Material

Chloroform, ethanol, methanol, span 60, Tween 60, cholesterol, dimethyl sulfoxide (DMSO), ultrapure sodium dodecyl sulfate (SDS), and Amicon® Ultra Centrifugal Filter Devices (Amicon Ultra-15 Membrane MWCO 30 KDa) were purchased from Merck company (Germany). Phosphate buffered saline tablet (PBS) provided by Bio Basic Company(Canada). The PX-12 (PubChem CID: 219,104), YC-1(PubChem CID: 5712), DEPC water, and dialysis membrane (MWCO 12 KDa) were obtained from Sigma Aldrich company(USA). Dulbecco's Modified Eagle's medium (DMEM), Trypsin–EDTA, and Penicillin–Streptomycin(pen-strep) were received fromBiosera company (France). Fetal bovine serum (FBS) was bought from Gibcocompany (USA)0.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Atocelcompany(Australia).ApoFlowEx FITC Kit (Annexin V/PI) was acquired from EXBIO company (Czech Republic). The flow cytometer used in this study is a FACScalibur belonging to BD Biosciencescompany (Canada). Sybr Green master mix real-time PCR was purchased from Ampliqoncompany (Denmark). Intestinal cancer cells (HT-29) and human foreskin fibroblast (HFF) were supplied from the National Center for Genetic and Biological Resources of Iran.

Optimization of noisome formulation

The central composition method (CCD) was used to optimize the niosomal formulations through the Box-Behnken design. To investigate the relationship between a set of independent variables and dependent variables by fitting the data using a polynomial equation, two numerical parameters lipid content (total surfactant and cholesterol) µmol and surfactant to cholesterol molar ratio, were selected for the study.

The effect of their concentration on niosomal particle size (nm), polydispersity index (PDI), entrapment efficiency percentage (EE%), and drug release percentage was investigated. The polynomial equation was obtained using Design-Expert software (Version 7.0.10, Stat-Ease, Inc., Minneapolis, MN, USA). Comparisons were made between the experimental data and the predicted responses. Using the point prediction method, the optimal formulation was chosen for further study. Table 1 represents these factors and their levels.

Table 1 Different levels for variables in the CCD optimizationaPreparation of niosomal formulations

The thin-layer hydration method was used for the synthesis of different formulations of niosomes containing PX-12 and YC-1. Due to the hydrophobic property of YC-1, it is added in the first step along with other nisome components. Briefly: YC-1, Cholesterol, span 60 and tween 60 are dissolved in 10 ml of organic solvent (chloroform /methanol with a ratio of 2:1). The solution was transferred to a 50 ml round bottom flask. The organic solvent was evaporated under vacuum using a rotary evaporator (Heidolph Instruments, Schwalbach, Germany) at 60°C and 150rpm for 30min until a thin dried film was formed in the bottom of the flask. Afterward, because PX-12 is hydrophilic, it is added to the solution in the last steps along with PBS. PBS containing PX-12(10 ml, PH 7.4) was used to hydrate dry, thin films, and the mixture was stirred at 60 °C and 150 rpm for 30 min. (Both drugs have concentration of 50 µg/ml). Finally, the sample was sonicated for 5 min (Hielscher UP50H ultrasonic processor, Germany, Amplitude: 25%, 200 w) to obtain the niosomal formulations with uniform size distribution. The samples were stored in a refrigerator (4°C) for future use.

Physicochemical characterization of niosomal formulations

Assessment of some critical parameters of formulations such as the particle size distribution, the polydispersity index (PDI), and zeta potential was performed by Dynamic Light Scattering technique (DLS) (Zetasizer Nano S90, Malvern Panalytical Ltd., Malvern, United Kingdom). All of these parameters were investigated in a nano-zeta sizer device equipped with a green laser with a wavelength of 633 nm at a temperature of 25 °C. Surface features and morphology of niosomes are evaluated by Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM). For examination by FESEM microscope (SSX-500, Shimadzu, Japan), about 1 ml of niosomal formulation solution was dried by freeze dryer. For TEM, a drop of 0.1% w/v niosomal solution was placed on a carbon-coated copper grid and stained with a 1% phosphotungstic acid. The stained niosomes were imaged with TEM (Model EM900, Zeiss Microscopy, Jena Germany). To determine the chemical composition of niosomal formulations and the possible interactions between the carrier and drugs Fourier Transform Infrared Spectrometer (FTIR) (Spectrum Two, USA) was used. Lyophilized samples were mixed individually in KBr, and the pellets were formed by placing the samples in a hydraulic press. FTIR analyses were accomplished in the scanning range of 4000 to 400 cm−1 at room temperature. UV–Visible absorption spectra were obtained on a UV–Visible spectrophotometer (UV-1700 PharmaSpec, Shimadzu, Kyoto, Japan).

