Construction of AC/DC magnetic syringe device for stimulated drug release, injection and ejection of nanocarriers and testing cytotoxicity in vitro

Iron nanoparticles are used as a targeted drug delivery system. The nanocarrier itself can be genotoxic, trigger oxidative stress or cell death. Therefore, we developed an AC/DC magnetic syringe for injecting, stimulating drug release and safe removing of the nanocarrier. Alongside we optimized the method for nanoparticles’ drug release kinetics and testing cytotoxicity in vitro.• This paper presents detailed instructions for construction of AC/DC magnetic syringe device for stimulated drug release, injection and ejection of magnetic nanoparticles; nanoparticles preparation; adsorbing methylene blue on nanoparticles; determination of drug release kinetics from nanocarriers on the example of methylene blue• Gomori´s Prussian blue reaction for differentiated SH-SY5Y human neuroblastoma cell line; MTT viability assay optimized for differentiated SH-SY5Y human neuroblastoma cell line and antioxidant enzymes activities assay and lipid peroxidation methods are optimized for cell analyses cell cultivation for nanoparticles cytotoxicity testing in vitro.• Those protocols are the first step toward further testing the effect of nanoparticles in vivo, on brain tissue.


Specifications
The syringe should be made preferably from permalloy to allow maximal induction and transfer of electromagnetic fields to the needle. The composition of permalloy varies but it can be described as an alloy containing 80% of nickel and 20% of iron what makes it highly magnetic [ 1 , 2 ]. Other materials suitable for this are supermalloy or soft iron. Since the majority of commercially available medical steel used in syringe production is steel 304 or steel 316, both austenitic and paramagnetic, the syringe has to be specially made [ 3 , 4 ]. Syringe body should be produced by turning 150 mm long, 5-8 mm in diameter wide permalloy rod on a lathe machine. The syringe length should be 100 mm, with an inner diameter of 3.25 mm and an outer diameter of 6.00 mm. The tip of the syringe must conform to the Luer tip (LT) specifications. Tip outer diameter can range from 3.924 -4.026 mm according to ISO 594-1 Luer standard. Tip inner diameter should be 2 mm. The piston should be turned on a lathe machine out of medical-grade PTFE with a final diameter of 3.24 mm. PTFE is a good material of choice because it is inert to most chemicals can and it has a hydrophobic property which makes it very good in creating seal if crafted to tight dimensional specifications [5] . Acceptable tolerances for both syringe body and piston should not exceed ±0.005 mm. The syringe and the needle are to be made from permalloy. This design is intended for use in ' in vitro' systems. Additional measures must be taken for use in ' in vivo' systems. For example, the inner chamber of the syringe could be immerse coated in a thin layer of medical-grade silicone to prevent an allergic reaction due to the high nickel concentration in permalloy [6] .

Needle preparation
The needle has to be specially ordered from manufacturers. It must have Luer type metal hub made from permalloy to properly fit onto the syringe. The needle length is 35 mm and the thickness of the needle is G26. Before use, the needle should be sterilized at 121 °C for 20 min.
1. To make the coil, use 23 AWG varnish insulated copper wire. The necessary length is around 4 m. 2. On the syringe body measure 6.5 cm length for the coil and mark the length on it. 3. Measure 15 cm of wire and bend it 90 °. 4. Place bent wire at one of the marked ends. (Note: Shorter part of wire must point outwards since it will become coil terminal.) 5. Start coiling the longer part of the wire tightly without spacing between turns. 6. When the first layer is finished, use a piece of Kapton tape to temporary fix the wire in position before start winding the second layer. 7. In the end, measure 15 cm of wire for the terminal and fix it temporarily. 8. Cover the entire coil with 2 layers of Kapton tape. 9. Slide the prepared coil from a mould and set it aside. 10. Once everything is prepared, connect the coil terminals to (silicone insulated) wire by soldering.
2 cm of terminals of coil wire must be burned off to remove the varnish and to allow proper soldering and securely isolate naked wires with shrink-wrap isolation sleeves. The wire must have on the other end safety-type banana jacks.
Assembly and testing 1. Slide the coil onto the syringe body. 2. Fit the needle onto the syringe. 3. Connect everything into the laboratory power supply with variable voltage, current and frequency. 4. Let current through the coil and test magnetization of the needle. 5. Move the coil up and down the syringe body until the best inductance is achieved. 6. Fix the coil in the place with fast curing epoxy resin. Apply resin in a dot-like manner. 2 dots per each side of the coil are sufficient. 7. After use, a syringe with a coil on it should be sterilized in an autoclave at 105 °C for a 20-min duration and dried in a laboratory oven at 50 °C for the 2-h duration.

