Aptamer functionalized nucleic acid nano drug for targeted synergistic therapy for colon cancer

Materials

The T4 DNA ligase, 10×T4 DNA ligase buffer, T7 RNA polymerase, and 10×T7 RNA polymerase buffer were purchased from Novoprotein Scientific Inc. (Suzhou, China). The phi29 DNA polymerase and 10×phi29 DNA polymerase buffer were obtained from New England Biolab (UK). The RNase inhibitor, ethidium bromide (EB), DNA Ladder, RNase-free H2O, TransScirpt® One-Step gDNA Removal and cDNA Synthesis SuperMix and RealMsterMix(SYBR Mix)were purchased from Tiangen Biotech Co. Ltd. (Beijing, China). The DTT and dNTP Mixture were obtained from Takara (Beijing, China), while the rNTP Mixture and SYBR Gold were purchased from Thermo Fisher Scientific (MA, US). The Tris-base and ethylene diamine tetraacetic acid (EDTA) were obtained from Biotopped Life Sciences (Beijing, China). The sodium chloride (NaCl), sodium monohydrogen phosphate (Na2HPO4), monopotassium phosphate (KH2PO4), and dimethylsulfoxide (DMSO) were supplied by Sigma-Aldrich (St. Louis, MO). The SW480 cells, human colon cancer cells, and L02 cells, normal human hepatocytes, were acquired from the National Experimental Cell Resource (BMCR) sharing platform (Beijing, China). The Dox and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Solarbio Life Sciences (Beijing, China), while the fetal bovine serum (FBS) and Trypsin-EDTA (0.25%) were purchased from Gibco (USA). The Cell Counting Kit-8 (CCK-8), Cell Cycle and Apoptosis Analysis Kit, Annexin V-FITC Cell Apoptosis Kit, MMP assay kit with JC-1, and ROS Assay Kit were obtained from Beyotime (Beijing, China). All DNA sequences were synthesized by Sangon Biotech (Shanghai, China).

Construction of circular template DNA

The hsa-miR-21 sequence was queried against the miRNA database, miRBase (http://www.mirbase.org/index.shtml). The AS1411 aptamer and T7 promoter sequences were obtained from available references [19, 21, 36] (Table S2). This study designed the DNA template, promoter, and Apt-DNA sequences. The cyclization of the single-stranded DNA template was then carried out. The DNA template, promoter, 10×T4 DNA ligase buffer, and RNase-free H2O were added to 200 µL EP tubes and mixed, placed in a PCR instrument at 95 °C for 10 min, and cooled to 25 °C at a 1 °C/min gradient. Next, 4.8 µL of T4 DNA ligase (40 U/µL) was added to the system, mixed thoroughly, and placed in a PCR machine overnight at 16 °C to obtain the ligated product. The product was then mixed with phi29 DNA polymerase, 10×phi29 DNA polymerase buffer, and RNase-free H2O and placed in a PCR machine for 2 h at 30 °C. The product was collected and stored at 4 °C. The circle template products were verified via 2% agarose gel electrophoresis.

Optimization of the RCT conditions

The circular template concentrations, T7 RNA polymerase, DTT, and PCR reaction time were optimized for maximum RCT results. Here, the rNTP, RNAase inhibitor, RNA polymerase buffer, and T7 RNA polymerase were mixed in an RNase-free 200 µL EP tube. Water was added to the final volume and vortexed gently to mix well. The mixture was placed in a PCR instrument at 37 °C, after which 6.4 µL DNA-Apt was added at 65 °C for 5 min and lowered to room temperature at a 2 °C/min gradient, followed by storage in a refrigerator at 4 °C for 2 h. The product was washed with RNase-free H2O via centrifugation (4 °C, 12,000 g, 30 min) and resuspended to its original volume with RNase-free H2O. The circle template concentrations were set to 0.25 µM, 0.5 µM, 1 µM, and 2.5 µM, while those of the T7 RNA polymerase was 2.5 U/µL and 5 U/µL, respectively. The final DTT concentrations in the T7 RNA polymerase buffer were 1 mM, 2.5 mM, and 5 mM, respectively, while the PCR reaction times were 8 h, 16 h, 24 h, and 48 h, respectively. Finally, the circle template products were analyzed via 2% agarose gel electrophoresis to determine the RCT conditions.

Verification of the RCT products

The SYBR Gold dye was diluted with 1 x TBE and incubated with the product for 10 min, protected from light, and washed via centrifugation with RNase-free H2O (4 °C, 12,000 g, 30 min). The process was finished by dropping the sample onto a slide and applying nail polish on the coverslip. After that, the item was examined using a fluorescence microscope and blue light excitation.

DLS analysis

The FND was diluted with RNase-free H2O in appropriate amounts and sonicated for 2 min for thorough dispersion. Particle size and potential cuvettes received the samples, accordingly. Using a Nano ZS90, the samples’ potential and particle size distribution were assessed.

