Exosome-mediated aptamer S58 reduces fibrosis in a rat glaucoma filtration surgery model

INTRODUCTION

In this study, we loaded S58 into exosomes (Exo-S58) and confirmed whether Exo-S58 has a better antifibrotic effect than naked S58 in human conjunctival fibroblast (HConF) cells and in rat GFS models.

Previous reports have demonstrated that transforming growth factor-β (TGF-β) ligands play a crucial role in cell proliferation, migration, conjunctival scarring and wound healing

. As one of the identified TGF-β isoforms, TGF-β

is closely related to the process of conjunctival scarring and fibrosis

. Consequently, TGF-β ligands or receptors are therapeutic targets in the process of postoperative fibrosis through antibody neutralization

, proteoglycan-like decorin inactivation and blockage of exogenous receptors

.Aptamers are single-stranded DNA (ssDNA) or RNA oligonucleotides that can bind to a target protein

and are characterized by low toxicity

, high selectivity and binding affinity

. In our previous study, we applied systematic evolution of ligands by exponential enrichment (SELEX)to identify and synthesize the DNA aptamer S58, which could bind to TGF-β receptor II (TβRII) with high affinity.Experiments

and

proved that aptamer S58 could reduce TGF-β

-induced fibrosis, and fewer myofibroblasts were observed in the S58 group compared with MMC groups

.However, the aptamer’s effective reaction time was limited by nuclease degradation. Thus, it deserves advanced investigation to determine whether a nontoxic and protective delivery vector can preserve the efficacy of aptamer S58.

To observe the change in scarring formation following GFS, the eyeballs from the rats were collected and fixed with 4% paraformaldehyde on postoperative day 14. After dehydration with graded ethanol, the conjunctival tissue was embedded in paraffin. Haematoxylineosin (HE) staining and Masson staining were used to detect the percentage of collagen after 3-μm-thick sections from the eyeballs were sliced. Furthermore, immunofluorescence staining was also performed to determine the tissue fibrosis level. Citrate acid was used to retrieve the antigen from the sections after deparaffination and rehydration. Then, 3%hydrogen peroxide was incubated with the sections in the dark for 25min. After blocking with 1% goat serum albumin for 30min, the sections were incubated with primary antibodies,including mouse anti-α-SMA (ab7817, Abcam) and rabbit anticollagen I (ab260043, Abcam), overnight at 4℃. The sections were incubated with specific secondary antibodies for 1h and stained with DAPI for 5min. Images were captured by a fluorescence microscope (Nikon Eclipse Ti-SR, Nikon, Japan).

All values are presented as the mean±SD.Statistical analysis for comparison of multiple columns or groups was performed by one-way ANOVA using GraphPad Prism 7 (GraphPad, San Diego, CA, USA), and a

＜0.05 was considered statistically significant.

现有文献鲜有学者关注新兴旅游部门的自我就业群体，并衍生研究大学生创业可行性。学者们基本都是从宏观或中观的角度分析旅游非正规就业及共享经济下的游客行为，鲜有学者从微观的角度去追踪考察旅游自我就业者现状与创业可行性。最后，随着失业问题的灵活就业弹性方法的提出，由于其主动性、家庭参与性等特征，旅游非正规就业在未来可能成为我国旅游地最重要的旅游就业方式之一，因而对于旅游非正规就业研究具有现实意义，所以以番禺沙湾特色小镇为主的旅游自我就业现状与创业可行性研究是本题研究的首要目标。

水结冰时其体积约增加9%，但水在变成冰的过程中，冰体的压力并不很大。随着气温的降低，冰的温度下降，体积变小。当温度再升高时冰则膨胀，如果发生阻碍冰体膨胀的情况，就产生了作用在钢闸门上的冰压力。冰压力分为静冰压力和动冰压力。静冰压力指冰盖层膨胀对钢闸门表面产生的冰压力，静冰压力的数值与冰原、开始升温时的气温及气温上升率有关。动冰压力是指冰盖层解冻后，冰块漂流撞击钢闸门时产生的撞击力。

Glaucoma is recognized as an irreversible progressive neurodegenerative eye disease that can cause optic atrophy, visual field defects and blindness

