Reagents
The 5ʹ polyA linker and 5ʹ-SH modified aptamer AS1411, with the sequence 5ʹ-SH-AAAAAA-GGTGGTGGTGGTTGTGGTGGTGGTGG, was synthesized by GENERAL BIOL Co., Ltd (Anhui, China). HSA-LDP and HSA-LDP-AE had been offered by Professor Zhen Yongsu’s workforce from the Institute of Medicinal Biotechnology, Chinese language Academy of Medical Sciences, Peking Union Medical Faculty. Tri (2-carboxyethyl) phosphine hydrochloride (TCEP) and 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt (Sulfo-SMCC) had been bought from Bide Pharmatech Co., Ltd (Shanghai, China). Annexin V-FITC/PI double staining apoptosis detection package was bought from BestBio (Shanghai, China). A reactive oxygen species (ROS) detection package, enhanced mitochondrial membrane potential (MMP) detection package (JC-1), Calcein/PI cell viability and cytotoxicity detection package, and BeyoClick™ EdU-488 cell proliferation detection package had been bought from Beyotime Biotechnology (Shanghai, China). Pierce™ BCA Protein Assay Equipment was bought from Thermo Fisher Scientific (Massachusetts, USA). Rabbit anti-NCL monoclonal antibody (Cat No. R27099), HRP-labeled goat anti-rabbit IgG (Cat No. 511203), and HRP-labeled goat anti-mouse IgG (Cat No. 511103) had been bought from ZENBIO (Chengdu, China). Mouse anti-Bax monoclonal antibody (Cat No. 60267-1-Ig) and mouse anti-PARP1 monoclonal antibody (Cat No. 66520-1-Ig) had been bought from Proteintech (Wuhan, China). Rabbit anti-Bcl-2 polyclonal antibody (Cat No. WL01556) was bought from Wanleibio (Shenyang, China). Rabbit anti-cytochrome c monoclonal antibody (Cat No. ET1610-60) was bought from HUABIO (Hangzhou, China). All different reagents had been of analytical grade.
Cell strains and tradition
The human esophageal most cancers cell strains KYSE520 and KYSE70 had been bought from Artistic Bioarray. EC109 was bought from the Nationwide Experimental Cell Useful resource Sharing Platform (Beijing, China). KYSE150 was bought from the Cell Financial institution of the Chinese language Academy of Sciences (Shanghai, China). All cells had been cultured in RPMI-1640 medium containing 1% penicillin-streptomycin resolution and 10% fetal bovine serum at 37℃ and 5% CO2.
Preparation and characterization of AS1411-HSA-LDP
To organize AS1411-HSA-LDP, the thiol-modified AS1411 aptamer was linked to the amino group of HSA-LDP by way of the generally used linker Sulfo-SMCC. Briefly, 10 mg HSA-LDP and 0.72 mg Sulfo-SMCC had been dissolved in 3 mL PBS and reacted on a shaker for 4 h beneath ice bathtub circumstances. Unreacted Sulfo-SMCC was eliminated by centrifugation utilizing an ultrafiltration tube (MWCO, 30 kDa) and resuspended in 500 µL PBS. 6 µL of 100 µM AS1411 resolution was added to 900 µL ultrapure water, and 55 µL of 800 mM TCEP resolution was added. The combination was positioned on a shaker for 30 min beneath ice bathtub circumstances. The AS1411 resolution was blended with the SMCC-treated HSA-LDP resolution and reacted on a shaker in a single day. The response product was ultrafiltered beneath an ultrafiltration tube (MWCO, 30 kDa) and resuspended in 1 mL PBS.
Agarose gel electrophoresis was used to detect whether or not AS1411 was efficiently conjugated to HSA-LDP 0.5 g agarose was precisely weighed and added to 25 mL 1×TAE electrophoresis buffer resolution and heated in a microwave to dissolve. Then Tremendous Pink nucleic acid dye was added to the gel. Free AS1411, AS1411-HSA-LDP, and HSA-LDP had been blended with the loading buffer after which dropped into the gel loading nicely. The pattern was electrophoresed at 120 V for 35 min. Beneath UV gentle, the gel imaging (Tanon-1600 Gel Picture) system was used to watch the gel imaging and take footage.
