Chemicals

MDA-MB-231 (RRID: CVCL_0062) and MCF7 (RRID: CVCL_0031) cells were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA). A kit for apoptosis detection using annexin V and fluorescein isothiocyanate (FITC) was bought from Cell signaling technology (Beverly, MA, USA). DSPE-PEG (2000) Maleimide, chitosan (purity 98%), RNase-free eppendorf tubes, acetic acid, 5-diphenyltetrazolium bromide (MTT), and protein assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following items were purchased from Gibco, Thermo Fisher Scientific, Inc. (Waltham, MA, USA): Dulbecco’s modified Eagle medium (DMEM), 0.25% trypsin–EDTA, penicillin–streptomycin, fetal bovine serum, ROS kit, and GE11 polypeptide. QIAzol Lysis Reagent (#79306) (QIAGEN, Hilden, Germany), miRNeasy Mini kit (QIAGEN, Inc, 217004), miScript® II RT Kit (QIAGEN, Inc, 21861), miScript Primer Assay (forward primer) (QIAGEN, 218300, MS00009079), miScript SYBR Green PCR Kit (QIAGEN, 218073), SensiFAST cDNA Synthesis Kit (BIO-65054), and SensiFAST SYBR Green No-ROX Kit (BIO-98005) (Meridian Bioscience, Cincinnati, OH, USA). RIPA lysis buffer, p-EGFR (3777), p-ERK (4377), p-Akt (Ser473) (4060), p-PI3K p85 (17,366), β-actin (4970), anti-rabbit IgG (7054), and U0126 (ERK inhibitor) were purchased from Cell Signaling Technology (Beverly, MA, USA). Alkaline phosphatase chromogen (BCIP/TNBT) (ab7413) was purchased from Abcam (Cambridge, UK).

Method of in-silico simulation studiesMolecular docking siRNA against miRNA-21

The computational (in-silico) technique has been widely used as an efficient tool for virtual biological screening. The 3D crystal structure of wild-type pre-miRNA-21 apical loop (Oncogenic miRNA-21 precursor) was downloaded from the Protein Data Bank, http://www.rcsb.org/pdb (PDB ID: 5UZT) and assumed for this study. At first, the crystalized water molecules were excluded from the downloaded crystal structure. Molecular preparation was done to add the hydrogen atoms. Energy minimization was performed by applying CHARMM force field. Then, the hydrogen atoms were hidden to make the areas of interaction clearer. The 3D structure of the tested siRNA/miRNA-21 inhibitor was generated from the next sequence (5′-UCAACAUCAGUCUGAUAAGCUA-3′) by using RNA-Composer server. Then, protonation and energy of inhibitor was minimized by applying CHARMM force field. The docking process was done using the Ligand scout 2.0 software (based on Autodock vina) using blind docking technique; about twenty poses were predicted and the best orientation was chosen, then the 3D binding modes were generated by BIOVIA Discovery Studio Visualizer (Tessaro and Scapozza 2020).

GE11 peptides de novo prediction

The 3D structure of the target peptide is not present in the protein data bank (PDB), so we used artificial intelligence (AI) to do the de novo prediction of 3D structure from the GE11 peptide sequence (peptide sequence: YHWYGYTPQWVI). 3D structures of peptides were prepared by the I-TASSER server and visualized by Biovia Discovery Studio 2019 software.

Structure validation of tested peptide

The best conformation (decoys) of 3D peptide structures selected by doing the homology modeling using the templet protein has structure similarity to detect the best configuration of alpha helix or beta sheets in predicted peptides. The I-TASSER modeling process commences with the structure templates that have been found by LOMETS from the PDB database. LOMETS is a metaserver threading method that consists of many threading programs. Each threading program has the ability to produce tens of thousands of template alignments. I-TASSER exclusively uses the templates with the utmost significance in the threading alignments, as determined by the Z-score. After the generation of the best conformation, I-TASSER algorithms selected the best one with a suitable C-score equal (C-score =  − 0.48) for molecular modeling.

