated by 638 nm laser lamp (power density of 100 mw/cm 2 ) for 5 min. Cells treated with free of drug or photosensitizer were used as control. After incubation 48 h, 20 µl of MTT in PBS solution with the concentration of 5 mg ml -1 was added and the plates were incubated at 37 C for another 4 h. After careful removal of the culture medium supernatant, 150 µl of dimethyl sulfoxide (DMS) was added to each well to dissolve the formed violet formazan crystals. Finally, the plates were shaken for 3 min, and the absorbance of violet product was quantified at 490 nm by a microplate reader. Calcein-AM/PI staining tests. To further demonstrate the chemotherapy and photodynamic therapy efficacy of M(TPC-PTX) and RBC(M(TPC-PTX)), HeLa cells were stained with the calcein-am/propidium iodide (PI) to identify dead (red) and live (green) cells. Shortly, Hela 6

7 cells were incubated with M(TPC-PTX) and RBC(M(TPC-PTX)) with the final TPC/PTX 2 -TK concentration of 13.75/0.325 µm for 6 h, and then illuminated by 638 nm laser lamp (power density of 100 mw/cm 2 ) for 5 min. M(TPC-PTX) and RBC(M(TPC-PTX))) without irradiation, PBS and PBS with irradiation were set as negative control. After additional incubation for 24 h, the medium was removed and cells were washed gently. Then cells were incubated with Calcein- AM/PI for 30 min at room temperature, subsequently imaged by a NikonC1si laser scanning confocal microscopy. Cell apoptosis and necrosis detection assays. The cell early and late apoptosis induced by PDT and chemotherapy of M(TPC-PTX) and RBC(M(TPC-PTX)) were quantified by FCM. Briefly, Hela cells were cultured with M(TPC-PTX) and RBC(M(TPC-PTX)) with the final TPC/PTX 2 - TK concentration of 13.75/0.325 µm for 6 h, and then illuminated by 638 nm laser lamp (power density of 100 mw/cm 2 ) for 5 min. The group PBS, M(TPC-PTX) and RBC(M(TPC-PTX)) without irradiation, PBS and M(TPC-PTX) with irradiation were set as negative control. After additional incubation for 24 h, cells were washed, harvested and collected, and stained with Annexin V-FITC and PI detection kit for about 20 min. Finally, the ratio analysis of apoptosis and necrosis were determined through flow cytometer. The pharmacokinetic and biodistribution study. Kunming Mice (20-25 g) with subcutaneous mouse cervical carcinoma U14 tumor xenografts were used as animal modal to investigate the pharmacokinetics and biodistribution of the prepared formulations. U14 cells were subcutaneously inoculated into the lateral aspect of the anterior right limb of mice ( cells in 0.1 ml PBS). After tumor volume achieved of about 500 mm 3, mice were randomly 7

8 divided into two groups (n = 4): (1) M(TPC-PTX) and (2) RBC(M(TPC-PTX)) (17.4 mg kg -1 equivalent of PTX 2 -TK). Nanoparticles were intravenously administrated through the tail vein. The blood samples (0.4 ml) were taken out from the orbit at 0 h, 1 h, 3.3 h, 6 h, 23 h and 47 h. The plasma was obtained by centrifugation at 3000 r/min for 5 min and then methyl tert-butyl ether (TBME, 1 ml) was added to extract the PTX 2 -TK. The extraction was repeated for three times. After removing TBME, 0.2 ml of acetontrile was added, and the samples were set for detection its concentration by HPLC. Then mice were sacrificed and the liver and tumor were excised and homogenated in PBS solution. The supertanant was obtained after homogenation and centrifugation at 3000 r for 5 min. After extraction the supertanant with TBME for three times, the concentration of PTX 2 -TK was detected by HPLC. Light-triggered extravasation of RBC(M(TPC-PTX)) and deep penetration. Kunming Mice (20-25 g) with subcutaneous mouse cervical carcinoma U14 tumor xenografts were used as animal modal to investigate light-triggered extravasation of RBC(M(TPC-PTX)) and deep penetration in vivo. U14 cells were subcutaneously inoculated into the lateral aspect of the anterior right limb of mice ( cells in 0.1 ml PBS). After tumor volume achieved of about 500 mm 3, mice were intravenously injected with RBC(M(TPC-PTX) at an identical TPC dose of 20 mg kg 1 through the tail vein. After injection 6 h, the tumors were locally irradiated (638 nm laser lamp, 200 mw/cm 2 ) for 15 min. Then the mice were sacrificed to collect the tumor tissues. The tumors were then frozen sectioned, blocked with goat serum, stained with immunofluorescence blood vessel marker CD31, stained with DAPI, and examined using CLSM. 8