Binding affinity of AS1411-HSA-LDP to most cancers cells
Movement cytometry was used to research the affinity exercise of various concentrations of AS1411-HSA-LDP labeled with fluorescein Cy5 to esophageal most cancers cells. Esophageal most cancers cells EC109, KYSE520, KYSE70, and KYSE150 within the logarithmic development part had been digested, centrifuged, and washed. Completely different teams had been arrange, and three × 105 cells had been incubated with various concentrations of Cy5-AS1411-HSA-LDP in an ice bathtub for 4 h. After incubation, the cells had been filtered by means of a 300-mesh nylon mesh and analyzed by movement cytometry (BD FACSCelesta).
Mobile uptake
EC109, KYSE520, and KYSE70 cells within the logarithmic development part had been seeded at 3 × 104 cells/nicely on 8-well chamber slides and cultured in a cell tradition incubator for twenty-four h. The outdated tradition medium was discarded, and 0.1 mg/mL Cy5-AS1411-HSA-LDP resolution diluted in full tradition medium was added, respectively, and incubated at 37℃ at midnight for 4 h. Beneath darkish circumstances, 200 µL of 4% paraformaldehyde resolution was added to every nicely to repair the cells for 20 min. Subsequently, an anti-fluorescence quencher (containing DAPI) was added to every nicely and reacted at room temperature at midnight for 15 min. They had been noticed and photographed beneath a confocal laser scanning microscope (200×).
Endocytic pathways
For the examine of endocytic pathways, EC109 and KYSE520 cells had been seeded in 6-well plates (3 × 105 cells per nicely) for twenty-four h and completely different endocytic pathway inhibitors had been used to check the uptake mechanism of AS1411-HSA-LDP by esophageal most cancers cells, together with chlorpromazine (CPZ, clathrin pathway inhibitor), methyl-β-cyclodextrin (MβCD, lipid raft-mediated endocytosis inhibitor), genistein (Gen, caveolin pathway inhibitor), and 5-N-ethyl-N-isoproamiloride (EIPA, macropinocytosis pathway inhibitor) to dam completely different endocytic pathways. For EC109 cells, completely different uptake pathways had been blocked for two h with genistein (200 µM), chlorpromazine (30 µM), amiloride (80 µM), and methyl-β-cyclodextrin (2.5 mM). Subsequently, 2 mL of Cy5-AS1411-HSA-LDP with a focus of 0.7 mg/mL was co-incubated with the cells for two h. For KYSE520 cells, 2 mL of Cy5-AS1411-HSA-LDP resolution diluted with full medium was added to the cells, which contained 200 µM genistein, 30 µM chlorpromazine, 80 µM amiloride, and a pair of.5 mM methyl-β-cyclodextrin, respectively, and incubated at midnight for 4 h. Esophageal most cancers cells had been washed with PBS after which detected by movement cytometry. The uptake effectivity was calculated in response to the imply fluorescence depth (MFI).
To confirm the precise endocytic pathway concerned in AS1411-HSA-LDP in two esophageal most cancers cells, EC109 and KYSE520 cells had been seeded in 8-well chamber slides (3 × 104 cells per nicely). For EC109 cells, completely different endocytic pathway inhibitors had been ready and incubated with cells for two h. After the incubation, Cy5-AS1411-HSA-LDP was added and incubated with EC109 cells for two h. For KYSE520 cells, after including Cy5-AS1411-HSA-LDP, it was instantly incubated with the 4 inhibitors for 4 h. Subsequently, 200 µL of 4% paraformaldehyde was added to repair the above EC109 and KYSE520 cells, and anti-fluorescence quencher containing DAPI was added to stain the cell nucleus. The cells had been imaged and photographed beneath a confocal laser scanning microscope (200×).
Biodistribution and imaging
EC109 and KYSE520 cells (5 × 106 cells /0.2 mL) had been inoculated subcutaneously into the fitting axilla of feminine BALB/c nude mice weighing 18–20 g to determine a mouse tumor xenograft mannequin. When the tumor measurement reached 500–600 mm3, the nude mice had been injected with Cy5-labeled AS1411-HSA-LDP at a dose of 20 mg/kg by way of the tail vein. On the predetermined time level, the nude mice had been anesthetized by inhalation of isoflurane, and the biodistribution of Cy5-AS1411-HSA-LDP within the nude mice was noticed and photographed utilizing an in vivo imaging system (Tanon ABL X5). After the experiment, the nude mice had been killed by CO2 technique. The guts, liver, spleen, lung, kidney, and tumor tissues had been obtained for in vitro imaging. Subsequently, the collected tumor tissues had been positioned in 4% paraformaldehyde. After dehydration with a 15-30% gradient sucrose resolution, the samples had been obtained and embedded in OCT. DAPI stain was added and incubated at room temperature at midnight for 10 min. After washing, an anti-fluorescence quenching sealing agent was added to seal the slides. Tumor tissue sections had been obtained and pictures had been collected beneath a confocal laser scanning microscope (200×).