Protein-GE11 molecular docking

Protein–protein docking was done to better understand the inhibitory functions of the tested peptide against the extracellular domain of EGFR TK. MOE software was used to perform the docking technique. First water molecules have been removed from the complex. Then, crystallographic disorders and unfilled valence atoms were corrected using protein reports, utility, and clean protein options. EGFR TK and tested peptide-energy were minimized by applying MMFF94 force fields. The docking process was done using the Ligand Scout 2.0 software (based on Autodock vina) using the blind docking technique, about twenty poses were predicted, and the best orientation was chosen, then the 3D and 2D binding modes were generated by the BIOVIA Discovery Studio Visualizer (Vakser 2014).

Preparation of targeted GE11-siRNA-CSNPs and non-targeted siRNA-CSNPs

First, 150 mg of chitosan was dissolved in 5 mL of acetic acid aqueous solution, and it was agitated for 20 min. The solution was mixed with 15 mL of distilled water, stirred (LCD Digital Hotplate Magnetic Stirrer, Laguna Hills, CA, USA), and left overnight before being vacuum-dried and kept at room temperature. After that, 50 mg of maleimide-poly-ethylene glycol-N-hydroxysuccinimide was combined with 50 mL of CSNPs solution for conjugation. The reaction was started and allowed to sit for three hours at room temperature and then stirred overnight. The solution was dialyzed using Milli-Q water, with half of the mixture being employed for an EGFR-binding peptide (Schäfer et al. 2011). In summary, PEG-CSNPs solution and GE11 were combined, and the mixture was constantly stirred for 4 h and allowed to react overnight at 4 ℃ under inert condition, so that the maleimide group and the peptide’s cysteine group could react to obtain GE11-targeted CSNPs. Lastly, siRNA was loaded using the produced nanoformulations. The mixture of 1 mL CSNPs or GE11-targeted CSNPs and 20 μL of siRNA (equivalent to 2.5 μg) was vortexed for 30 s at 3000 rpm and then allowed to sit at room temperature for 2 h in order to create non-targeted siRNA-CSNPs and GE11-siRNA-CSNPs (Malhotra et al. 2013; Joshi et al. 2021). Then the formation of GE11-siRNA-CSNPs complex was examined using gel electrophoresis. An agarose gel was loaded with 20 μL of the GE11-siRNA-CSNPs suspension using a loading dye dilution of 5:1. For 30 min, electrophoresis was run at 80 mA and a ChemiDoc Imaging Systems (Bio-Rad, Hercules, CA, USA) was utilized to visualize the siRNA bands, with free siRNA serving as the control.

Characterization of prepared nanoparticles

The size, charge, shape, and encapsulation efficiency of the produced nanoparticles were evaluated. Dynamic light scattering (DLS) (Malvern Instruments, Malvern, UK) was used to calculate the polydispersive index (PDI), zeta potentials, and mean particle size of nanoparticles. The transmission electron microscope (TEM) (JEOL JEM-1400Flash, Tokyo, Japan) was used to study the morphological traits. Then, the formula was used to determine the effectiveness of siRNA encapsulation in the supernatant of nanoparticle formulation at 260 nm wavelength using UV-spectrophotometer (U.V-1601; Shimadzu, Japan) after centrifugation (Optima L-100 XP Ultracentrifuge with a rotor NV 70.1, Beckman-Coulter, USA) (18,000 rpm, 20 min, 4 ℃): Encapsulation Efficiency (%) = ((total amount of siRNA − free siRNA)/ (total amount of siRNA)) × 100.

In vitro release profile and stability of synthesized nanoparticles

In RNase-free Eppendorf tubes, the GE11-siRNA-CSNPs are dissolved in 4 mL of PBS buffer at pH 7.4 or 6 while being shaken at 100 rpm and 37 ℃ (MS MP8 Wise Stir Wertheim, Germany). The entire material was collected by centrifugation at 14,000 rpm for 30 min at specified intervals of 0, 0.5, 1, 2, 4, 6, 8, 12, 20, 20, 24, 36, 48, and 72 h. Using a UV spectrophotometer, the amount of siRNA in supernatant at each time point was measured at 260 nm and substituted with the same volume of fresh buffer solution (Katas et al. 2013). In addition, gel electrophoresis was utilized to evaluate the serum stability of EGFR targeted siRNA-CSNPs. Briefly, a total of 200 μL of GE11-siRNA-CSNPs (containing 5 μg of siRNA) and 100 μL of FBS were combined and gently agitated at room temperature. As a control, naked siRNA received the same treatment. Subsequently, a precise volume of the samples (20 μL) was collected and stored at – 20 ℃, and their stability was assessed by subjecting them to analysis using a 4% agarose gel electrophoresis using 0.5 × TBE buffer, for 30 min at 4, 8, 16, 24, 36, and 48 h following incubation. After that, siRNA bands were visualized (ChemiDoc Imaging Systems, Bio-Rad, Hercules, CA, USA) (Salehi Khesht et al. 2021).