. According to previous studies, glaucoma is also a multifactorial disease accompanied by pathologically elevated intraocular pressure(IOP) as the most important risk factor

. As such, glaucoma filtration surgery (GFS) has been an integral part of glaucoma control strategies through drainage of the aqueous humour to decrease IOP

. However, surgical failure may occur because of excessive subconjunctival fibrosis and scar formation caused by wound vascular reaction, exudate stimulation and hormones

. Mitomycin C (MMC) and 5-fluorouracil (5-FU)have been generally used to avoid unexpected filtering bleb fibrosis in clinical practice and have side effects such as bleb leakage, endophthalmitis and corneal epithelial toxicity

.Therefore, it is necessary to identify safe and effective antifibrotic alternatives that can be used to prevent scar formation and preserve filtering bleb function after GFS.

MATERIALS AND METHODS

Animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University.All animal procedures complied with the ARVO Statement for the Use of Animals in Ophthalmic and Visual Research.

The human embryonic kidney cell line HEK293T was cultured in complete medium containing 10% exosome-free foetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel), Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries), and 1%cyan-streptomycin (Biological Industries) at 37℃ and 5%CO

. Primary HConFs were cultured in complete medium containing 10% FBS, DMEM and 1% cyan-streptomycin at 37℃ and 5% CO

. In the S58 treatment group, aptamer S58 was first diluted in ddH

O to a final concentration of 50 nmol/L.Exosomes (3.75 μg) loaded with 50 nmol/L S58 were used in the Exo-S58 treatment group. For exosome treatment, 3.75 μg of unloaded exosomes were used. TGF-β

(5 ng/mL, PeproTech,Rocky Hill, NJ, USA) was used for all cell experiments.

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HEK293T cell culture medium was obtained every other day followed by centrifugation at 300×g for 10min and 2000×g for 10min. After further centrifugation at 10 000×g for 45min, the supernatant was filtered through a 0.22-μm filter membrane. Exosome pellets can be obtained through two runs of ultracentrifugation(CP100MX, Hitachi, Japan) at 100 000×g for 70min

. The collected pellets were resuspended in phosphate-buffered saline (PBS) and stored at -80℃ for further use.

For the transmission electron microscopy (TEM) assay,exosome pellets were added to copper grids for fixation. A 2%uranyl acetate solution was then put on grids for 5min at room temperature. Images were taken by an electron microscope(HT7700, Hitachi, Japan) after the grids were washed and dried. Nano flow cytometry (NanoFCM; Fujian, China) was employed to measure the size distribution of the exosome pellets

. The expression level of marker proteins of purified exosomes was detected by Western blot containing the primary antibodies rabbit anti-CD63 (ab134045, Abcam, Cambridge,MA, USA), rabbit anti-CD81 (ab109201, Abcam), and rabbit anti-TSG101 (ab125011, Abcam).

For aptamer loading, 7.5 μg of exosomes was incubated with the aptamer mixture after a 20-min incubation of 100 nmol/L S58 (Sangon Biotech,Shanghai, China) and 1 mg/mL polyethyleneimine (PEI)in Hepes buffered saline (HBS) according to the PEItransferrinfection kit protocol (Thermo Scientific, Carlsbad,MA, USA). Ultracentrifugation at 100 000×g for 90min was performed to remove the redundant aptamers and PEI after incubation at 4℃. The resulting pellet was rinsed and then resuspended in PBS. Nano flow cytometry was applied to measure the loading efficiency of aptamer S58.

Exosomes were stained with a PKH26 red fluorescent cell labelling kit (Ur52302, Umibio, Shanghai,China) according to the manufacturer’s protocol

. In brief,the PKH26 linker was diluted with 100 μmol/L Diluent C.Then, 10 μg of exosomes was added to 50 μL of dye solution and mixed by pipetting for 1min. After 10min of incubation at 37℃, the staining solution was resuspended in 10 mL of PBS.Ultracentrifugation at 100 000×g for 90min was performed to remove redundant dye at 4℃. The exosomes were resuspended in PBS for further use. The fluorescence intensity of PKH26-labelled exosomes was detected by confocal microscopy.