Preparation and characterization of AS1411-HSA-LDP-AE
8.4 mg HSA-LDP-AE and 0.75 mg Sulfo-SMCC had been added to three mL PBS and reacted on a shaker for 4 h beneath ice bathtub circumstances. The unreacted Sulfo-SMCC was eliminated by centrifugation in an ultrafiltration tube (MWCO, 30 kDa) and resuspended in 500 µL PBS. 5 µL of 100 µM AS1411 resolution was added to 750 µL ultrapure water, and 50 µL of 800 mM TCEP resolution was added. Beneath the situation of an ice bathtub, the response was carried out on a shaker for 30 min. The AS1411 resolution was blended with the SMCC-reacted HSA-LDP-AE resolution and reacted on a shaker in a single day. The ultimate product was positioned in an ultrafiltration tube (MWCO, 30 kDa) and resuspended in 1 mL PBS.
Agarose gel electrophoresis was used to detect whether or not AS1411 was efficiently coupled to HSA-LDP-AE. The gel was ready with 1×TAE buffer resolution (containing Tremendous Pink dye) containing 2% agarose. Free AS1411, AS1411-HSA-LDP-AE, and HSA-LDP-AE had been blended with the loading buffer on an ice field after which dropped into the gel loading nicely. The samples had been electrophoresed at 120 V for 35 min. The gel imaging was noticed and photographed beneath UV gentle utilizing the Tanon-1600 Gel Picture system.
Cell viability assay
In line with the reagent directions, the viability of esophageal most cancers cells was detected utilizing the cell counting kit-8 (CCK-8) assay. EC109, KYSE520, and KYSE70 cells had been seeded in 96-well plates at 4 × 103 cells/nicely and cultured in RPMI-1640 medium containing 10% FBS for 12 h. The cells had been then incubated with completely different concentrations of AS1411-HSA-LDP-AE. After incubation for twenty-four and 48 h, 10 µL CCK-8 resolution was added to the wells, and incubated at 37 °C for two h, and the optical density (OD450) was measured utilizing a microplate reader. Based mostly on the info obtained, the cell survival curve was plotted and the IC50 worth (50% inhibition focus) was calculated.
5-Ethynyl-2’-deoxyuridine (EdU) assay
In line with the provider’s directions, the EdU detection package was used to guage the impact of medication on the proliferation of esophageal most cancers cells. EC109 and KYSE520 cells within the logarithmic development part had been inoculated in 96-well plates at 4 × 103 cells/nicely and cultured for twenty-four h. Then AS1411-HSA-LDP-AE (400 ng/mL) and AS1411-HSA-LDP-AE (800 ng/mL) had been added and cultured for twenty-four h. 10 µM EdU reagent (Beyotime, C0071S) was added to every nicely and incubated for two h. After washing with PBS containing 3% BSA for 3 instances, 1 mL of 4% paraformaldehyde was added to every nicely and glued at room temperature for 15 min. After washing with 3% BSA in PBS for 3 instances, 100 µL of immunostaining robust permeabilization resolution was added and incubated at room temperature for 15 min. Click on response reagent was added and incubated at room temperature at midnight for 30 min. 35 µL of Hoechst 33,342 (1:1000) resolution was added to every nicely and incubated at room temperature at midnight for 10 min. After the incubation, the washing resolution was washed 3 instances and the staining outcomes had been noticed and photographed by fluorescence microscope (100×).
Colony formation assay
Human esophageal most cancers cells EC109 and KYSE520 within the logarithmic development part had been seeded into six-well plates at 5 × 102 cells per nicely. Cultured at 37℃, 5% CO2 for twenty-four h. EC109 and KYSE520 cells had been cultured with AS1411-HSA-LDP-AE (1 ng/mL, 12.5 ng/mL, and 100 ng/mL) respectively. The tradition lasted for about 1 week. The recent cell tradition medium was changed each 2 days. Lastly, the colonies had been fastened with 4% paraformaldehyde for 15 min and stained with crystal violet for 30 min, then washed with water, air-dried, and photographed.