SEM analysis

The product was washed with RNase-free H2O, centrifuged (4 °C, 12,000 g, 30 min), resuspended in RNase-free H2O, and sonicated for 2 min to disperse well. A drop of 3 µL was added to the center of the smooth surface of a clean 5 × 5 mm single-polished silicon wafer and placed in a metal bath at 37 °C to dry. Prior to detection, the sample was gold-sprayed onto the wafer. The product morphology and size were evaluated using 2 kV electron microscopy scanning.

EDS analysis

An SEM fitted with an EDS detector was used to carry out the EDS characterization. The samples were made by adding each component dropwise to the middle of a clean, single-throw silicon wafer, then drying them at 37 °C in a metal bath. A non-standard quantitative method was employed to determine the relative atomic ratio normalized to each particle’s Mg content for the EDS examination.

Preparation of the FND

The product was mixed with different Dox concentrations (1 µM, 5 µM, 10 µM, 20 µM, 50 µM, and 100 µM). At the same time, the same volume of RNase-free water was mixed with Dox concentrations (1 µM, 5 µM, 10 µM, 20 µM, 50 µM, and 100 µM). Then, these samples were incubated on a shaker in a cold room at 4 °C for 2 h. The samples were then centrifuged at high speed (4 °C, 12,000 g, 30 min), and the supernatant was used to determine fluorescence intensity using a microplate reader (ELx808, USA). The difference between the two groups of fluorescence intensity was taken to make a line chart. When the difference value is in a relatively stable stage, it indicated that the loading of products on Dox has reached a relatively saturated state.

Encapsulation efficiency and loading capacity of the FND

To characterize the stability of Dox loading in RNase-free H2O and PBS, free Dox and products loaded with Dox were resuspended in RNase-free H2O or PBS, respectively, while the fluorescence intensity was monitored at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h, respectively, using a microplate reader (ELx808, USA).

A fluorescence spectrophotometer was employed to measure the fluorescence intensity of the different Dox concentrations (0.01 µM, 0.1 µM, 1 µM, 5 µM, 10 µM, 100 µM, and 200 µM) and create a standard curve. The Dox encapsulation rate was calculated using the following formula: Loading capacity of Dox= (total Dox amount- Dox amount in the supernatant)/total Dox amount×100.

Characterization of the FND

The FND was characterized using DLS and TEM. The DLS procedure was the same as mentioned above. The FND was washed with RNase-free H2O, centrifuged (4 °C, 12,000 g, 30 min), resuspended in RNase-free H2O, and sonicated for 2 min to disperse well. A small amount of the sample solution was dipped in a copper mesh, blown dry with nitrogen, and observed via TEM.

Cell culture

The SW480 and L02 cell medium consisted of DMEM basal medium containing 10% (v/v) FBS, 100 U/mL penicillin, and 100 U/m streptomycin. The cells were cultured at 37 °C in 5% CO2 and 95% saturated atmospheric humidity conditions. Cell passaging was performed via 0.25% trypsin digestion. For cell seeding, the cells were inoculated into 96-well, 6-well, or 24-well plates for 24 h, according to the experimental requirements. When the cells reached 70% confluence, the cells were treated with FND or Dox, followed by subsequent experiments.

FND targeting

The FND was prepared using a FAM-labeled aptamer (FAM-Apt) to obtain fluorescently labeled FAM-FND. The SW480 and L02 cells were seeded into 6-well plates, while control and FAM-FND groups were established, with three replicate wells in each group. The cells were treated with FAM-FND for 8 h when they reached 70% confluence. Finally, the cells were resuspended in PBS, filtered through a 40 μm cell strainer into a flow tube, and examined via flow cytometry.

FND uptake

The SW480 and L02 cells were seeded into 24-well plates and treated with FAM-FND for 8 h when they reached 70% confluence. The culture medium was discarded, after which the cells were washed once with PBS and incubated with DAPI at 37 °C for 5 min. The working solution was discarded, and the cells were washed, after which they were examined via fluorescence microscopy and flow cytometry for detection.

Cell viability

The SW480 cells were seeded into a 96-well plate for 24 h. When they reached 70% confluence, different FND or Dox concentration gradients (Dox concentration: 0 µM, 0.02 µM, 0.2 µM, 0.37 µM, 0.75 µM, 1 µM, 1.5 µM, 3 µM, and 4.5 µM) of 100 µL culture solution was used to treat the cells for 24 h. 10 µL of CCK8 was added into each well, mixed, and incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using a microplate reader (ELx808, USA). The cell viability was calculated using the following formula: Cell viability (%) = (ODsam-ODblank)/(ODck-ODblank) ×100.

Cell cycle

The SW480 cells were seeded into 6-well plates. CK, Dox, and FND groups were established. Cells were treated with FND or Dox for 24 h once they had achieved 70% confluence, then collected after being washed and digested. The cells were then rinsed with pre-cooled PBS and centrifuged once more. The precipitated cell material was then resuspended in 1 mL of pre-cooled 70% ethanol and fixed for an overnight fixation period at 4 °C. The following day, fixed cells were collected by washing in PBS that had already been chilled. To collect the cells, the washing procedure was repeated. The cells were then incubated for 30 min at 37 °C in the dark and subjected to flow cytometry analysis.

