Photodynamic Therapy with Liposomes Encapsulating

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Photodynamic therapy with liposomes encapsulating photosensitizers with aggregation-induced emission Yang Yang, Lei Wang, Hongqian Cao, Qi Li, Ying Li, Mingjuan Han, Hao Wang, and Junbai Li Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b04875 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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Photodynamic therapy with liposomes encapsulating photosensitizers with aggregationinduced emission Yang Yang, †, ‖ Lei Wang, †, ‖ Hongqian Cao, † Qi Li, ‡ Ying Li, †, § Mingjuan Han, § Hao Wang,*, † Junbai Li*, ‡ †CAS

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National

Center for Nanoscience and Technology, Beijing 100190, China. ‡Beijing

National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Colloid

and Interface Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. §College

of Chemistry and Molecular Engineering, Nanjing Tech University, Nanjing,

China.

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ABSTRACT: As a non-invasive treatment, photodynamic therapy (PDT) is a promising strategy against tumors. It is based on photosensitizers (PSs) induced photo-toxicity after irradiation. However, most clinically-approved PSs will be widely distributed in normal tissues, especially in the skin, where they will induce photo-toxicity on exposure to light. Therefore, patients have to remain in a dark room for up to several weeks during or after a PDT. Herein, we proposed a strategy of aggregation-induced emission PSs (AIE-PSs) entrapped in liposomes with controlled photosensitization. The AIE-PSs become lose their photosensitivity when entrapped in liposomes. After liposomes carried AIE-PSs into tumor tissues, the AIE-PSs will be released and immediately re-aggregate in a targeted area as the liposomes are decomposed. Their photosensitivity can be triggered at turn-on state and induce cytotoxicity. Two different types of AIE molecules were synthesized and entrapped by liposomes, respectively, to verify the PDT features against tumors in vitro and in vivo. The results indicate that using this strategy, the photosensitivity of AIE-PS can be controlled and PDT can be treated under normal work condition, not necessarily in a dark room. KEYWORDS: liposome, aggregation-induced emission, self-assembly, photodynamic therapy, bis(pyrene) Compared with traditional treatment options, photodynamic therapy (PDT) has obvious advantages on against tumors for its unique selectivity, high spatiotemporal precision and good controllability.1-3 Basically, PDT is based on produced reactive oxygen species (ROS) resulting in toxicity after irradiating photosensitizers (PSs). 2 ACS Paragon Plus Environment

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However, after injection, traditional PSs are inevitably distributed into normal tissues, especially skins, and also cause photo-toxicity to them. Therefore, traditional PDT must be operated in a darkroom. The patients should also take a long rest in darkroom after PDT, which leads to low tolerance for them. (Table S1) Except for the caused photo-toxicity to normal tissues, the traditional PSs, such as porphyrin and chlorin derivatives, also suffer from an aggregation-caused quenching (ACQ) process, which result in low ROS productivity and low PDT efficiency.4,5 Although considerable progress has been made to overcome the problems associated with the PSs, the situation is far from satisfactory. Dispersed PSs will become distributed around the whole body and cause photo-toxicity to normal tissues. Developing aggregationinduced emission PSs (AIE-PSs) might solve the issue because aggregation-induced emission (AIE) reagents present very weak emission in molecular state but strong emission in aggregated state.6-9 Therefore, some researchers developed AIE-PSs for improving photosensitivity in PDT.10-12 However, how to control the photosensitivity of their PSs in living system is still a challenge. Herein, we proposed AIE-PSs can be entrapped in liposomes for controlling their photosensitization through co-assembled method. After co-assembly with lipid molecules, AIE-PS can be embedded into lipid bilayer to form AIE-PS@liposomes complex. In this case, the photosensitivity/aggregation of AIE-PS molecules would be inhabited, which presents low photo-toxicity for normal tissues after injection. When AIE-PS@liposomes reach tumor sites with passive and active mechanism, AIE-PSs