Entrapment Efficacy (%EE)

The ultrafiltration method has been used to investigate entrapment efficiency. For this, niosomal formulations of drugs were filtered at 4°C and 4000rpm for 20 min in a centrifugal filter tube (Amicon Ultra-15-Membrane MWCO 30000 DA). The free drug passes through the filter pores, and what remains on the filter is the niosome containing the drugs. The absorbance of the sample passing through the filter is read at 250 and 270 nm for PX-12 and YC-1, respectively, using a UV–Visible spectrophotometer. The percentage of encapsulation efficiency is calculated according to Eq. 1:

$$\mathrm=\frac-\mathrm}}\;\mathrm\;100$$

(1)

In vitro drug release study

In vitro release profile of PX-12 and YC-1 from niosomal formulation (NIO/PX-YC) was conducted using a semipermeable acetate cellulose dialysis bag (MWCO 12kDa) in phosphate buffer saline containing 0.5% w/v SDS. Briefly, two dialysis bags containing a fixed amount of drug-loaded niosomal solution were placed in 25 ml of PBS-SDS solution with pH 5.4 and 7.4 and incubated at 37°C with a gentle shaking of 50 rpm. At predetermined time intervals (1, 2, 4, 6, 8, 24, 48, and 72h), 1 ml of the release medium was withdrawn and replenished with the same volume of fresh PBS-SDS. Finally, the amount of PX-12 and YC-1 released from the niosomal formulations was determined by UV–Visible spectrophotometer at 250 and 270 nm, respectively and the cumulative release of drugs was calculated. For the comparative study, the experiment was repeated for the free drugs (PX-12, YC-1) as a control with the same concentration of their niosomal formulation. The experiments for each group were conducted three times and the results were represented as mean ± SD. The amount of cumulative release percentage (CR) was calculated according to the following equation (Eq. 2, 3) [48]:

$$CR \left(\%\right)= \frac _+ _$$

(2)

$$_= \frac_V} \times 100$$

(3)

where \(Vs\) is the volume of sample withdrawn (ml), \(V\) is the bath volume (ml), \(_\) is drug concentration at time t (µg/ml), \(M\) is the total amount of drug (µg), \(_\) and \(_\) represents drug release percentage at times t and t-1. The experiment was repeated using free drugs as a control with the same concentration of their niosomal formulation.

Release kinetics study

To describe the release kinetics of PX-12 and YC-1 from noisome various mathematical models were used. Zero-order(Eq. 4), where the drug release rate does not depend on its concentration [49]; the first order (Eq. 5), where the drug release rate is concentration-dependent [50]. Korsmeyer-Peppas (Eq. 6) describes the drug release from a polymeric system [51]. Higuchi (Eq. 7) describes the release of drugs from the insoluble matrix as a square root of a time-dependent process based on the Fickian diffusion [52].

where C is drug concentration at time t, C0 is the initial drug concentration, t is the time, and k0 is the zero-order rate constant expressed in units of concentration/time.

$$\mathrmC-\mathrm_= _t/2.303$$

(5)

where k1 is the first-order rate constant.

where Mt/M∞ is the fraction of drug released at time t, KKP is the rate constant, and n is the release exponent.

where constant KH reflects system design variables.

Stability

The NIO/PX-YC was stored under two different storage conditions room temperature (25 ± 2 °C) and refrigerator temperatures (4 ± 2 °C) for two months to evaluate its stability. During the storage time, their physicochemical properties, including particle size, PDI, and EE%, were analyzed at certain time intervals (0, 30, and 60 days).

MTT assay

HT-29 and HFF cells were cultured in DMEM/F12 medium which was supplemented by adding 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin in an incubator having humidified atmosphere. The gas content and tempratue of incubator were 5% CO2 and 37°C respectively.

The cells were seeded into 96-well plates at a density of about 104 cells per well and incubated for 24 h. Subsequently, the culture medium was replaced by the fresh medium containing different concentrations of drug formulations (free PX-12, free YC-1, free drugs combination, NIO/PX-YC). YC-1 dissolved in cell culture safe range of DMSO (< 0.1%) due to its hydrophobic property, and PX-12 dissolved in medium because of its hydrophilicity and enough water solubility. To assess the biocompatibility of the niosomes, the cells were treated with non-loaded niosome. The untreated cells with culture medium were used as a control group. After 24, 48, and 72 h incubation, the culture medium was aspirated and replaced with MTT solution (5 mg/ml, 20 µl) and was incubated for 4 h. Incubation for HFF cells was performed for 48 and 72 h. Following incubation, the medium was removed, and 100 µl DMSO was added to dissolve the obtained formazan crystals. Finally, absorbance was measured at 540 nm using an ELISA plate reader (Multiskan MK3, Thermo Electron Corporation, USA). The viability percentage was calculated according to Eq. 8:

$$\mathrm(\mathrm)=\frac}}\mathrm100$$

(8)