Reside-dead cell assay
Reside/lifeless cells had been evaluated and analyzed utilizing Calcein-AM and PI dyes. EC109, KYSE520, and KYSE70 cells (4 × 103 cells/nicely) had been incubated in 96-well plates for twenty-four h. AS1411-HSA-LDP-AE with a closing focus of 400 ng/mL and 800 ng/mL was added to the corresponding wells within the drug-treated teams. After 24 h, 100 µL of the ready Calcein-AM/PI detection working resolution (Beyotime, C2015S) was added, and incubated at 37℃ for 30 min. Noticed and photographed with a fluorescence microscope (100×).
Migration and invasion
Transwell experiment was used to detect cell migration and invasion means. The experiment was primarily carried out utilizing transwell chambers (Corning, Cat No. 3422). For migration experiments, the chambers added with 200 µL of basal tradition medium had been incubated in an incubator for 30 min to realize the impact of balancing the polycarbonate membrane. After the incubation, 600 µL of full tradition medium resolution containing 20% fetal bovine serum was added to the decrease chamber, and completely different concentrations of AS1411-HSA-LDP-AE cell suspension (1 × 105 cells per nicely) ready with serum-free tradition medium had been added to the higher chamber. For invasion experiments, the matrix gel was diluted with serum-free tradition medium and added to the higher chamber of the cell chamber, and gently shaken to unfold the gel evenly on the membrane. Incubate at 37℃ for two h. Then a serum-free cell suspension containing AS1411-HSA-LDP-AE (1 × 105 cells per nicely) was added to the higher chamber, and a development medium resolution containing 20% fetal bovine serum was added to the decrease chamber. After 24 h, the cells had been fastened with 4% paraformaldehyde and stained with crystal violet. After washing with PBS, all cells that migrated to the decrease floor of the filter had been noticed beneath a fluorescence microscope. Random fields of view had been chosen and photographed (100×). After the pictures, the cells had been washed with 33% acetic acid, and the eluate was blended and added to a 96-well plate. The sunshine absorption worth was measured at a wavelength of 570 nm utilizing a microplate reader to research the variety of cells that migrated and invaded the decrease floor of the filter.
Cell apoptosis evaluation
The impact of AS1411-HSA-LDP-AE on inducing apoptosis was detected utilizing the Annexin V-FITC/PI cell apoptosis detection package. EC109 and KYSE520 cells had been seeded into 6-well plates at 3 × 105 cells/nicely and cultured to stick to the wall. The tradition medium was discarded, and completely different concentrations of AS1411-HSA-LDP-AE ready with full tradition medium had been added and incubated for twenty-four h. The cells had been washed with PBS, and all of the cell pellet samples after centrifugation had been collected in Annexin V binding resolution (400 µL), Annexin V-FITC resolution (5 µL) was added, and incubated at midnight for 15 min, adopted by staining with PI resolution (10 µL) and incubation at midnight for two–5 min. Lastly, all samples had been detected by movement cytometry (BD FACSCelesta) to visualise the modifications in cell apoptosis.
Mitochondrial membrane potential measurement
JC-1 staining was used to detect MMP ranges. Briefly, EC109 cells and KYSE520 cells had been seeded in 6-well plates at 3 × 105 cells per nicely and cultured for twenty-four h. Subsequently, the cells had been handled with completely different closing concentrations of AS1411-HSA-LDP-AE (400 ng/mL, 800 ng/mL) for twenty-four h. Then, the cells had been stained with JC-1 (1:200 dilution) at 37 °C for 20 min. After washing with PBS, 2 mL of cell tradition medium was added to every nicely, and fluorescence evaluation was carried out beneath a fluorescence microscope and photographed (100×).
ROS stage evaluation
In line with the producer’s directions, the DCFH-DA probe was used to detect the ROS stage in esophageal most cancers cells. EC109 and KYSE520 cells had been seeded in 6-well plates at 3 × 105 cells per nicely and cultured for twenty-four h. Subsequently, a clean management group and an AS1411-HSA-LDP-AE group had been arrange, and the concentrations of the drug-treated teams had been 400 ng/mL and 800 ng/mL, respectively. After 24 h of incubation, 2 mL DCFH-DA diluted with serum-free medium at a closing focus of 10 µM was added to every nicely. Incubated at midnight at 37 °C for 20 min, washed 3 times with serum-free tradition medium, and noticed inexperienced fluorescence utilizing a fluorescence microscope to guage ROS ranges and photographed (100×).