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are delivered and re-aggregated due to liposome degradation, which results in phototoxicity for target tissues (as Figure 1a illustrated). Therefore, the system can reduce the photo-toxicity of PSs to normal tissues during PDT according to in-situ biological response. In addition, although liposome based drug complexes have been widely used, it is still difficult to monitor their bio-distribution and research their degradable mechanism due to the relative scarcity of in-situ detection methods. AIE@liposomes complex also provide a chance to solve the problem. Two kinds of novel AIE reagents, bis(pyrene) (BP) and MC4, were co-assembled with lipid to construct AIE@liposomes complex for confirming the proposed theory and methods. Especially for BP molecules, they not only present good AIE activity and photosensitivity for PDT, but also possess large two-photon absorption cross-sections (TPAC) and can be excited by two-photon laser for improving treatment depth in PDT. We believe AIE@liposomes is an excellent platform for accommodating newly developed AIE-active photosensitizers for controlling their photosensitivity on in-situ biological response.


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Figure 1. a) Illustration of co-assembled AIE-PS@liposomes composed of AIE-PS and lipid for anti-tumor in PDT; b) TEM images and the inset of DLS of liposomes, c) nanoBP and d) BP@liposomes; e) The contrast of fluorescence intensity between BP dispersion and the corresponding amount of BP in liposomes complex. The excitation wavelength was 405 nm. Preparation and Fluorescent Property of BP@liposomes. Liposomes composed of lipid mixture were prepared by typical thin lipid film hydration method. The lipid mixture contained DMPC, DSPE-PEG and DSPE-PEG-Folate in a certain proportion. Two kinds of AIE reagents, BP and MC4 were used as AIE model molecules to coassemble with lipid to form BP@liposome or MC4@liposome complex. The molecular structures of reagents were listed in Figure S1. Typical morphologies of liposomes and 5 ACS Paragon Plus Environment

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BP@liposomes were showed in Figure 1b, d. Both of them presented vesicle structures dispersed with 160-200 nm. There were no significant size and topography changes for them. According to our recent work, pure BP molecules are distinctly hydrophobic and will form nano-aggregates (nano-BP) in water (Figure 1c).13-15 However, no aggregates existed in transmission electron microscopy (TEM) of BP@liposomes, which indicated that BP molecules had been embedded into lipid bilayer. BP@liposomes vesicle structures were formed based on interaction between lipid hydrophobic chain and BP molecules. Dynamic light scattering (DLS) data (the inset image in TEM) and statistical analysis result according to TEMs (Figure S2) confirmed that the products dispersed well in solution with low polydispersity index. BP molecules possess obvious AIE activity and present strong fluorescence at aggregated state (Figure S3a). Conversely, BP@liposomes presented faint fluorescence when the same amount of BP was embedded into lipid bilayers (Figure S3b), which proved that BP existed in lipid bilayer with dispersed state. Furthermore, a series of BP@liposome samples loading different amount of BP were prepared and their fluorescent properties were investigated. The corresponding amount of pure BP was dispersed into solution and assembled into nano-BP for comparing their fluorescent intensity with those of BP@liposomes. As shown in Figure 1e, the fluorescence intensity of nano-BP gradually boosted followed by increasing BP concentrations. On the contrary, the fluorescence intensity of liposomes entrapped with same amount of BP was dropped rapidly (Figure S4). The BP@liposome samples containing less than 18 μg mL-1 BP showed scarcely fluorescence, which proved that 6 ACS Paragon Plus Environment

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most of BP was embedded into lipid bilayers. BP@liposomes were enough stable as BP was hardly released from liposomes for more than ten days (Figure S5). Furthermore, BP@liposomes were broke by surfactant (Triton X-100) and the fluorescence was tested by fluorescence spectrum and flow cytometry. The fluorescence intensity of BP in liposomes was almost restored its level of aggregated state after adding Triton X-100 (Figure S6). TEM results proved that BP@liposome structures were broken and BP was released and re-aggregated for emission (Figure S7).