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HConFs were seeded into 96-well plates at a density of 4×10

cells per well and cultured in 200 μL of fresh complete medium with different treatments for 24, 48, and 72h. Cell cytotoxicity was detected with an LDH cytotoxicity assay kit (C0017, Beyotime, Shanghai,China). Twelve microlitres of LDH release reagent was added per well and incubated with cells for 1h. After centrifugation at 400×g for 5min, 120 μL of supernatant from each well was collected in new 96-well plates. The absorbance values at 490 nm of each well were determined by Multiscan Spectrum(BioTek, Winooski, VT, USA), and the LDH release rate was statistically evaluated.

HConFs were plated into 24-well plates at a density of 1×10

cells per well and treated with PKH26-labelled exosomes loaded with Alexa 488-labelled S58. The cells were rinsed and fixed with 4%paraformaldehyde solution for 15min at room temperature immediately after permeabilization with 0.1% Triton X-100 for 15min and blocking with 1% bovine serum albumin (BSA)for 1h at room temperature. 4’,6-diamidino-2-phenylindole(DAPI) staining was performed for 10 min. Images were taken by an inverted fluorescence microscope (Ti2-U, Nikon, Japan)and analysed.

HConFs were plated into 96-well plates at a density of 4×10

cells per well and cultured in 100 μL of fresh complete medium with different treatments for 24, 48,and 72h. Ten microlitres of CCK-8 reagent (Biosharp, Beijing,China) were added per well and incubated with the cells for 2h.The absorbance values at 450 nm of each well were identified by Multiscan Spectrum (BioTek), and the cell viability rate was statistically analysed.

HConFs were plated at a density of 4×10

cells per well and cultured in 100 μL of serum-free medium in 24-well migration chambers. Then, 600 μL of fresh complete medium with 20% FBS was added below the chambers. Cell medium was removed from the chambers after cells were incubated with different treatments for 24h at 37℃. Then, the cells were fixed and stained with crystal violet for 15min at room temperature followed by three washes with PBS.Photographs were taken by an inverted microscope (Nikon).

一、当代雕塑观念的变异性和先锋观，直接依附本体语言方可“拨苗助长”。除了意识高度和品质境界以及形态外，最直接的“快感”，通过具有阻断力和颠覆性的媒材来完成思考的更移。

Isolated HConFs and exosomes were lysed for 40min on ice with radioimmunoprecipitation (RIPA) buffer(Beyotime) containing protease inhibitors (Thermo Scientific,IL, USA). The supernatant was extracted after centrifugation at 12 000×g for 15min, and a bicinchoninic acid kit (BCA;Beyotime) was used to quantify the protein according to the manufacturer’s protocol. The extracted samples were separated by 6%-15% SDS-polyacrylamide gel electrophoresis (PAGE)and transferred onto a polyvinylidene fluoride (PVDF)membrane (Millipore, Billerica, MA, USA). Following blocking in Western blocking buffer (Beyotime) for 1h,membranes were incubated with primary antibodies including rabbit anti-collagen I (ab260043, Abcam), rabbit anti-vimentin(ab92547, Abcam), mouse anti-α-SMA (ab7817, Abcam),rabbit anti-fibronectin (ab268020, Abcam), rabbit anti-CD63(ab134045, Abcam), rabbit anti-CD81 (ab109201, Abcam),rabbit anti-TSG101 (ab125011, Abcam) and mouse anti-GAPDH (AG019, Beyotime, China) overnight at 4℃. After rinsing three times with Tris-buffered saline Tween (TBST) for 10min, the protein membranes were incubated with the specific HRP-linked secondary antibody for 1h at room temperature.The protein membranes were visualized with a gel imaging analysis system (Bio-Rad, Hercules, CA, USA).

Totally 24 Adult male Sprague-Dawley rats that weighed 200-300 g were purchased from the Laboratory Animal Center of Chongqing Medical University. The rats were anaesthetised by an intraperitoneal injection of 5 mL/kg 7% chloral hydrate(Sangon Biotech) followed by ocular surface anaesthesia using 0.5% oxybuprocaine hydrochloride eye drops (Santen Pharmaceutical Co., Ltd. Osaka, Japan). GFS in the rats was performed on bilateral eyes based on a previous technique

.Then, the rats were treated with subconjunctival injection containing saline, S58 (50 nmol/L) or Exo-S58 (3.75 μg)randomly every other week after surgery (

=6/group). The effects of different interventions against scar formation through subconjunctival injection were evaluated at days 0, 7, and 14.Additionally, rats operated eyes were tested and evaluated for IOP and filtering bleb using a slit lamp (CARL ZEISS,Germany) and tenonometer (TonoPen; Medtronic Solan, FL,USA), respectively, at specific times.