Cell culture conditions

MDA-MB-231 and MCF-7 were grown to confluence at 37 ℃ in 5% CO2. High-glucose Dulbecco modified Eagle’s medium (DMEM) was used to sustain MDA-MB-231 and MCF-7. It also contained 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin for antibiotics. The freezing media consisted of dimethyl sulfoxide (DMSO) diluted with FBS at a concentration of 10%. Cells were sub-cultured when they had achieved 80% confluence by aspirating the media, rinsing the cells in PBS, and then adding 10X trypsin/EDTA for 2 min. FBS was added to full media after the cells were separated from the flask to inactivate the trypsin. The cells were then moved and divided into several flasks for more study. Every 3 days, the media was replaced, and the cells were divided once a week.

Assessment of EGFR expression by Western blotting

About 1 × 105 MDA-MB-231 or MCF7 cells were added to 6-well growth plates for Western blotting. Before being lysed with RIPA cell lysis solution and a protease inhibitor cocktail, the medium was removed, and the cells were washed twice with ice-cold PBS. Following the removal of the lysates from the plates, centrifugation was performed at 12,000 g for 5 min at 4 ℃. The BCA protein assay technique was used to measure protein concentrations. Briefly, 2X loading buffer (130 mM Tris–HCl, pH 8.0, 30% (v/v) Glycerol, 4.6% (w/v) SDS, 0.02% Bromophenol blue, and 2% DTT) was mixed with 50 µg of protein lysates from each sample, boiled for 5 min, and then chilled at 4 °C. Samples were run at 120 v while being separated on a 12% SDS-PAGE mini-gel (Mini-PROTEAN System, Bio-Rad, Hercules, CA, USA). Proteins were transferred to a nitrocellulose membrane (Trans-Blot® Turbo™ Transfer System, Bio-Rad, Hercules, CA, USA). The membrane was washed three times in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20), blocked for an hour at room temperature with TBST that contains 5% nonfat dry milk, and then incubated overnight with primary antibodies (EGFR and β-actin) diluted in TBST. The membrane was treated with a secondary antibody for 1 h at room temperature following three TBST washes. Bands were detected using an alkaline phosphatase solution (Burnette 1981).

Cytotoxicity evaluation using viability assay

The MDA-MB-231 cells were grown on DMEM medium supplemented with 10% FBS and 50 µg/mL gentamycin, at a concentration of 2 × 106 cell/well in Corning® 96-well tissue culture plates, for the cytotoxicity assay. The cells were then incubated for 24 h at 37 ℃ in a humidified atmosphere with 5% CO2. To achieve six different concentrations for each formula, 100 µL of the nanoparticle solution was added onto 96-well plates (three replicates). These concentrations were 300, 100, 30, 10, 3, 1, 0.3, 0.1, and 0.03 μg/mL. MTT prepared in medium was added and incubated for 4 h after the media had been removed after 48 h of incubation. The final concentration of MTT was 0.5 mg/mL. Following the removal of the solution, DMSO was added, and it was incubated at 37 ℃ for 10 min. Cells that were not treated acted as a negative control. The absorbance of formazan solutions was measured at 570 nm using multi-well plate reader (BMGLABTECH®FLUOStar Omega, Germany). Using the GraphPad Prism program, the IC50 was determined (Mosmann 1983).

miRNA-21 expression detection by RT-PCR

For evaluating the feasibility of miRNA-21 inhibition by GE11-siRNA-CSNPs, RT-PCR of miRNAs was carried out. Total RNA including miRNAs was extracted from MDA-MB-231 cells using miRNeasy Mini kit. The concentration and purity of total RNA was quantified by Thermo Scientific NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and expressed as µg/µL. Also, a reverse transcription reaction was then performed using miScript® II RT Kit. For detection of mature miRNA, cDNA prepared in a reverse transcription reaction using miScript HiSpec Buffer serves as the template for RT-PCR analysis using an miRNA21-specific miScript primer assay (forward primer) and the miScript SYBR Green PCR Kit, which contains the miScript universal primer (reverse primer) and QuantiTect SYBR Green PCR Master Mix (CFX96 Touch Real-Time PCR Detection System, Bio-Rad, Hercules, CA, USA). Finally, the relative miRNA-21 expression was measured based on 2−ΔΔCT method.

