Materials
Folic acid, thioglycolic acid, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxy succinimide (NHS), polyethylene glycol (PEG), Cetyltrimethylammonium chloride (CTAB), tetraethyl orthosilicate (TEOS), 1,3,5-trimethylbenzene (TMB), and (3-aminopropyl) triethoxysilane (APTES) were purchased from Sigma-Aldrich. The Human ABCB1 siRNA and the negative control siRNA were purchased from Bioneer Corporation (South Korea). Optimized TAT peptide was ordered from TAG-Copenhagen (Copenhagen, Denmark). All other reagents and solvents were of analytical grade and provided by Sigma-Aldrich (Deisenhofen, Germany). HeLa cells (MDR1 negative and high folate receptor-expressing line) and EPG85.257-RDB (high MDR1 and trace folate receptor-expressing line) were kindly supplied by Professor Herman Lage (Universitäts medizin Berlin, Germany).
MSN synthesis and surface amination
NH2-MSNs with large pores were prepared in the presence of TMB as a micelle swelling agent. Initially, 1 g CTAB was dissolved in 480 ml deionized water, the temperature of the mixture was increased to 60 °C, 7 ml TMB was added, and the mixture was stirred for 5 h. The solution was then poured with 3.5 ml of 2 M sodium hydroxide. Following that, the temperature was increased to 80 °C, and 5 ml TEOS and 0.2 ml APTES were simultaneously inset dropwise into the solution for 5 min, followed by another 2 h of stirring. After cooling, the white precipitate was washed with generous amounts of 96% ethanol, deionized water, and freeze-dried. By incorporating APTES into the silanization process, the pores and surface were functionalized with amine groups. Additionally, the outer surface of the nanoparticles was covalently aminated. Afterward, 1 g of nanoparticles was dispersed in 100 ml ethanol containing 2% v/v APTES and stirred for 12 h at 60 °C under reflux conditions. NH2-MSNs were collected with centrifugation at 14000 rpm for 15 min at ambient temperature. Finally, 1 g nanoparticle was resuspended in 200 ml 96% ethanol containing 2 g NH4NO3, and the suspension was refluxed at 60 °C for 12 h. The precipitate was collected via centrifugation, and the process was repeated at least three times to remove the surfactant from the pores completely. For future use, the surfactant-free NH2-MSNs were freeze-dried and stored in 96% ethanol47. A transmission electron microscope (TEM, Zeiss, Germany), a scanning electron microscope (SEM, JEOL, Japan), X-ray diffraction (XRD, STOE, Germany), and Fourier transform infrared spectroscopy were used to physiochemically examine the nanoparticles (FTIR, Bruker, Germany). A nitrogen adsorption analyzer was used to determine the surface area (Belsorp-mini, Japan). All samples’ particle size and zeta potential were determined in deionized water using a DLS (Malvern, Worcestershire, England).
siRNA loading and gel retardation assay
Through RNA retardation assay, the siRNA loading capacity was optimized. For 30 min at room temperature, siRNA and aminated-MSN were mixed at various ratios (naked siRNA, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 μg/μg in 50 μl water). The RNA-MSN complex was loaded with a dye, and electrophoresis was performed on a 1.5% agarose gel containing Gel-Red at a constant 80 V for 30 min. The migration patterns were recorded at 254 nm irradiation27. The loading capacity was determined using the following formula:
$${text{Loading}};{text{capacity}};% = frac{{Weight;of;loaded;siRNA}}{{Weight;of;the;nanoparticles}} times 100$$
Covalent pegylation of TAT and folate
First, folate was thiolated and activated for nucleophilic substitution using 2-mercaptoacetic acid. Then, 2 mg SH-folate and 7 mg MAL-PEG(3000)-NHS was stirred for 24 h at room temperature in the dark under argon protection in 1 ml anhydrous DMSO (1:1 molar ratio).This reaction results in the formation of folate-MAL-PEG(3000)-NHS. Second, a cysteine modified version of TAT peptide (HS-CYGRKKRRQRRR-NH2) was linked to MAL-PEG(3000)-NHS to form TAT-MAL-PEG(3000)-NHS. Briefly, 1 mg TAT and 2 mg MAL-PEG(3000)-NHS (5:1 molar ratio) was stirred in 1 ml deionized water for 24 h at 4 °C. The reaction was controlled on TLC (silica gel-60 F254) using methanol, chloroform, and water (45:9:1). TLC was stained with Dragendorff’sreagent and ninhydrin. For both reactions, unreacted substrates were dialyzed (3.5 kD, Sigma-Aldrich) over deionized water32. Conjugation efficiency for the reactions was calculated through the Ellman assay for free sulfhydryl measurement: 100 × ((substrate-SH) − (PEG-S-substrate)) ÷ substrate-SH). Finally, pure conjugates were lyophilized and maintained at a temperature of 4 °C48.