Recently, exosome drug delivery systems have received great attention due to their high delivery efficiency, long circulation times and great biocompatibility

. Furthermore, exosomes have multiple advantages compared to viruses and existing synthetic carrier systems in terms of immunogenicity, blood stability and tissue penetration

. Several studies have shown that exosomes carrying short interfering ribonucleic acid (siRNA)sequences could access target tissues for drug delivery

.

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RESULTS

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The exosomes were purified from HEK293T cell culture medium by ultracentrifugation, and characterization of the exosomes was successfully validated by TEM, NanoFCM and Western blot. The TEM results showed that the exosome particles were integral and hemispheric with a double membrane less than 100 nm in diameter (Figure 1A). NanoFCM measured that the average diameter of exosome particles was approximately 73 nm at a concentration of 5.32×10

/mL (Figure 1B). Western blot analysis showed that the expressions of the exosome marker proteins CD63, CD81, and TSG101 were positive (Figure 1C).

To improve the therapeutic effect of S58, aptamer S58 was loaded into exosomes by PEI transfection according to the protocol.After transfection, excess S58 and PEI were removed by ultracentrifugation. In addition, we determined the loading efficiency of exosome-encapsulated S58 by using NanoFCM analysis. The results of the NanoFCM analysis showed that incubation with exosomes for 3h reached the greatest aptamer retention rate at 88.83%±1.06% (Figure 2A). Furthermore,TEM showed that the morphological structure of exosomes loaded with S58 did not change after transfection with PEI(Figure 2B). Then, NanoFCM measured that the average diameter of loaded exosome particles was approximately 78 nm at a concentration of 1.34×10

/mL (Figure 2C). Therefore,incubation with exosomes for 3h was performed to load aptamer S58. To determine whether exosomes can deliver S58 into HConFs, the fluorescence of PKH26-labelled exosomes and Alexa 488-labelled aptamer was detected after incubation with cells for 24h and 48h by confocal fluorescence imaging(Figure 2D). The confocal imaging results showed that HConFs could take up Exo-S58, and incubation with exosomes for 48h reached the highest exosome uptake efficiency.

The rats were treated with subconjunctival injection containing saline, S58 or Exo-S58 randomly after surgery. To better observe the change after GFS, IOP and filtering bleb areas of the rats were detected on operative days 0, 7 and 14.Results showed that the Exo-S58 and S58 treatment prolonged the filtering bleb retention significantly, while Exo-S58 treatment prolonged bleb retention more effectively than S58 treatment (Figure 5A, 5B). Statistical analysis of mean IOP in the surgical eyes showed that the IOP of rats in the Exo-S58 group was lower than that of rats in the S58 group on operative day 14, while IOP of the control group was significantly higher than that in both groups (Figure 5C).

Exo-S58 Inhibited TGF-β-induced HConFs Fibrosis

Western blot was used to determine the antifibrotic effect of Exo-S58 after the HConFs were incubated with different treatments for 48h. The expression levels of the proteins fibronectin, collagen I, vimentin, α-SMA in the TGF-β

group were increased significantly, while fibrosis protein expression levels in the Exo-S58 group were decreased significantly compared with those in the naked S58 group (Figure 4). The result indicated that Exo-S58 could reduce TGF-β

-induced fibrosis significantly in HConFs compared with naked S58.

Exo-S58 Inhibited TGF-β-induced HConFs Cell Proliferation and Migration

Next, to investigate the cytotoxicity of Exo-S58, HConFs were incubated with different treatments for 24, 48, and 72h. The LDH release rate showed that Exo-S58 did not cause cytotoxicity to the cells (Figure 3A). To investigate the effect of Exo-S58 on HConFs cell proliferation and migration, they were cultured with different treatments.The data showed that there were no significant differences in cell viability among the different groups at 24h. TGF-β

significantly promoted HConFs viability, while the cell viability of the Exo-S58 group was decreased significantly compared with the naked S58 group at 48 and 72h (Figure 3B).The result also showed that TGF-β

treatment significantly promoted HConFs migration, while the migration of Exo-S58 treatment was decreased significantly compared with naked S58 treatment (Figure 3C). These results suggested that Exo-S58 could inhibit cell proliferation and migration caused by TGF-β

more effectively compared with naked S58 in HConFs.