Colony formation assay

As previously mentioned, colony development tests were conducted. Then, 500 MDA-MB-231 cells were cultured into 6-well plates, and the cells were allowed to develop overnight. The cells were subsequently exposed to GE11-siRNA-CSNPs for 2 h. The cells were washed in PBS after the drug-containing media was removed, and then they were cultivated for a further 10 days in a complete medium to form colonies. The cells were then fixed with 2% paraformaldehyde and washed twice with PBS. After that, each well of 6-well plates containing fixed colonies received 1 mL of (1%, 25 mM) crystal violet and was incubated at room temperature for 30 min. After the dye was removed, the wells were washed three times with dH2O and then twice with PBS. In five magnification fields chosen at random, cell colonies were calculated using ImageJ 1.47v software (http://imagej.nih.gov/ij) (Franken et al. 2006).

Wound healing assay

In 6-well culture plates, MDA-MB-231 cells were cultured at a density of 2 × 105 cells/well and expanded until 100% confluence. In a sterile setting, a 200 µL pipette tip was used to forcefully press against the tissue culture plate’s top, create a vertical wound through the confluent cell monolayer, and wash each well with PBS to remove non-adherent cells. The medium and cell debris were thoroughly aspirated. Then, enough culture media was added to the well wall to cover the bottom of the well and stop further cell detachment. The first image was then captured. At 37 ℃ and 5% CO2, the tissue culture plate was incubated. GE11-siRNA-CSNPs were applied to the cells at concentrations (0, 6, 12, 30 µg/mL), and they were subsequently incubated at the indicated time intervals. Under a microscope, the border of the center cell-free zone was verified. Using a microscope set at ×100 magnification, images were taken at 0, 24, 48, 72, and 96 h, and the wound area was calculated using ImageJ 1.47v software (http://imagej.nih.gov/ij) (Justus et al. 2014). Using MII ImageView 3.7v software, wound width was calculated as the average distance between the edges of the scratches.

Cell cycle analysis

The MDA-MB-231 cell line was subjected to GE11-siRNA-CSNPs at concentrations of 12 µg/mL or 30 µg/mL or control media. The treatments were administered for a duration of 48 h. Following administration of treatment, trypsinization was used to gather cells (105). Ice-cold PBS (pH 7.4) was then used twice to wash the cells. After being resuspended in two milliliters of 60% ice-cold ethanol, the cells were fixed for 1 h at 4 ℃. After repeatedly washing the fixed cells in PBS (pH 7.4), they were again suspended in 1 mL of PBS containing 10 µg/mL propidium iodide (PI) and 50 µg/mL RNAase A. Following a 20-min dark incubation period at 37 ℃, cells were subjected to flow cytometry analysis utilizing a FL2 (λex/em 535/617 nm) signal detector (ACEA NovocyteTM flowcytometer, ACEA Biosciences Inc., San Diego, CA, USA) to determine the DNA content of the cells. We collected 12,000 events for every sample. The ACEA NovoExpressTM program (ACEA Biosciences Inc., San Diego, CA, USA) was used to compute cell cycle distribution.

Measurement of intracellular ROS

MDA-MB-231 cells were isolated, centrifuged twice, and suspended in PBS. The cells were then stained using the ROS detection kit as directed by the manufacturer at 37 ℃ for 30 min. After that, the cells were incubated with GE11-siRNA-CSNPs and non-targeted siRNA-CSNPs (100 µL/well) for varying lengths of time at 37 ℃. Using a microplate reader to determine intracellular ROS levels, cell fluorescence intensity was evaluated (excitation and emission wavelength set as 485/535 nm) (Bass et al. 1983).

