Materials
Low molecular weight chitosan (MW 50–190 kDa, 75–85% degree of deacetylation) was purchased from Sigma Aldrich (St. Louis, MO, USD). Sodium alginate (low viscosity, 1.56 ± 0.01 cP, 2% in distilled H2O (25°C)) was obtained from İlko Pharmaceuticals (İstanbul, Turkey). Human CTNNB1 ELISA Kit and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Cell Proliferation Kit were purchased from BT-LAB (Birmingham, UK) and Roche (Basel, Swiss), respectively. Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were brought from PAN Biotech (Aidenbach, Germany). All other chemicals used were of molecular grade.
Transformation and isolation of shRNA plasmid
pshRNA-CTNNB1 (GenePharma, Shangai, China) transcribes stem-loop structured shRNA targeting CTNNB1 mRNA under the control of the SV40 promoter in eukaryotic cells. The type of shRNA expression vector is pGPU6/GFP/Neo. The plasmid structure, which is 5117 bp in size, contains the Kanamycin/Neomycin antibiotic resistance gene and the GFP reporter gene. The target sequence of pshRNA-CTNNB1 is GCTTGGAATGAGACTGCTGAT. The target sequence was selected against human CTNNB1 mRNA (Accession number NM_001098210.2). pshRNA-NC (target sequence: TTCTCCGAACGTGTCACGT) as a negative control was also synthesized and purified by Genepharma (Shangai, China).
pshRNA-CTNNB1 and pshRNA-NC were transformed into E.coli DH5-alpha bacterial strain. Transformed cells were grown in LB broth containing Kanamycin (100 µg/ml). The plasmid was isolated according to protocol using the PureLink HiPure Plasmid Maxiprep Kit (Invitrogen, USA). DNA concentration was measured by UV-vis spectrophotometer (Shimadzu Biospec 1601, Shimadzu Scientific Instruments, Japan) at 260 nm. The purity of DNA was controlled by agarose gel electrophoresis after ethidium bromide staining.
Preparation of alginate-chitosan hydrogels
Alginate-chitosan hydrogels were prepared at the concentrations shown in Table 1 using sodium alginate and chitosan polymers. Sodium alginate was dissolved in bidistilled water. Chitosan in powder form was added to the sodium alginate solution and dispersed. A polyionic gel form was obtained by adding 8% acetic solution to the resulting mixture. Afterward, the pH was adjusted to 5–7 with 1 M NaOH solution. While preparing hydrogels containing pshRNA, the plasmid was added to the chitosan-alginate mixture at a concentration of 50µg pshRNA/1gr hydrogel. To check whether the hydrogels contained pshRNA, samples were loaded into agarose gel at 0.8% (a/h) concentration and the gels were imaged on a UV transilluminator (Vilber Lourmat, France) after electrophoresis procedure.
Table 1
Polymer content (w/v) and pH values of alginate-chitosan hydrogels.
Hydrogel formulation
|
Concentration of sodium alginate
|
Concentration of chitosan
|
pH value
|
Nac1
|
2%
|
2%
|
5.59 ± 0.07
|
Nac2
|
2.5%
|
2.5%
|
5.98 ± 0.05
|
Nac3
|
3%
|
3%
|
6.21 ± 0.04
|
Characterization of Hydrogels
The chemical structure of hydrogels was studied by FTIR analysis. The measurements were performed using a Shimadzu FTIR-8400S spectrophotometer (Shimadzu Scientific Instruments, Japan) at a wavelength range of 4000 − 750 cm− 1. After the lyophilized hydrogels were palladium-gold plated, surface morphology and physical structure were examined with SEM (Zeiss EVO MA10, Jena, German). The viscosities of the hydrogels were determined using a cylindrical spindle with the Brookfield DV-E Viscometer (Toronto, Canada) at different rotational speeds at room temperature. Measurements were performed three times for each sample.
To determine the swelling capacity of the hydrogels, the hydrogels were dried in an oven at 37°C until the constant weight of the hydrogels. The dried hydrogels were weighed and immersed in PBS (pH 7.4). At regular time intervals, the samples were taken out and weighed after excessing water was removed using filter paper. The experiment continued until the hydrogels reached a constant weight. The experiment was performed in triplicate and at room temperature. The swelling ratio of the hydrogels was calculated according to the following formula (Salehi et al. 2019).
$$\text{Swelling}\text{ }\text{ratio}\text{ }\left(\text{%}\right)\text{= }\frac{\left(\text{W}\text{s}\text{-W}\text{d}\right)}{\text{W}\text{d}}\text{ × 100}$$
where Ws and Wd are the weight of the swollen and dried hydrogel, respectively.