Chitosan modification and targeting
Four sequential syntheses were used to generate MSN targeting coats. Amine moieties on chitosan were determined using 1H-NMR in a solution containing 2% deuterated acetic acid.The pH was adjusted to 6.0 after dissolving 10 mg chitosan (equivalent to 40 mol NH2 groups) in 2 ml of 2% acetic acid. Polyethylene glycol, folate, TAT, or double folate/TAT were used to conjugate chitosan. Around 0.8 mol of MAL-PEG(3000)-NHS, TAT-PEG(3000)-NHS, folate-PEG(3000)-NHS, or a mixture of TAT-PEG(3000)-NHS and folate-PEG(3000)-NHS was added to 2 ml chitosan solution (1:50 ratio to amine group) and stirred in the dark at room temperature. After 3 h, the pH was raised to 7 for another 24 h. For 48 h, the solution was dialyzed against deionized water (cutoff 12 kD, SLS, UK). The modified chitosans included chitosan-PEG(3000), chitosan-PEG(3000)-folate, chitosan-PEG(3000)-TAT, chitosan-PEG(3000)-folate/TAT and were collected after freeze-drying and stored at 4 °C. The conjugations were confirmed and quantified using NMR49,50,51.
Formulation and evaluation of MSNs coated with functionalized chitosan
NH2-MSNs loaded with optimized RNA were briefly coated with various functionalized chitosans, and 5 µl of the MSN stock (0.1% w/v) plus 25ρmol of siRNA were stirred gently for 30 min to form the NH2-MSN-siRNA complex. Then, 2 µl of each functionalized-chitosan solution (0.5% w/v in 2% acetic acid, pH: 6) was added to the RNA-loaded NH2-MSNs and stirred for 24 h. The chitosan-coated NH2-MSN-siRNA were collected by centrifugation (14000 rpm, 15 min) and washed twice with deionized water52. Zeta potential was measured to verify the chitosan coating on the surface of the MSN structures. Finally, the protective role of chitosan coats was evaluated by incubating the nanostructures with 0.25% RNase-A at optimal conditions for varying times. The enzyme was inactivated at 60 °C for 5 min. Heparin (200 IU/µl) was added to the complexes for 10 min to release the siRNA, and agarose gel electrophoresis was performed to identify the siRNA degradation level27,42,53. For the stability of nanoparticles, siRNA leakage from nanoparticles was studied for 4 days in PBS by UV-spectroscopy at 260 nm. Briefly, the siRNA loaded nanoparticles were dispersed in PBS at 4 °C. At a predetermined sampling time (1, 2, 3, and 4 day), the suspension was centrifuged and samples were taken from the supernatant. siRNA quantities of the supernatant were determined by UV-spectroscopy at 260 nm.
HeLa transfection with multidrug-resistant pumps
The cell lines were cultivated in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, and 100 IU/ml penicillin at 37 °C in a 5% CO2 atmosphere. Multidrug-resistant HeLa-RDB was generated by transducing HeLa cells using engineered lentiviral particles with the human ABCB1 (NM 000927.4) gene (pReceiver-Lv152 plasmid, CMV promoter, hygromycin resistant, Genecopoeia Company). HeLa cells (5 × 104 cells/well) were cultured in 24-well plates. After achieving 75% confluence, recombinant lentiviruses were added at multiplicity of infection (MOI) values ranging from 0.5 to 10. After 24 h, the virus-containing medium was removed from the wells and replaced with a fresh medium. The cells were treated with an optimum hygromycin-B concentration that kills untransfected cells to select stably transduced cells. The drug-resistant colonies of HeLa cells became visible after 10 days. EPG85.257, HeLa, and their MDR1 resistant counterparts were evaluated for folate receptor and MDR1 pumps using real-time PCR48.