Cell apoptosis

The SW480 cells were seeded into 6-well plates. CK, Dox, and FND groups were established, with three replicate wells per group. When the cells reached 70% confluence, they were treated with FND or Dox for 24 h and washed with PBS, followed by cell digestion and centrifugation to collect the cell precipitate. The cell precipitates were then resuspended using 195 µL Annexin V-FITC conjugate from the Annexin V-FITC assay kit, followed by the addition of 5 µL Annexin V-FITC and 10 µL PI, and mixed well. The cells were incubated for 25 min at room temperature, protected from light, and detected via flow cytometry or observed via fluorescence microscopy.

ROS content

The SW480 cells were seeded into 6-well plates. CK, H2O2 positive, Dox, and FND groups were established, with three replicates in each group. After 21 h of treatment, the medium in the positive wells was replaced with fresh medium containing 500 µM H2O2, and the cells were treated for another 3 h. After collection, the cells were suspended in a fluorescent DCFH-DA probe (10 µM) diluted in serum-free culture, incubated for 20 min at 37 °C, protected from light, washed three times with serum-free culture medium, and prepared for flow cytometry or fluorescence microscopy.

MMP

The SW480 cells were seeded into 24-well plates. CK, CCCP positive control, Dox, and FND groups were established. After 23 h 40 min of treatment, the medium in the positive control wells was replaced with fresh medium containing CCCP (10 µM), and the cells were treated for 20 min. After treatment, the medium was removed, and the cells were washed once with PBS, followed by the addition of 250 µL of cell culture medium and 250 µL of JC-1 staining solution and incubation at 37 °C for 20 min. After staining, the supernatant was removed, and the cells were washed twice with 1× JC-1 staining buffer, after which 500 µL of cell culture solution was added. The cells were directly observed using a fluorescent microscope.

Quantitative real-time PCR (qRT-PCR)

After discarding the media from the 6-well plate, 1 mL of Trizol reagent was added, and samples were collected in RNase-free 1.5 mL centrifuge tubes after 10 min of lysis on ice. Each sample tube received 200 µL of trichloromethane after the samples were lysed on ice for an additional 10 min. The samples were well mixed, vortexed, and placed on ice for 15 min before being centrifuged at 4 °C and 12,000 g for that same period of time. Each tube was then filled with 500 µL of isopropanol, mixed, and kept on ice for 20 min before being centrifuged at 4 °C at 12,000 g for 20 min and being allowed to air dry at ambient temperature. The RNA was dissolved with RNase-free water, and its concentration was determined using Nanodrop. Finally, the RNA was stored at -80 °C.

U6 and Oligo(dT) were selected as the reference genes for miRNA and mRNA quantification (Table S3). Here, the 20 µL reverse transcription system for miRNA consisted of 500 ng RNA, 0.5 µL RT-primer (10 µM), 10 µL 2×TS Reaction Mix, 1 µL TransScript RT/RI Enzyme mix, 1 µL gDNA Remover, and RNase-free H2O to 20 µL. The 20 µL reverse transcription system for mRNA consisted of 2 µg RNA, 0.5 µL oligo (dT), 10 µL 2×TS Reaction Mix, 1 µL TransScript RT/RI Enzyme mix, 1 µL gDNA Remover, and RNase-free H2O to 20 µL. The reverse transcription program consisted of 42 °C for 15 min and 85 °C for 5 s.

The 20 µL PCR system for miRNA and mRNA included 2x SuperReal PreMix Plus (SYBR GREEN), a forward primer (10 µM), a reverse primer (10 µM), cDNA, and RNase-free H2O. The PCR program comprised the following 95 °C for 5 min, 95 °C for 10 s, 58 °C for 40 s, and 72 °C for 10 s, with 40 cycles. The melting curve was detected, and β-actin was used for mRNA normalization. The RT-PCR primers of all the mRNAs are listed in Table S4. The relative gene expression was calculated as 2−ΔΔCt. Each sample was detected in triplicate.

mRNA library construction and sequencing

The mRNA was isolated from the total RNA using RNA binding buffer, wash buffer, Tris buffer, and Mix I, after which cDNA synthesis was performed according to the protocol. The concentration was measured using Qubit, while the subsequent library was constructed using a KAPA Hyper Prep Kit for Illumina. End repair was performed first, followed by junction ligation and finally elution with 30 µL nuclease-free H2O, after which the concentration was measured using Qubit. The constructed libraries were sequenced on the Illumina HiSeq X Ten platform.

Bioinformatics analysis

After sequencing, the raw data was filtered and processed. Following quality evaluation and qualification, each one was compared with a particular reference gene before being matched with the genome to provide mapped reads. Following that, base region distribution and homogeneity analyses were used to gauge the quality of the library sequences. After analyzing the differentially significant genes, the DEGs were screened using fold changes ≥ 1.2 or ≤ 1.2 and q < 0.05. In order to annotate the DEGs and correct p < 0.05 as the threshold of significance, the genomes were examined using the GO, KEGG, Reactome, and Metascape databases.

Statistical analysis

The data were shown as mean ± standard deviation (SD). In this study, *P < 0.05 and **P < 0.01 were considered statistically significant by using Student’s t-test (two-tailed) analysis.

留言 (0)

沒有登入
gif