Figure 2. a) CLSM image of MCF-7 cells incubated with BP@liposome suspensions for 4 h. The corresponding image was the 3D reconstruction image of the cells; green was from cell membrane dye (Alexa Fluor® 488), blue was from cell nuclei dye (Hoechst 33342) and red was from Texas Red® labeled lipid (Texas Red-DHPE) in BP@liposomes; b) Flow cytometry analysis (Ex405 nm) of MCF-7 cells after 7 ACS Paragon Plus Environment

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incubation with BP@liposomes (no folate, blue curve), BP@liposomes-Folate (with folate, cyan curve) for 30 h and cells without any treatment as control group (red curve); c) CLSM image of MCF-7 cells cultured with BP@liposomes for 30 h and irradiated with two-photon laser (Ex810 nm) in location, green fluorescence from Calcein-AM and red fluorescence from PI; d) CLSM images of cells co-cultured with BP@liposomes doping with folate lipid (Ex810 nm with two-photon laser) and e) nano-BP (Ex810 nm with two-photon laser). Investigation of Fluorescent Property and Phototoxicity of BP@liposomes in Cells. MCF-7 cells were used as models to investigate the assembly and disassembly of BP@liposomes in cells. Firstly, BP@liposomes were co-cultured with cells for 4 h and observed immediately with confocal laser scanning microscopy (CLSM). Figure 2a showed that BP@liposomes were easily internalized into cancer cells. In addition, as shown in Figure S8, the fluorescence signals from BP@liposomes in cells were enhanced gradually within 30 h. It was reported that liposomes could be degraded by intracellular phospholipase.16 Therefore, BP@liposomes were degraded gradually and BP molecules were released and re-aggregated in cells. Similar result was obtained with two-photon laser as imaging source (810 nm, Figure 2d). It was because that nano-BP has extremely large TPAC (2.4 × 105 GM),14 which is helpful for the following work in improving treatment depth in PDT. In addition, it was found that BP signal decreased linearly in time when cells internalized nano-BP (Figure 2e, which


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was contrary to the phenomenon of BP@liposomes in cells. This result indirectly validated BP’s re-aggregated mechanism in cells after BP@liposomes degradation. The cells were also co-cultured with BP@liposomes for 4 h, washed and then monitored the fluorescence change of BP inside cells by in-situ observation using CLSM. Although the increasing fluorescent signals in cells were observed, most of cells died gradually for more than 30 h observation (Figure S9). We hypothesized that the cell apoptosis is caused by photosensitivity of BP under real-time imaging and irradiating condition. It means BP could produce ROS after laser irradiation. We verified this in different ways, such as using ROS sensors (ABDA and DCFH-DA) and phosphorescence spectra. Absorbance of ABDA at 400 nm will be decreased with 1O2 concentration increases in solution.17 As shown in Figure S10a, the absorbance of ABDA in nano-BP dispersion decreased gradually over time under light irradiation (480 nm). On the contrary, there was little decrease of ABDA absorbance for BP@liposomes system when same irradiation was applied (Figure S10b). Furthermore, BP@liposomes were broke by Triton X-100 and irradiated with same condition, and the decrease of ABDA absorbance was observed again (Figure S10c). As blank sample, the absorbance of ABDA solution (without any samples) nearly had little decrease with the same condition (Figure S10d). Phosphorescence spectrum was used to directly detect ROS due to characteristic phosphorescence of singlet oxygen at around 1270 nm.18 As we expected, nano-BP presented singlet oxygen phosphorescence maximum at 1277 nm after light irradiation, while BP@liposomes