Gene expression of apoptotic markers by quantitative real-time-polymerase chain reaction

About 1 × 105 cells/mL of MDA-MB-231 cells were cultured into 25 cm2 TPP-Swiss flasks. Different concentrations of prepared GE11-siRNA-CSNPs were applied to cancer cells for 48 h while also considering an untreated flask. The afflicted cells were separated from the remaining cells, which were then trypsinized and centrifuged at 4 ℃. PBS was used to rinse the pelted cells before transferring them to Eppendorf tubes. Using QIAzol Lysis reagent and following the manufacturer’s instructions, total RNA was extracted. Following the manufacturer’s instructions, cDNA products were produced from RNA using the SensiFAST cDNA synthesis kit. A thermal cycler was used to operate the reaction mixture (CFX96 Touch Real-Time PCR Detection System, Bio-Rad, Hercules, CA, USA). The RNA concentration was determined from the threshold cycle (Ct) values. The mRNA expression levels were calculated relative to the GAPDH gene’s mRNA levels using 2−ΔΔCT method (Livak and Schmittgen 2001). Primer sequence: BCL2 (NM_000657.3) F: 5ʹ-ATGTGTGTGGAGACCGTCAA-3ʹ and R: 5ʹ-GCCGTACAGTTCCACAAAGGG-3ʹ. The Bax (NM_138763.4) F: 5ʹ-ATGTTTTCTGACGGCAACTTC-3ʹ and R: 5ʹ-AGTCCAATGTCCAGCCCAT-3ʹ. Caspase-9 (NM_001229.5) F: 5ʹ-CATTTCATGGTG-GAGGTGAAG-3ʹ and R: 5ʹ-GGGAACTG-CAGGTGGCTG-3ʹ. Caspase-3 (NM_004346.4) F: 5ʹ-TGTTTGTGTGCTTCTGAGCC-3ʹ and R: 5ʹ-CACGCCATGTCATCATCAAC-3ʹ. GAPDH (NM_001256799.3) F: 5ʹ-GGCACAGTCAAGGCTGAGAATG-3ʹ and 5′ R: 5ʹ- ATGGTGGTGAAGACGCCAGTA-3ʹ.

Western blotting

After 48 h of treatment with different concentrations of GE11-siRNA-CSNPs, levels of the proteins BCL2, Bax, p-AKT, p-PI3K p58, and p-ERK1/2 was measured by Western blot analysis. As already described above, following the primary antibodies incubation, alkaline phosphatase chromogen was used to produce the membrane (Burnette 1981). U0126 treatment, TNBC cells were divided into four groups: control TNBC, U0126 (ERK1/2 inhibitor) (10 µM), GE11-siRNA-CSNPs (10 μM), and U0126 + GE11-siRNA-CSNPs treated cells. After 24 h, the cells were collected for western blot assay and detection of p-ERK1/2.

Molecular docking of miRNA-21 and AKT

The blind docking technique was used to dock miRNA-21 to the target site of AKT. This was done to evaluate the potential affinity and inhibitory activity of miRNA-21 toward this target protein. Applying the above-described molecular docking method for the interaction between EGFR and GE11 peptide.

Hypoxic cytotoxicity assay

A hypoxic environment with 0.5% O2 and 5% CO2 was produced with the use of hypoxic chambers (Anaero Pack, Mitsubishi Gas Chemical Co., Tokyo, Japan) according to the manufacturer’s recommendations, and the SRB assay was used to measure cell viability. In 96-well plates, aliquots of 100 μL cell suspension (5 × 103 cells) were incubated for 24 h in full medium. Another aliquot of 100 μL medium containing medications at different concentrations (0.01, 0.1, 1, 10, and 100 μg/mL) were added to treat the cells. After drug treatment, cells were fixed by replacing medium with 150 μL of 10% TCA and incubated at 4 ℃ for 1 h. After removing the TCA solution, the cells underwent five rounds of distilled water washing. Once added, aliquots of 70 μL SRB solution (0.4% w/v) were incubated for 10 min at room temperature in a dark area. After three 1% acetic acid washes, the plates were left to air dry for the entire night. After that, 150 μL of TRIS (10 mM) was added to dissolve the protein-bound SRB stain, and an Infinite F50 microplate reader (TECAN, Männedorf, Switzerland) was used to measure the absorbance at 540 nm (Skehan et al. 1990).

Statistical analyses

Statistical analyses were performed with GraphPad Prism v8 (GraphPad Software Inc.), and SPSS Predictive Analytics Software (IBM, Version 26). Multiple comparisons were made using one-way ANOVA with Tukey’s post-hoc test, and differences with a significance level of p < 0.01 were considered to be statistically significant.