In vitro degradation assay
After the hydrogel samples were weighed into tared containers, they were incubated in PBS at 37°C in a shaker at 75 rpm. The samples were taken out at certain time intervals, dried and their weight measured. The degradation was calculated using the following equation. W0 and Wd are weights before and after incubation, respectively (Saygılı et al. 2021).
$$\text{Degradation}\text{ (%) = }\frac{\text{W}\text{0}\text{-W}\text{d}}{\text{W}\text{0}}\text{ x 100}$$
Serum stability assay
The serum stability assay was performed according to the experiment reported by Salva and Akbuğa (2010). The hydrogels containing 10µg of pshRNA were suspended in 1ml of PBS (pH 7.4) with 10% FBS and incubated at 37°C. 100µl samples were taken at certain time intervals (0, 15, 30 min; 1, 4, 24, 48, and 72 h) and the reaction was inhibited with 0.5M EDTA. The integrity of pshRNA was controlled by agarose gel retardation assay. The serum stability of free pshRNA was also studied as described above.
In vitro release study
Hydrogels containing 20µg of pshRNA were weighed into sealed sample tubes and incubated in PBS (pH 7.4) at 37°C with shaking at 75 rpm. At predetermined intervals, the samples were centrifuged at 14000 rpm for 30 min. The supernatants were collected and replaced with the same volume of fresh buffer (Gao et al. 2020). The amount of pshRNA released in the supernatant was measured spectrophotometrically at 260 nm. The supernatant of hydrogel without pshRNA was used as blank control.
In vitro transfection study
MDA-MB-231 cells were cultured in DMEM with 10% FBS, 100mM L-glutamine, and 100mM antibiotic solution (penicillin/streptomycin) in an incubator (Sanyo, Japan) with 5% CO2 at 37°C. The cells were seeded in a 24-well plate at a cell density of 2.5 x 105 cells/well and incubated overnight. When the cells reached approximately 70% confluence, the medium on the cells was removed and hydrogels with 1µg pshRNA and without pshRNA were administered to cells with serum-free medium. Nine groups were constituted: control group (untransfected cells), free pshRNA CTNNB1 group, blank Nac1, Nac2, and Nac3 hydrogel groups, pshRNA CTNNB1-loaded Nac1, Nac2, and Nac3 hydrogel groups, pshRNA NC-loaded hydrogel group. After the cells were incubated for 4–6 hours, a fresh medium with 10% serum was added to the cells. After 72h from transfection of the hydrogels containing pshRNA, the cells were observed using a fluorescence microscope (Olympus CKX41, Japan) to image GFP expression.
Determination of CTNNB1 expression
CTNNB1 levels in cells after transfection were determined using the ELISA method. The ELISA assay was performed according to the manufacturer's protocol. CTNNB1 levels were determined using absorbance values spectrophotometrically measured at 450 nm. The decrease in the concentration of CTNNB1 in transfected cells was determined relative to untransfected cells. Three samples were studied for each group.
Cell viability assay
The effect of prepared alginate-chitosan hydrogels on cell proliferation was determined by MTT assay. MDA-MB-231 cells seeded into 96-well cell culture plates at a concentration of 4x103 were incubated overnight at 37°C and 5% CO2. After removing the medium from the cells, the prepared hydrogels were applied to the cells with a serum-free medium. After 4–6 h of incubation, fresh medium with 10% serum was added to each well. After 72 hours, 10 µl of MTT reagent was added to the wells, and the cells were incubated for 4–6 hours at 37°C and 5% CO2. Formazan crystals formed by living cells in MTT-treated cells were dissolved with 10% SDS (sodium dodecyl sulfate) solution. Absorbances of the samples were measured spectrophotometrically at 550 nm and 690 nm. Cell viability (%) of the hydrogel-treated groups was determined compared to the control group (untreated cells).
Statistical analysis
Results were expressed as the mean ± standard deviation (SD). The treatment groups were analyzed using One-way ANOVA with Tukey's multiple comparisons test. All analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, San Diego, CA). P < 0.05 was considered to be statistically significant.