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showed nothing there with same conditions (Figure S11). It directly proved that aggregated BP produced singlet oxygen after irradiation, while BP@liposomes did not. In addition, DCFH-DA was used as intracellular ROS sensor.19 Cells internalized nanoBP presents strong DCF fluorescence after irradiation, which proves high ROS level induced by nano-BP. Under the same light irradiation condition, cells internalized BP@liposomes present low DCF fluorescence in their initial stage, while high DCF fluorescence after 30 h incubation with BP@liposomes (Figure S12). The above results clearly indicated that ROS came from aggregated BP rather than dispersed BP. It was entirely different from ACQ type of PSs, such as porphyrin and chlorin derivatives et al. Therefore, we predict that AIE-PS@liposomes based system would hardly produce ROS whether light irradiate them or not. AIE-PS would aggregate again in biological environment after AIE-PS@liposomes degradation and then produce ROS after irradiation. Then, photo-toxicity of BP@liposomes against MCF-7 cells was investigated by MTT assay with different sample doses and co-culture times. As shown in Figure S10e, the cell viability decreased with increasing both BP@liposomes concentration and coculture time. Especially with increasing co-culture time from 0 to 30 h and then irradiation, the cell viability decreased from 100% to 30.8% for BP@liposomes (700 μg mL-1) co-cultured with cells. The cell viability has no significant changes with sample doses and co-culture times under the same experimental conditions in dark (Figure S10f). These results indicated that BP@liposomes gradually degraded inside cells over time; BP aggregated again and induced photo-toxicity. The cytotoxicity of 10 ACS Paragon Plus Environment

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BP@liposomes to MCF-7 cells induced by two-photon was also evaluated through CLSM equipped with two-photon laser. To observe the apoptosis of cells visually, propidium iodide (PI) and Calcein-AM were added to stain population of necrotic and viable cells, respectively.11 Compared with blank cells, there was almost no change for those cells treated with BP@liposomes for 3 h and irradiated with two-photon laser in location (Figure S13). However, when the cells were incubated with BP@liposomes for 30 h and then irradiated with two-photon with same condition, the cells in twophoton irradiated area were apoptotic obviously (Figure 2c and Figure S13b). The results indicated that the dead cells were induced by re-aggregated BP after twophoton irradiation according to the mechanism mentioned above.

Figure 3. Fluorescent property of AIE@liposomes in vivo. a) 3D reconstruction of CLSM images of tumor in situ at different post-injection times, scale bar=100 μm,


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Ex810 nm with two-photon laser. b) Fluorescence images of the major organs (heart, liver, spleen, lung and kidney) resected from mice after post-injection at 3, 15, 30, 60 h point (Ex520 nm). c) Average fluorescent intensity of tumor after post-injection for 3, 15, 30, 60 h. d) Relative intensity of organs after 30 h post-injection. Data collected were expressed as mean ± SD. (n = 3). Investigation of Fluorescent Property and Phototoxicity of AIE@liposomes in vivo. Folate conjugated lipids were added into AIE@liposomes forming BP@liposomesFolate for improving their tumor selectivity. The effect had been validated primitively in cellular level as shown in Figure 2b. The cancer cells internalized more BP@liposomes containing Folate-lipid than those no Folate-lipid doped sample. Some PEG-lipids were also added into AIE@liposomes for prolonging their residence time.20 The biodegradation and fluorescent properties of AIE@liposomes in vivo were studied in model of tumor bearing mice. After intravenous injection with BP@liposomes, the tumor tissues were observed in situ and in vivo by CLSM equipped with two photon laser. As shown in Figure 3a, the fluorescence from BP in tumors gradually increased with time and reached maximum after 30 h post-injection. The result meant that BP@liposomes

reached

tumor

location

and

BP

accumulated

there

after

BP@liposomes decomposition. As expected, deeper tissues were observed with twophoton CLSM than with one-photon CLSM (Figure S14), which would be very helpful for the following anti-tumor PDT research. In addition, another AIE type of moleclule, MC4 (Figure S1b), was also assembled with lipid as MC4@liposomes with same