9 Antitumor efficacy and safety evaluation. Male nude mice were obtained and maintained under required conditions. All animal procedures were approved and controlled by the local ethics committee and carried out according to the guidelines of Chinese law concerning the protection of animal life. To evaluate antitumor effects, mice with subcutaneous human cervical carcinoma HeLa tumor xenografts were utilized as animal modal. HeLa cells were subcutaneously inoculated into the lateral aspect of the anterior right limb of mice (5*10 6 cells in 0.1 ml PBS). The tumor-bearing mice were randomly divided into seven groups (n=6): (1) normal saline (blank control), (2) PBS with irradiation (PBS (L+)), (3) M(PTX), (4) M(TPC) (L+), (5) M(TPC-PTX), (6) M(TPC-PTX) (L+) (7) RBC(M(TPC-PTX)) and (8) RBC(M(TPC- PTX)) (L+) groups. Before treatment, mice were marked, weighed and measured. Then experimental groups were intravenously injected into the tumor-bearing mice at PTX/TPC equivalent dose of 30 mg/kg and 10 mg/kg (drug weight/body weight), respectively, on day 1. After injection 6 h, tumor was irradiated by 638 nm laser lamp (power density of 200 mw/cm 2 ) for 15 min. At designed time (1, 3, 5, 7, 10, 12, 15, 18, 21 and 24 d), the body weight of mice and tumor volume were measured. At day 24, mice were sacrificed, and tumor was excised to intuitionally evaluate the tumor inhibition. In order to investigate the safety of formulations, the potential hepatic and renal toxicity was evaluated. The blood of mice was taken out for testing the level of serum alanine transaminase (ALT), aspartate transaminase (AST), uric acid (UA), urea (UREA) and creatinine (CREA). Main organs (heart, liver, spleen, lung, kidney) and tumor were collected, fixed in 4% paraformaldehyde solution, and then embedded in paraffin, sliced and stained with hematoxylin and eosin (H&E) to evaluate potential toxicity for main organs and apoptosis degree for cancer cells. 9

10 Physicochemical characterization. Proton nuclear magnetic resonance ( 1 H NMR) spectra was recorded on a Bruker AV400 M in CDCl 3 at 25 o C. Chemical shifts were given in parts per million from that of tetramethylsilane (TMS) as an internal reference. The mass spectrum (MS) analyses were performed on a LTQ ion trap mass spectrometer (Finnigan, USA) equipped with an electrospray source. Size, size distribution and zeta-potential of the nanoparticles were determined by Malvern Zeta-sizer Nano. The scattering angle was fixed at 90 o and the measurement was carried out at 25 o C. The morphology of the nanoparticles was measured by transmission electron microscopy (TEM) performed on a JEL JEM-1011 electron microscope operating at an acceleration voltage of 100 kv. To prepare specimens for TEM, a drop of nanoparticles solution (0.1 mg/ml) was deposited onto a copper grid with a carbon coating. The specimens were air-dried and measured at room temperature. UV-vis absorption spectra were obtained using a Shimadzu UV-2450 PC UV-vis spectrophotometer. Fluorescence intensity tests were performed using Perkin Elmer LS-55 fluorospectrophotometer. The cellular localization was visualized under a confocal laser scanning microscope (CLSM) (Zeiss LSM 700, Zurich, Switzerland). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were measured at 490 nm by a microplate reader (BioTek, EXL808). Flow cytometry analysis was performed by a flow cytometer (Beckman, USA) which collected gated events for each sample. 10

11 Supplementary Figures a H SH + HCl H S S H thioketal linker (TK) b N H H H 3 C H H H H S S H EDCHCl, DMAP H CH 3 H NH H S S H HN H H 3 C H PTX dimer (PTX 2 -TK) Figure S1. Synthetic route of RS-cleavable thioketal linker (TK) (a) and PTX dimer PTX 2 -TK (b). 11

12 Figure S2. 1 H NMR spectra of PTX (up) and PTX 2 -TK (down) in CDCl 3. 12

13 Figure S3. Linear ion trap mass spectrum of PTX 2 -TK. Figure S4.The purity of PTX 2 -TK detected by HPLC. 13

14 Figure S5. 1 H NMR spectrum of TPC in CDCl 3. Figure S6. Linear ion trap mass spectrum of TPC. 14

15 Figure S7. Representative normalized UV vis absorption spectra (a) and fluorescence (b) of TPC in DMF/H 2 = 9:1 (v/v) and M(TPC-PTX)/RBC(M(TPC-PTX)) in aqueous solution. 15

