Tumor exocytosis bodies / AIEgen hybrid nanovesicle, promote tumor infiltration and photodynamic therapy

Photodynamic therapy (PDT) is a clinical strategy, which is the use of a photosensitizer (PS) exposure to a particular wavelength of the compound in an oxygen (O 2 ) the presence of light, and then generates free radicals and cytotoxicity reactive oxygen species (of ROS), such as singlet oxygen, thereby treating or localized superficial tumors and other diseases. PS development of drug-induced aggregation has a characteristic emission (AIE) of the new strategy PDT can be achieved. When the AIE fluorophore groups present in the solution in the form of isolated molecules, the minimum emissivity which, when they are present in the form of aggregates, it will produce more intense emission. AIE luminescent agent (AIEgens) is ideal for bio-imaging applications, because they have a high degree of light stability, biocompatibility, and can achieve a high contrast imaging. Importantly, many AIEgens can easily generate ROS and based therapeutic intervention for PDT. Unfortunately, most of the AIE-PS compound is very hydrophobic, and therefore can not easily be used in vivo biological environment. Before drug loading based AIEgen system (DLC) is low, encapsulation efficiency (EE) is low, the limited ability of the particular tumor, tissue penetration ability, therefore, to develop new platforms based AIEgen as a more effective penetration It means the tumor is very necessary. In addition to these limitations, common solid tumors of hypoxic tumor microenvironment (TME) due to lack of O2 and affect the efficacy of PDT, while O2 is the key to the effectiveness of these treatment strategies. Tumor exocytosis bodies (exosomes, EXO) over a potential way to enhance penetration and AIE-PS tumor efficacy. EXO is a small particles (50-200 nm), secreted by cells, derived from multivesicular bodies. EXO has many features, including a lack of immunogenicity, good biocompatibility, and the ability due to the endogenous origin and circulation for long periods, making it a promising candidate methods of drug delivery. Also able to cross the blood brain barrier and deep into the structure or other dense tissue types. Is important, EXO may be internalized by the cell, and may be based on the specific EXO membrane protein profile and cell surface-specific manner attributable to a particular tissue or cell type. EXO can not only increase the solubility of water-insoluble compounds, but also very suitable for clinical drug delivery applications, therefore, EXO / AIEgen nanovesicle hybrid has great potential to achieveEffective PDT treatment of cancer. Recently, Tang Benzhong Fellow Hong Kong University of Science and Technology in \” Angewandte Chemie International Edition \” published a paper entitled \”Tumor-exocytosed exosome / AIEgen hybrid nano-vesicles facilitate efficient tumor penetration and photodynamic therapy\” article, the use of electricity Preparation of tumor perforation exocytosis bodies / AIEgen hybrid nanovesicle (DES), the vesicles permeable to efficient tumor. Then dexamethasone function in blood vessels TME normalization, in order to reduce local hypoxia, thereby significantly enhancing the effect of PDT DES nanovesicles, it is possible to effectively inhibit tumor growth. They first time AIEgen tumor exocytosis and biological hybrid bodies, and the PDT method for normalizing blood vessels within the tumor in combination with, as a means of reducing local tissue hypoxia.

Photo Introduction [123 ]

1. preparation and characterization of DES

tumor exocytosis bodies / AIEgen hybrid nanovesicle (DES) was prepared by Method electroporation, wherein AIEgen employed DCPy (BZ Tang previous studies, [ 123] ACS Nano

2018, 12, 8145-8159). The film subjected to a fluorescent probe DiO dye can be observed under a confocal microscope to bright green and red fluorescence DCPy DiO fluorescence (FIG. 1C), confirmed the success of the coupling in these DCPy of DES. Further DES nanovesicle ultraviolet-visible absorption spectrum exhibits characteristic absorption peaks DCPy (FIG. 1D) at at 452nm. The efficiency value DCPy electroporation, DCPy with increasing initial concentration, coating efficiency increased slowly until a maximum value reached 88.2%, than the commonly used polymeric nanoparticles much higher. In the PB 4 ℃S stored for 3 days, DES nanovesicle size and no significant change in the zeta potential, indicating that these particles are highly stable (Fig. 1F and 1G). Photoluminescence (PL) spectra show, the nature of the AIE and PL properties DCPy DCPy blue shift occurs in the formation of DES, once again demonstrates the successful preparation of nano hybrid vesicles.

FIG. 1 (A) EXO and (B) TEM images DES nanovesicles. (C) was observed within DES nanovesicle DiO (green) and DCPy (red) co-positioned with a confocal microscope. (D) PBS in DCPy, EXO DES nanovesicles and UV-vis spectroscopy, the illustration shows DCPy (DMSO) is, EXO (PBS), and DES nanovesicle (PBS) is a color. (E) EXO, Zeta potential value DCPy DES and nanovesicles. (F) measuring the hydrodynamic diameter of EXO and DES nanovesicles by DLS method. (G) are measured on days 1 to 3 was suspended in PBS DES nanovesicle zeta potential. (H) containing 10% DMSO, 100% DMSO solution, and DES-PBS suspension polymerization DCPy PL spectrum. Illustration: DCPy fluorescence photograph taken at 365 nm UV light. (I) Western blotting detection CD9 (i) and CD63 (ii) labeled substandard exon. 2. In vitro testing

the DES target cancer cells in vitro tests. Since DES nanovesicle are prepared from tumor cells, so they are easy of target tumor cells. DCPy in the body of the living cells capable of specifically staining mitochondria, so this can easily be tracked internalization. They were incubated with 4T1 cells nanovesicle DES, and commercial mitochondria Mito-tracker Green FM dye probe (FIGS. 2A and 2E). After 30 minutes incubation period, DES nanovesicle visible adhered to the 4T1 cells, after 2 hours, DCPy staining seen in tumors DES nanovesicle mitochondria. These results demonstrated that DES is an ideal platform for targeting tumor cells and mitochondria.

