3.1. Materials
A mixture of olive oil 64% (v/v), hexadecane 30% (v/v) and hexane 6% (v/v) is used as the continuous phase with 2wt% Span80 surfactant which is added to avoid droplet merging in the downstream channel.
Both of the dispersed phase fluids are composed of the monomer Poly(ethylene glycol) diacrylate (PEGDA 575) 80.6% (v/v), photo initiator -hydroxy-2-methylpropiophe- (HMP) 13% (v/v) and Tween 20 3.2% (v/v). One of the dispersed phases contains ferrofluid with 0.25g/mL SPIONs suspended in deionized water 3.2% (v/v) and the other contains titanium dioxide nanoparticles (sigma Aldrich-914576) 0.2% (v/v).
Poly(ethylene glycol) diacrylate (PEGDA) average molecular weight (Mn) 575, n-hexane, hexadecane Reagent Plus 99%, Tween 20, Span 80 surfactant, and 2-hydroxy-2-methylpropiophe-(HMP), TiO2 nanoparticles were purchased from Sigma-Aldrich. A polydimethylsiloxane (PDMS) SYLGARD 184 silicone elastomer kit was purchased from Dow Corning GmbH, Germany. Photoresist SU-8 2050 and the SU-8 developer were purchased from Microresist Technology Germany.
3.2. Methods
The microfluidic chip was created using the traditional soft lithography method. Initially, an SU-8 master mold was produced through photolithography and subsequently developed with a resist developer. A mixture of PDMS base and curing agent was prepared in a 10:1 ratio. This blend was then poured onto the SU-8 mold and subjected to vacuum degassing to eliminate air bubbles. The mixture was cured in an 80°C oven for 2 hours. After curing, the PDMS layer was detached from the SU-8 mold, and inlet and outlet holes were created using a 2.0 mm microfluidic chip hole puncher. To seal the channels, the PDMS was bonded to another flat PDMS surface using oxygen plasma treatment for 60 seconds. The finished devices were placed in an 80°C oven overnight to restore the hydrophobic properties of the channel walls.
The experimental setup consisted of an optical microscope, two syringe pumps, a microfluidic device, and an ultraviolet source. The Ultraviolet radiation exposure setup consisted of a UV lamp and an aperture placed on top of the device to expose only the specific parts of the device to UV radiation. After the particles were collected in a centrifuge tube, excess oil was removed, and the particles were washed with pure ethanol (to remove oil) multiple times (at least five times) and DI water (to remove uncured monomer) alternately at a centrifuge speed of 500rpm. The particles were transferred on top of a SEM stub covered with conducting tape using a micropipette. The sample was then coated with around 5nm of gold and then observed under the SEM.
3.3. In vitro Characterizations of the TiO2/Fe3O4 Janus Particles
L-929 fibroblasts (Şap Enstitüsü, 92123004) were used to assess biological characterizations of the Janus particles, in accordance with ISO 10993 standards. The cells were cultured in DMEM (Dulbecco's Modified Eagle F-12) medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under standard cell culture conditions (37°C, 5% CO2). Simultaneously, TiO2/Fe3O4 Janus particles were sterilized in 70% ethanol and UV-C irradiation each for 1 h. Then, these particles were weighed and incubated in the culture medium at 37°C for 72 hours to obtain extracts having 1 and 0.01 mg/ml concentrations. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase (LDH), and total protein assays, as well as morphological analysis were performed.
To assess cellular proliferation, fibroblasts were seeded in 96-well plates at a density of 10,000 cells/well and cultured under standard conditions for 24 h. At the end of 24 h, the culture medium was replaced with the Janus particle extracts (0.01 and 1 mg/ml) and fresh culture medium as the control group. Experiments were performed for 5 days, and the media were changed every 2 days. At the end of the 1st, 3rd, and 5th days, the media was removed, and the cells were rinsed with 1xPBS. MTT solution was added to the cells and incubated for 4 h (37°C, 5% CO2). Then, formazan crystals formed by the reduction of MTT by metabolically active cells were dissolved with 37% isopropyl alcohol containing 0.77% HCl, and optical density (OD) values were measured at 570 nm. Standard curves were drawn to correlate the optical density values to cellular proliferation. The experiments were repeated three times.
The cytotoxic of the Janus particles was assessed using an LDH assay kit. For these experiments, cells were seeded in 96-well plates at a density of 10,000 cells/well and interacted with the regular culture medium for 24 h. Then, the culture medium was replaced with the Janus particle extracts (0.01 and 1 mg/ml), and fresh culture medium for the control group. LDH measurements were performed at 24, 48, and 72 h of culture. For low control measurements, the amount of LDH released from the healthy cells using standard culture medium was measured. For high control measurements, standard media were replaced with media containing 1% Triton-X. Absorbance measurements were made at 492 nm following the manufacturers’ instruction (Cytotoxicity Detection Kit (LDH)-11644793001 Roche). Experiments were repeated three times.
Total protein measurements indirectly assessed the Janus particles’ effect on cellular activity. Similar to MTT and LDH experiments, cells were seeded in 96-well plates using a standard medium at a density of 5,000 cells/well, and cells were allowed to adhere and proliferate for 24 h. Then, the culture medium was replaced with the Janus particle extracts (0.01 and 1 mg/ml), and fresh culture medium for the control group. At the end of the 3rd day, the cells were rinsed with 1xPBS. Total protein measurements were carried out using the Bradford kit (Ozbiosciences) according to the manufacturers’ instruction. The experiments were repeated three times.
To examine the effect of Janus particles on cellular morphology, cells were seeded in 96-well plates at a density of 2,000 cells/well and cultured for 24 h. Then, the cells were exposed to the Janus particle extracts at 1 mg/ml concentration for 3 days. At the end of the 3rd day, optical microscope images of the cells were captured. Then, the media was removed, and the cells were rinsed with 1xPBS, followed by fixing them with 4% paraformaldehyde solution for 20 min. Afterward, they were permeabilized with 1xPBS containing 0.1% Triton-X for 10 min. Then, the cells were stained with rhodamine phalloidin (stains f-actin filaments) and DAPI (stains cell nuclei), respectively. Stained cells were examined under confocal microscope (Leica TCS SP8 DMI8) and cellular spreading was quantified using ImageJ software.