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method. The properties of fluorescence and degradation in vitro for MC4@liposomes were extremely similar with those for BP@liposomes. The corresponding results were listed in supporting information (Figure S15-S17). Furthermore, MC4@liposomes were used to research their biodistribution in vivo due to MC4’s deep red emission.21 While it is difficult to do this with BP@lipsome due to BP’s green emission. After postinjection of MC4@liposomes into tumor bearing mice at 3, 15, 30 and 60 h, major organs and tumors were resected and observed with small-animal imaging system. As shown in Figure 3b-d, the fluorescence from MC4 was detected in tumor and reached maximum after 30 h post injection, indicating that MC4 was accumulated in tumor after 15-30 h post-injection. The high effective accumulation of AIE@liposomes in tumors might attribute to positive targeting from folate species and passive targeting from enhanced permeability and retention (EPR) effect.


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Figure 4. BP@liposome induced photo-toxicity to normal and tumor tissues. a) The body weight of tumor-bearing mice injected with BP@liposomes (drug) then irradiated or not within 20 days observation; b) the corresponding tumor volumes after treatment. N = 5, **P < 0.01. Blood vessels in ears being injected with c) nano-BP; d) nothing (as control) and e) BP@liposomes followed by two-photon laser irradiation treatment (middle and right images) or without irradiation treatment (left image). The ears in mice who exposed their whole body under sunlight for 2 h after injection with f) Chlorin e6; g) BP@liposomes.


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The promising targeting tumor imaging and photo-toxicity of BP@liposomes in vitro encouraged us to perform antitumor PDT test in vivo. An infrared femtosecond laser (800 nm) was used as light source with the help of its deep penetrating ability and large TPAC of BP. To evaluate the antitumor efficiency in vivo, BP@liposomes were intravenously injected into tumor bearing mice and the tumors were irradiated at different times. Mice were separated into four groups randomly as follows: mice only injected with saline as negative control (group 1), mice injected with BP@liposomes without irradiation (group 2), mice injected nothing but treated with radiation (group 3) and mice injected with BP@liposomes followed by irradiation (group 4), respectively. The weights and tumor sizes were measured every other day and the results were showed in Figure S18 and Figure 4a, b. Within 20 days monitoring, less body weight changes were observed in these groups, while group 4 exhibited a much higher antitumor efficiency than the other groups. After 20 days, the mean volume of tumors in these groups were 250 mm3 (group 4), 650 mm3 (group 3), 980 mm3 (group 2) and 1058 mm3 (group 1), respectively. The data in group 4 is only 25% of that in negative control group (P < 0.01), which indicated that BP@liposomes presented obvious antitumor PDT effect in vivo. Furthermore, the photo-toxicity of BP@liposomes and nano-BP to normal tissues were researched with representative ear tissues of mice. As we know that ears are rich in blood vessels which are located in superficial tissues. PSs would induce photo-toxicity more easily for them than other tissues. Three groups of mice were injected with PBS, BP@liposomes and nano-BP, respectively, and irradiated with two-photon laser immediately under same conditions. As shown in 15 ACS Paragon Plus Environment

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Figure 4c, mice receiving nano-BP presented severe burns in the irradiated location and the skin shrinked and damaged obviously. Both mice receiving PBS and mice receiving BP@liposomes showed nearly no changes after the irradiation (Figure 4d, e). The results demonstrated that nano-BP itself exhibited apparent photo-toxicity to normal tissues, while BP@liposomes had little effect to them. The results were consistent with those results in vitro. The mice were also exposed to the sunlight after being injected BP@liposomes or commercial photosensitizer, Chlorin e6 (Ce6). As expected, BP@liposomes present much lower photo-toxicity to normal tissues than Ce6. The blood vessels of the mice began to turn brown and became damaged after injecting Ce6 followed by exposure of their whole bodies to sunlight (Figure 4f). Those injected with BP@liposomes presented no clear changes after exposure to sunlight, as was also the case for those receiving PBS followed by sunlight (Figure 4g, Figure S19). In summary, we have thus assembled AIE type of PSs with lipid into AIEPS@liposomes complex for improving the selectivity of traditional PSs in PDT. The results present that liposomes may be the most suitable carriers for AIE type of PSs. It benefits from the consistency of ROS productivity and aggregation degree of AIEPS. Low ROS productivity induced low photo-toxicity when AIE species were embedded into liposomes bilayer with dispersed state, while significant photo-toxicity would be turned on when AIE-PS@liposomes target into tumors and degrade there. The targeting lipid species in liposomes and EPR effect improved targeting ability to