16 Figure S8. Change of size (a) and polydispersity index (PDI) (b) of all NPs when stored at -4 o C for two weeks. Figure S9. (a) Flow cytometry analysis in RAW264.7 cells showing the cellular uptake of M(TPC-PTX) and RBC(M(TPC-PTX)). Cells were cultured with M(TPC-PTX) and RBC(M(TPC-PTX)) for 6 h, respectively. (b) The positive percent summarized from flow cytometry analysis. 16

17 Figure S10. Mass spectrum of PTX 2 -TK after 120 h treatment without H 2 2. Figure S11. The degradation of PTX 2 -TK triggered by H 2 2. (a) The degradation of PTX 2 -TK in the presence of 100 µm H 2 2 monitored by HPLC. (b) The change of integral area of PTX 2 -TK and its degradation product PTX-S 3 H over time summarized from HPLC. 17

18 Figure S12. UV-vis spectra of pure ICG solution (9 µg/ml, in PBS) under irradiation with a 638 nm laser lamp (power density of 100 mw/cm 2 ) for 7 min. Figure S13. UV-vis spectra of mixture solution of (a) M(TPC-PTX) (7.5 µm) +VC (4 mm) +ICG (9 µg/ml) and (b) RBC(M(TPC-PTX)) (7.5 µm) +VC (4 mm) +ICG (9 µg/ml) under irradiation with a 638 nm laser lamp (power density of 100 mw/cm 2 ) for 7 min. 18

19 Figure S14. Photostability of (a) M(TPC), (b) M(TPC-PTX), (c) RBC(M(TPC-PTX)) by testing relative absorbance A/A 0 as a function of irradiation time under irradiation with a 638 nm laser lamp (power density of 100 mw/cm 2 ) for 7 min. A = absorbance of nanoparticle solution at respective maximum absorption wavelength (431 nm for M(TPC) and 437 nm for M(TPC-PTX) and RBC(M(TPC-PTX))) and at time t, A 0 = absorbance of the mixture solution at time 0. Figure S15. TEM image of RBC(M(TPC-PTX)) before (a) and after (b) being irradiated by 638 nm laser lamp (100 mw/cm 2 ). 19

20 Figure S16. Cellular uptake of M(TPC-PTX) detected by CLSM (a) and FCM (b) for different cultured time. Cellular uptake of RBC(M(TPC-PTX)) detected by CLSM (c) and FCM (d) for different cultured time. 20

21 Figure S17. CLSM images of HeLa cells cultured with RBC(M(TPC-PTX)) at 37 o C for 6 h. From left to right: pictures correspond to hoechst (blue), RBC(M(TPC-PTX)) (green), Lyso- Tracker red (red) and merged images. Scale bar, 20 µm. 21

22 Figure S18. (a) CLSM images of HeLa cells cultured with RBC(M(TPC-PTX)) at 37 o C for 6 h, and subsequently irradiated HeLa cells with 638 nm laser lamp (power density of 100 mw/cm 2 ) for 20 min. From left to right: pictures correspond to hoechst (blue), RBC(M(TPC-PTX)) (green), Lyso-Tracker red (red) and merged images. Scale bar, 20 µm. Corresponding fluorescence intensity profiles upon treatment without (b) and with (c) irradiation. 22

23 Figure S19. Intravital CLSM images of light-triggered extravasation of RBC(M(TPC-PTX)) from tumorous vasculature to intratumor in vivo. Scale bars, 20 µm. Figure S20. Histologic assessments of H&E staining in different organs (heart, liver, spleen, lung and kidney) of mice. Scale bar stands for 100 µm. 23

24 Figure S21. Serum biochemical analysis of kidney and liver function parameters. (n=6) Reference (1) Yuan, Y.; Liu, J.; Liu, B., Conjugated-Polyelectrolyte-Based Polyprodrug: Targeted and Image-Guided Photodynamic and Chemotherapy with n-demand Drug Release upon Irradiation with a Single Light Source. Angew. Chem., Int. Ed. 2014, 53, (2) Lu, K.; He, C.; Guo, N.; Chan, C.; Ni, K.; Weichselbaum, R. R.; Lin, W., Chlorin-Based Nanoscale Metal-rganic Framework Systemically Rejects Colorectal Cancers via Synergistic Photodynamic Therapy and Checkpoint Blockade Immunotherapy. J. Am. Chem. Soc. 2016, 138, (3) Han, K.; Lei, Q.; Wang, S. B.; Hu, J. J.; Qiu, W. X.; Zhu, J. Y.; Yin, W. N.; Luo, X.; Zhang, X. Z., Cancer Treatment: Dual-Stage-Light-Guided Tumor Inhibition by Mitochondria-Targeted Photodynamic Therapy. Adv. Funct. Mater.2015, 25,