Rats from different groups were sacrificed on operative day 14. HE staining,Masson staining and immunofluorescence staining were performed on the sections to observe the pathological changes.Representative Masson staining images of sections showed that the control group had more collagen deposition in the surgical site than the Exo-S58 group and S58 group, while the Exo-S58 group exhibited significantly less collagen deposition than the S58 group (Figure 6A). Immunofluorescence staining showed that the expression of collagen-I and α-SMA in the control group was higher than that in the Exo-S58 group and S58 group, while Exo-S58 significantly decreased fibrosis protein expression relative to that in the S58 group (Figure 6B).The above results indicated that Exo-S58 reduces fibrosis in rats after GFS.

DISCUSSION

GFS is an effective therapeutic strategy for glaucoma

. The aqueous humor can be drained through the subconjunctival outflow channel, which is established through filtration surgery. However, excessive postoperative subconjunctival fibrosis would often reduce aqueous humor drainage and even lead to surgery failure

. MMC and 5-FU are widely used in the clinic to prevent subconjunctival excessive fibrosis,accompanied by many side effects such as bleb leakage,endophthalmitis and corneal epithelial toxicity

. A key molecule in fibrosis development is TGF-β, which accelerates cell differentiation, migration, and proliferation

. As one of the identified TGF-β isoforms, TGF-β

is closely related to the process of conjunctival scarring and fibrosis

. To inhibit TGF-β

stimulation, we developed aptamer S58 to specifically bind to TβRII. Based on the conformational flexibility and targeting specificity, aptamers can be considered as alternatives to an antibody to inhibit protein-protein interactions

.Although we have proved that aptamer S58 could reduce TGFβ

-induced fibrosis

and

, the valid time of naked S58 is limited by several factors. Naked, single-stranded RNA could easily suffer from nuclease degradation and have poor circulation time

. The process of wound healing is long lasting. Therefore, it is necessary to identify a new drug delivery system to prolong or enhance the effect of aptamer S58 for successful application to the conjunctiva after GFS.

Nano-carrier drug delivery systems can significantly improve bioavailability and efficacy of the drug in the eye. Chitosan nanomicelles carrying dexamethasone exhibited good ocular tolerance and provided a relatively longer retention time

. Liposomes containing betaxolol hydrochloride were more efficient than the betaxolol hydrochloride solution on decreasing IOP in rabbits

. For RNA drug delivery, viral vectors

, cationic polymers

, liposomes

and exosomes

have been used. Among all the nano-based drug delivery systems, liposomes as the most common and extensively studied vehicle that have shown therapeutic potential in many biomedical areas

. Despite liposomes having the advantages of biocompatibility, bio-degradability and low toxicity

, there are many limitations, including rapid clearance of liposomes,low targeting efficiency and potential immunogenicity

.Exosomes as the natural carrier possess some advantages over liposomes and polymeric nanoparticles in active targeting and any immunogenicity

. Exosomes are cellderived nanosized membrane vesicles, which transfer their components such as proteins, RNA, and DNA to mediate cell-to-cell communication

. Exosome natural biological properties contribute to high targeting efficiency, cell adhesion,cell fusion and cellular delivery of cargo

. Exosomes have been suggested as novel nanomaterials for treating fibrosis related diseases. Recent study explored that exosomes isolated from human umbilical cord-derived mesenchymal stem cell inhibited subretinal fibrosis by delivering miR-27b

. Guiot

suggested that macrophage-derived exosomes may reduce pulmonary fibrosis progression

the delivery of antifibrotic miR-142-3p. Similarly, miRNA-loaded human peripheral blood derived-exosomes may be used as a therapeutic tool to prevent cardiac fibrosis

. However, research about the application of exosomes on excessive subconjunctival fibrosis after GFS is lacking. Thus, we used exosomes to deliver the aptamer S58 in our study and to identify whether it would enhance or prolong the effect of the naked aptamer.