Apoptosis assay

To evaluate the apoptosis/necrosis ratio, Annexin V-FITC dual staining method was used. First, the IC50concentration of free drugs and drug formulations was used to treat the cells for 24 and 48 h, then Annexin V/Propidium Iodide (PI) kit was used to differentiate apoptotic and normal cells according to the manufacturer's instructions. The cells were washed twice with cold PBS, and the pellet was re-suspended in 100 µl of 1 × Annexin binding buffer (5 × 10 5 cells/well). Next, 5μl of Annexin V- FITC and PI was added to each sample. After 15 min of incubation (25°C), the cell suspension was centrifuged, and the resulting pellet was re-suspended in 100 µL of 1 × Annexin V Binding Buffer. The cells without any treatment were considered a control group. Finally, the levels of apoptotic/necrotic cells were evaluated using flow cytometry. The samples were analyzed in three replications using the BD FACSDiva instrument and Flow Jo software (BD Biosciences company, Canada).

Cell cycle analysis

The proliferation of cells and the cell cycle process were evaluated by propidium iodide (PI) staining. Cells were seeded in the complete medium in 6-well plates at a density of about 1 × 106 cells/well and incubated for 24 h. The cells were then treated with the IC50 concentration of free drugs and drug formulations for 24 and 48 h. Following incubation, the cells were fixed with cold ethanol 70% and stored overnight at 4°C. Then, they were collected by centrifuging (300rpm, 5 min), washed with PBS, and stained with 1 ml of PI master mix (containing 40μl PI, 10μlRnase, 950μlPBS) in the dark. The cells were then incubated for 30 min at room temperature and evaluated by flow cytometry. The data were analyzed by FlowJo software.

Gene expression analysis by q RT-PCR

The cells (1 × 108 cells/well in a 6-well plate) were treated with IC50 concentration of free drugs and drug formulations for 72 h. Un treated cells were considered as a control group. Trizol reagent was used to extract total RNA according to the manufacturer's procedures and quantified with gel electrophoresis and nanodrop (Thermo Scientific, Waltham, MA, USA). Revert Aid First Strand cDNA transcription Kit (Fermentas, Vilnius, Lithuania) was used to transcript extracted total RNA to cDNA. The mixture contained reaction buffer (2µl, 2X), total RNA (5 µg), reverse transcriptase enzyme (1 µl), deoxynucleotide triphosphate mixture (1µl), random hexamer (1 µl), DEPC water (up to the final volume of 20 µl). The mixture was incubated at 25 °C (10 min), 47 °C (60 min), and 85 °C (5 min). subsequently, it was kept on ice until use. The primers sequence for target genes (HIF-1α and β-actin) is presented in Table 2. β-actin gene was used as an internal control. Finally, qRT-PCR was done by SYBR Green Master Mix (Applied Ampliqon, Danmark) using Bioneer Real-Time PCR equipment (Korea). The cDNAs, primer, and master mix cocktail were incubated according to the following temperature program: 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s and 20 s at 60 °C and 72°C at 20 secs. The results were reported as fold changes that were calculated by the ΔΔCт method.

Table 2 The sequence of primers used in PCRWestern blot analysis

Western blot analysis was performed to investigate the HIF-1α protein in HT-29 cell line in hypoxia conditions. For this purpose, 2 × 105cells were seeded in each well of 6-well plates. After 24 h, the cells were treated with IC50 concentrations, similar to the previous tests. The plate was transferred to the hypoxia incubator (1% O2, 5% CO2, 94% N2) for 24 h. Following incubation, the cells were harvested, washed with ice-cold PBS, and then scraped and centrifuged at 1500 rpm for 5 min. The harvested cells were lysed using RIPA buffer having the following reagents: Tris–HCL, SDS, Protease inhibitor cocktail EDTA, sodium deoxycholate, NaCl, and 1% Triton X-100 or NP40, then centrifuged (10,000 rpm, 10 min). The proteins in the supernatant were collected and stored at -20°C. After quantification by Bradford assay, round 10 µg of proteins were separated by SDS-PAGE and subsequently transferred to nitrocellulose membrane overnight. Blocking of the membranes was performed with 0.5% Tween-PBS and 2% low-fat dried milk. The blocked membranes were then incubated with primary antibodies against β-actin (Santa Cruz Biotechnology Inc. Dallas, USA) or HIF-1α (Santa Cruz Biotechnology Inc. Dallas, USA) for 16–18 h at 4°C followed by washing and incubation with secondary antibodies (Santa Cruz Biotechnology) mouse anti-rabbit IgG-HRP at a dilution of 1:10,000 (Santa Cruz Biotechnology, Inc, Dallas, USA), for 2h. Finally, a chemiluminescence reagent, ECL (Elabscience company, USA), was added, and the bands emerged in the dark room detection system. After the films appear, the appeared protein bands are analyzed with Image J software (USA).

Statistical analysis

Data were reported as mean ± SD, and the graphs were plotted using GraphPad Prism version 8 and Design-Expert software (Version 7.0.10.). Data were statistically analyzed using analysis of variances (ANOVA), and a p-value less than 0.05 was considered a significant difference.