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tumors.22,23 Therefore, it can take the initiative to reduce photo-toxicity of PSs to normal tissues and organs. Two kinds of AIE type of molecules were assembled with lipid into AIE@liposomes to test the mechanism in vitro and in vivo. We believe that this model is also suitable to other AIE type of PSs reported recently.10,24-29 It is promising that patients would be liberated from dark room during and after PDT. ASSOCIATED CONTENT Supporting Information Available: The Supporting Information is available free of charge on the ACS Publications website. This file includes materials, experimental methods and Figure S1 to S19. AUTHOR INFORMATION Corresponding Author Hao Wang and Junbai Li E-mail: [email protected]; [email protected]; Fax: +86 10 82612629; Tel: +86 10 82614087 ORCID Junbai Li: 0000-0001-9575-3125 Hao Wang: 0000-0002-1961-0787 Yang Yang: 0000-0002-1535-718X Author Contributions


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Y. Y. and L. W. contributed equally to this work. Y. Y., L. W., H. C., Q. L., Y. L., M. H. and H. W. performed the experiments. Y. Y. and J. L. designed the system and advised on manuscript preparation, Y. Y., H. W. and J. L. wrote the paper. Notes The authors declare no competing financial interest. ACKNOWLEGGMENTS This work is financially supported by the National Nature Science Foundation of China (21673056, 21433010, 51573031, 51725302 and 21003074) and the open project of the CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety (NSKF201801). M. H. particularly thanks the fund from Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (No.20161002). We also thank Dr. Lei Chen and Dr. Xiaofu Wu from Changchun Institute of Applied Chemistry, CAS, for kindly providing the MC4 chemical. REFERENCES (1) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3 (5), 380-387. (2) Lucky, S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015, 115 (4), 1990-2042. (3) Abbas, M.; Zou, Q.; Li, S.; Yan, X. Self-Assembled Peptide- and Protein-Based Nanomaterials for Antitumor Photodynamic and Photothermal Therapy. Adv. Mater. 2017, 29 (12), 1605021. (4) Singh, S.; Aggarwal, A.; Bhupathiraju, N. V. S. D. K.; Arianna, G.; Tiwari, K.; Drain, C. M. Glycosylated Porphyrins, Phthalocyanines, and Other Porphyrinoids for Diagnostics and Therapeutics. Chem. Rev. 2015, 115 (18), 10261-10306. (5) Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R. K. The role of porphyrin chemistry in tumor imaging and photodynamic therapy. Chem. Soc. Rev. 2011, 40 (1), 340-362. (6) Luo, J.; Xie, Z.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang, B. Z.; Tang, B. Z.; Tang, B. Z. Aggregation-induced emission of 18 ACS Paragon Plus Environment

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Table of contents Aggregation-Induced Emission Reagents Entrapped in Liposomes with Controlled Photosensitization Yang Yang, Lei Wang, Hongqian Cao, Qi Li, Ying Li, Mingjuan Han, Hao Wang, and Junbai Li


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Fig TOC 380x256mm (85 x 85 DPI)

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Fig 1 224x342mm (150 x 150 DPI)


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Fig 2 226x207mm (150 x 150 DPI)


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Fig 3 346x372mm (150 x 150 DPI)


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Fig 4 325x328mm (150 x 150 DPI)


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Photodynamic Therapy with Liposomes Encapsulating

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