Our research suggests that Exo-S58 could reduce excessive fibrosis more effectively than naked S58 in HConF cells and in rat GFS models. Recent studies have employed exosomes purified from HEK293T cells as a new method for the

delivery of siRNA

. In this study, we used exosomes derived from HEK293T cells to deliver aptamer S58. The obtained exosomes showed normal morphological characteristics by TEM, and aptamer S58 was loaded into exosomes by transfection efficiently without cytotoxicity.Furthermore, it was verified that Exo-S58 could be taken up by HConFs. In the HConFs treated with Exo-S58, cell proliferation, migration and the expression of fibrosis marker proteins were decreased significantly compared with those in the S58 group. In GFS rats subjected to subconjunctival injection of Exo-S58, Exo-S58 treatment prolonged filtering bleb retention and reduced the mean IOP compared with naked S58 treatment. The results of Masson staining and immunofluorescence staining indicated that Exo-S58 treatment significantly reduced fibrosis compared with S58 treatment.The loosely organized subconjunctival matrix in Exo-S58 treated eyes may contribute to the prolonged retention time of filtering blebs. Taken together, our study demonstrated that exosomes are safe and valid carriers to deliver aptamers,and Exo-S58 reduced excessive fibrosis in HConFs and in rat GFS models more effectively than naked S58. Our results are consistent with previous studies which suggested that anticancer drugs encapsulated in exosomes demonstrated enhanced anticancer properties

compared to free drugs

. We suppose that the exosome membrane’s lipid bilayer structure can protect the aptamer from degradation caused by direct contact with ribonuclease. It is also possible that exosomes as natural carriers yield a longer circulation and a reduced clearance rate increasing the circulation time of aptamer S58 without toxicity. In addition, exosomes may be internalized through interaction with the plasma membrane

. Indeed, there exists another hypothesis that exosomes can fuse with endosomal membranes

. As a result, exosomes loaded with aptamers S58 could easily be taken up by HConFs and improve the antifibrotic effect in the process. However, there were several limitations in our study.Although exosome-mediated S58 delivery in our study did not show obvious side effects and demonstrated the expected therapeutic effect, the possible impact of other cell-derived exosomes will need to be further studied and explained.Finally, the combination of exosomes and sustained-release material such as chitosan hydrogel may contribute to treating patients for long periods of time. Further studies are required to improve exosomes as drug carriers and the application of aptamer S58.

党的十八大以来，以习近平同志为核心的党中央坚持把解决好“三农”问题作为全党工作重中之重，坚持农业农村优先发展，两个“坚持”掷地有声、落地见效，“三农”发展开创出崭新局面。综观十八大以来的6个中央“一号文件”，逻辑上一脉相承、层层递进、浑然一体，实践中加速了城乡融合发展的历史进程。乡村振兴战略的提出，更是奏响了加快实现农业强、农村美、农民富的气势磅礴的主旋律，书写了我国城乡社会向现代化转型过程中致力于协调发展、共同繁荣的浓墨重彩的新篇章。

In conclusion, the study presents the evidence that exosomes are safe and valid carriers to deliver the aptamer S58. The exosome-mediated delivery of aptamer S58 can significantly reduce cell proliferation, migration and fibrosis in TGF-β

-induced HConFs. Moreover, the exosome-mediated delivery of aptamer S58 exhibited superior antifibrotic effect compared to naked aptamer S58 in rat GFS models. The current study provided the possible therapeutic value for preventing scar tissue formation after GFS and other tissue fibrosis.

Lin QY designed the study,performed experiments, interpreted the data, and wrote the manuscript. Xie L participated in foundation acquisition,planning experiments and revising the manuscript. Li XJ,Leng Y and Zhu XM conceived and performed the animal experiments. Tang M, Lin Y participated in experiments plan, and manuscript writing. Luo WD, Jiang BC and Chen X supplied materials and assisted experiments. All authors discussed the results and critically reviewed the manuscript.

Supported by the National Natural Science Foundation of China (No.81700836; No.81470629;No.81670860); Chongqing Natural Research Foundation (No.cstc 2018jcyjAX0034).

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