FIG. 2 (A) over time, 4T1 tumor cells Mito-trackerGreen FM Probe (green) and DCPy (red) or DES colocalization. (B) at normal or hypoxic conditions, PBS, DCPy or DES treatment and subsequent relative tumor cell viability after laser irradiation (0.5W / cm2,5min) 532nm, assessed by the MTT assay. And a fluorescence image (D) DCFH-A fluorescence intensity of tumor cells (C) treatment. (E) using ImageJ software DCPy fluorescence intensity A quantitative analysis 1

O ² for generating a PDT effect is important. Accordingly, they use ABDA assessed their ability to produce ₁ O ² , which may be ₁ O ² to generate the oxide peroxide, resulting in reducing the absorption of ABDA. In 0.5W / cm _{at 2} The dose, the presence of absorption ABDA DCPy DES and a sharp decline. Further, even at high concentrations DCPy, they also have satisfactory biocompatibility. Then assessed the efficacy of PDT DES nanovesicles (FIG. 2B) under normal and hypoxic conditions. Under normoxic conditions, DES or treatment DCPy significant phototoxic appropriate laser irradiation. However, under hypoxic conditions, this effect is significantly reduced. When the active oxygen of these cells using quantitative probe DCFH-DA is generated, fluorescence intensity significantly lower than the normal conditions under hypoxic conditions (Figure 2C). It confirmed that oxygen levels can profoundly affect the efficacy of anti-tumor PDT treatment regimen DES or DCPy mediated. To overcome this limitation, use of the (DEX) up TME as modifier, as a means of improving the supply of available oxygen of these tumors. To confirm the validity of the method, from 11-14 days after tumor implantation, with a daily DEX (3mg / kg) or PBS (n = 3 / group) were injected subcutaneously BALB / c mice with a primary tumor 4T1. The results showed that, compared to PBS treatment, of DEX treatment significantly reduced the hypoxia inducible factor HIF-1 staining intensity, it was confirmed that the therapeutic interventions sufficient to significantly alleviate the tumor hypoxia. ^{3. Test vivo} in vivo pharmacokinetics of the drug DES, biodistribution and tumor tissue permeability evaluation. In addition to having a high degree of cytotoxicity and are easily within the tumor cells completely apart, over the internet antitumor further required to have good bio-distribution system. To highlight the DES in vivo biodistribution and pharmacokinetic characteristics of drugs, they polylactic acid – glycolic acid (PLGA) to embedded DCPy, and successfully PLGA / DCPy hybrid nanoparticles (the DPS), compared with DES prepared. (N = 3 / group) and in vivo DES DPS found by observing the distribution of 4T1 tumor-bearing mice, DES DPS accumulate in the liver and is relatively strong, and exhibit more effective than DES DPS tumor predisposition in an animal ( Figure 3D). It is noteworthy that, after treatment mediated DES (Fig. 3E and 3F), can be detected from the blood vessel of about 200μm DCPy fluorescence was nanovesicle DES can be easily leaking from the vessel, so that there is or depth of tumor tissue without DEX case.

FIG. 3 (A) PBS, and DEX treatment after typical CD31 (red) and aSMA (green) Immunofluorescence images. (B) PBS and post-treatment with DEX representative HIF-1α Immunofluorescence images. (C) 4T1 tumor-bearing mice in vivo imaging and fluorescence imaging major organs 48h after injection. White dotted line represents a tumor. Quantitative study tissue biodistribution 12h DCPy after injection (D) DCPy + DEX, DES nanovesicle, DPS + DEX or the like DES + DEX dose (5mg / kg). (E) in the tumor tissue sections 4T1 tumor-bearing mice after treatment of 12h, DCPy (red) and CD31 labeled endothelial cells (green) co-localized. Distribution (F) DCPy in blood vessels and tumor tissues. Evaluation of 2

, 20 minutes 4 irradiation spot, the irradiation point of 5 minutes each); 3) DEX; 4) DES; 5) DES + L; and 6) DES + DEX + L group (FIG. 4A). DES administration and laser irradiation mediates tumor growthThe partial inhibition, whereas when the animals treated with of DEX, this inhibition is more effective (FIGS. 4B, 4C). Hematoxylin and eosin (H & E) stained tissue sections confirmed, DES + DEX + L treatment significantly large tumor necrosis or apoptosis of tumor cell death (FIG. 4D) about the simultaneous loss. Treatment of these mice generate tumor reactive oxygen species (ROS) was measured using DCFH-DA, the display DES + DEX + L tumor staining combined treatment of mice significantly enhanced. This confirms the enhanced efficacy observed in these animals and to improve the related free radical generation. Summary

FIG. 4 (A) DEX + DES + L Experimental Design. (B) tumor weight mean tumor volume values ​​after treatment over time (C). (D) H & E staining of tumor sections assigned treatment groups, and measuring the activity of oxygen in tumor sections stained by DCFH-DA.

Highlights Summary

In summary, the authors developed a tumor exocytosis bodies / AIEgen hybrid nanovesicle. The DES nanovesicle easily penetrate the tumor in vivo, and when used in combination of DEX, as a means of normalization TME vascular hypoxia, is ideal for PDT applications. First, the nano system overcomes many of the limitations of using AIEgens in PDT. Further, these bionic nanovesicle DEX treatment with normalization of tumor angiogenesis mediated by combining laser radiation as a means of enhancing the generation of ROS within the tumor, thereby improving the performance of PDT. In short, this work emphasizes a new tumor PDT methods, and highlights the potential clinical application of AIEgens. The full text link: https: //onlinelibrary.wiley.com/doi/10.1002/anie.202003672