Growth pattern and functional cytotoxicity kinetics
Recombinant and parental cell lines were seeded at a density of 1000 cells per well in 96-well plates to measure the growth kinetics during 7 days. Cellular proliferation was monitored daily using MTT. A trend line was depicted on natural logarithms of the cell number (dependent variant) versus times (independent variant). According to the Monod equation in the logarithmic phase, the cell-specific growth rate (μmax) was considered the slope of the line. The cell cytotoxicity of the final nanostructures was determined on HeLa-RDB cells and EPG85.257-RDB cells. In 96-well plates, 3000 cells were seeded per well. Cells were treated with various concentrations of nanoparticles (0, 20, 60, 100, 140, and 180 µg/ml) after 24 h. After 72 h of treatment, optical density at 570 nm was used to determine the viable cell fraction in the MTT assay. Finally, daunorubicin toxicity (0–10000 nm) was determined in the presence of siRNA-loaded nanoparticles (10 µg/ml) to assess whether the multidrug-resistant cells were chemosensitized. The IC50 value was defined as the concentration of daunorubicin required to inhibit cell growth by 50% in the presence of silencing RNA39,48.
Nanoparticle endocytosis and competition analysis
Cellular uptake was assessed by flow cytometry (CyFlow Space®, Munster, Germany) using the FL-2 channel. HeLa-RDB and EPG85.257-RDB cells (2 × 105 cells per well) were seeded into 12-well plates. Initially, 10 μl of DiI fluorescent dye (1 mg/ml in ethanol) was loaded into 10 mg of NH2-MSN. The DiI-MSNs were washed three times with ethanol and centrifuged to collect them. The fluorescent nanosilica particles were then coated with various modified chitosans, and cells were treated for 2 h at 37 °C with 100 μg/ml of the modified-fluorescence nanovehicles39. After washing the cells with PBS, the mean intracellular fluorescence intensity indicated the constructs’ endocytosis activity. The same procedure was used to conduct a competitive uptake assay in the presence and absence of 5 µM folate as the competitor. Data were analyzed using FlowJo software (version 7.6.1) and compared to the control samples (treated cells with uncoated DiI-NH2-MSN)54.
Molecular evaluation of gene silencing
Real-time PCR and Western blotting were used to determine the MDR1 level. In 6-well plates, HeLa-RDB and EPG85.257-RDB cells were seeded. After 24 h, cells were transfected with optimized si-MDR1-loaded nanovehicles (25 pmol si-MDR1/5 µg MSNs) coated with specific chitosans. After 48 h, RNA was extracted, cDNA was synthesized, and real-time PCR was performed according to the Pfaffl method on a Rotor-GeneQ instrument (Qiagen, Germany) using the SYBR Green kit (Qiagen kits, Germany). Simultaneously, proteins were extracted using a lysis buffer (8 M urea, 2 M thiourea, Tris 10 mM, pH = 8.0), and the Bradford method was used to quantify the proteins colorimetrically. Proteins were run in a 12% SDS-PAGE according to the Laemmli method and then electroblotted to the nitrocellulose membrane using a semi-dry Trans-Blot apparatus (Bio-Rad, Richmond, CA). Blots were briefly blocked by 5% skimmed milk, incubated with a primary anti-Pgp mouse monoclonal IgG (1:500 v/v) overnight at 4 °C, treated with an HRP-conjugated secondary antibody (1:5000 v/v) for 2 h, and photos were taken using the luminal system on a blot scanner (LiCor, Lincoln, NE). For quantification procedures, β-actin was considered as the internal normalizer for both quantifications48.
Statistical analyses
Results were expressed as mean ± standard deviation of at least three independent experiments and analyzed using graph pad prism. P values < 0.05 were considered statistically significant.
Ethical statement
The authors have read and adhered to the journal’s ethical standards for manuscript submissions. This article does not contain any studies with human participants or animals performed by any of the authors. We declare that the submitted manuscript does not contain previously published materials and is not considered for publication elsewhere. All the authors have contributed to conception and design, collection, analysis, and interpretation of data, writing or revising the manuscript, or providing guidance on the research’s execution.