Western blot evaluation
Western blot was used to detect the expression ranges of NCL, PARP1, BAX, Bcl-2, and cytochrome c (Cyto C) proteins. Cells had been lysed with RIPA lysis buffer containing protease inhibitors for 1 h beneath ice bathtub circumstances. Protein focus was decided utilizing the BCA protein detection package. Samples with equal protein concentrations had been loaded onto a protein separation system, protein samples had been separated by SDS-PAGE, after which the proteins had been transferred to polyvinylidene fluoride (PVDF) membranes at 200 mA for two h. The PVDF membranes had been blocked with 5% milk for two h at room temperature. After washing 3 times with TBST buffer, the membranes had been incubated with diluted corresponding major antibodies at 4℃ in a single day. After washing with TBST buffer, the membranes had been incubated with HRP-conjugated secondary antibodies on a shaker at room temperature for 1 h. The PVDF membranes had been visualized utilizing a chemiluminescent detection package and imaged utilizing a gel imaging system.
In vivo anti-tumor efficacy
Anti-tumor examine of AS1411-HSA-LDP-AE in KYSE520 tumor-bearing mice
Feminine thymus-free BALB/c nude mice had been bought from Hangzhou Ziyuan Laboratory Animal Expertise Co., Ltd (Hangzhou, China). To review the impact of AS1411-HSA-LDP-AE on tumor development, KYSE520 cells (4.5 × 106 wells) had been subcutaneously implanted in the fitting axilla of 18–20 g feminine BALB/c nude mice. When the tumor quantity reached 60–80 mm3, the nude mice had been randomly divided into 3 teams, with 5 mice in every group. The management group didn’t obtain any therapy; the drug-treated group was given completely different concentrations of AS1411-HSA-LDP-AE (0.6 mg/kg, 1.2 mg/kg) by means of the tail vein as soon as per week for a complete of two instances. The physique weight and tumor quantity of mice had been recorded each different day. The formulation for calculating tumor quantity is V=(a×b2)/2 (V is the tumor quantity, a is the tumor’s lengthy diameter, and b is the tumor’s brief diameter). The tumor development inhibition fee was calculated as [1-(tumor terminal volume of the drug-treated group-tumor initial volume of the drug-treated group)/(tumor terminal volume of control group-tumor initial volume of control group)]×100%. On the finish of therapy, all nude mice had been killed by the CO2 technique, and tumor tissues had been remoted, photographed, and weighed. The principle organ tissues and tumor tissues had been eliminated had been additional analyzed. For the histological evaluation, the center, liver, spleen, lung, kidney, abdomen, and small gut had been fastened in 4% paraformaldehyde for hematoxylin and eosin (H&E) staining. Tumor apoptosis and proliferation had been studied by immunohistochemical (IHC) staining evaluation in tumor sections, and nucleolin expression in numerous teams was analyzed.
Anti-tumor exercise in EC109 cell xenograft in athymic mice
EC109 cells (5 × 106 cells) had been suspended in 200 µL PBS and injected subcutaneously into the fitting axilla of 18–20 g feminine BALB/c nude mice. When the tumor quantity reached 60–80 mm3, the nude mice had been randomly divided into 2 teams, 6 nude mice in every group, together with a management group and a drug-treated group. The management group didn’t obtain any therapy, and the drug-treated group was injected with 200 µL AS1411-HSA-LDP-AE (1.6 mg/kg) by way of the tail vein as soon as per week for a complete of two instances in two weeks. The tumor quantity and weight of the mice had been monitored each 2 days. After 2 weeks of drug administration, the nude mice had been killed, and the tumor tissues had been collected, photographed, and weighed. The principle organs of the nude mice had been eliminated for histological evaluation. The formulation for calculating tumor quantity and tumor development inhibition fee is identical as above.
Statistical evaluation
All information had been obtained from no less than 3 unbiased experiments and introduced as imply ± commonplace deviation. SPSS software program was used for statistical testing and Graphpad Prism software program was used for plotting. The t-test was used to match between two teams, and a one-way evaluation of variance was used to match between a number of teams. P < 0.05 was thought-about statistically important.