Material
Note: Materials are listed in the order that they appear in procedure steps.

Procedure
The preparation of nanoparticles is described by Mustapi ć et. All in a previous publication [7] . A slight deviation from the synthesis protocol is to be made to achieve the highest gains.
1. Prepare a detergent solution by mixing 1.5 g of the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) and 1.5 g of the anionic surfactant sodium dodecyl sulphate (SDS) with 30 ml of water. 2. In detergent solution add 60 ml of cyclohexane place on a magnetic stirrer until detergents are dissolved remove from stirrer and precede with degassing in N 2 atmosphere by gently blowing a nitrogen gas through the solution.
3. Gas purging equipment is assembled by drilling two holes in a rubber stopper and pushing two metal or glass 4 mm wide tubes through the holes and placing everything on a round bottom flask. One tube is longer and should be immersed in the solution, the second tube is short and bent 180 °on the outer end. 4. Gas is blown through a long tube in the solution and released through short out from the flask. 5. During the period of degassing of surfactants prepare 20 ml of 0.0015 mol/L of iron (II) chloride solution in type 1 water under argon or another inert gas atmosphere. (Note: The inert gas atmosphere is to prevent further oxidation of iron (II) chloride). 6. Prepare 20 ml of 0.003 mol/L of iron (III) chloride solution in type 1 water. (Note: The inert gas atmosphere is not required for iron (III) chloride because it is in the highest oxidation state already.) 7. Stir both solutions on a magnetic stirrer for half an hour. 8. In an inert gas atmosphere in a separate flask mix prepared iron chloride solutions and start adding into it 5 ml of 30% ammonia and do so for 1 min. 9. After 1 min of a reaction pour the detergent solution in the flask and gently mix by hand vortexing. 10. After 5 min reaction has finished and formed nanoparticles can be collected from the bottom of the flask by using an external permanent magnet. 11. Wash NP several times -alternatively first with type 1 water and then with an acetone, the washing procedure must end with an acetone. (Note: in this step centrifuge can be used, minimum 5 min at 10 0 0 0 g). 12. Dry NP overnight in a laboratory oven at 60 °C. 13. Collect black powder and place it in a laboratory oven at 250 °C in 2-h duration to remove detergent under an inert atmosphere.

Material
Note: Materials are listed in the order that they appear in procedure steps.

Procedure
In this protocol, we have chosen to adsorb methylene blue on the nanoparticles, but any active and easily measurable substance can be used. When using other substances expect protocol adjustment to be made. 6. During pressure, release nitrogen or other inert gas to devoid solution out of residual oxygen.
(Note: It serves to prevent additional oxidations due to prolonged exposure to water.) 7. Take out the flask and cap it with a corresponding ground glass cap. 8. Wrap cap and neck of the flask with laboratory paraffin film. 9. Leave NP in degassed MB solution overnight to saturate at room temperature. 10. The next day gathers nanoparticles on one spot by using a rare earth magnet. 11. Wash NP 4 times with type 1 grade water for 10 min each, or until water runs clear. Use at least 50 ml of water per wash. (Note: This step serves to remove any loosely bound MB.) 12. Remove water and freeze-dry prepared nanoparticles. (Note: If freeze dry apparatus is not available, at the bottom of 50 ml Falcon tube place 50 g of anhydrous calcium sulphate or silica gel. On top of it place a wad of cotton wool 1 cm thick. Place open Eppendorf tube into the cotton wool and fix it firmly. Close the Falcon tube and place everything in -80 °C freezer for 7 days to completely dry NP) [7] .

Material
Note: Materials are listed in the order that they appear in procedure steps.  Sample every 10 min for the first hour and once after two hours upon needle immersing. (Note: Same procedure can be utilized for different current settings. In the assessment of the methylene release behaviour, the cumulative amount of released MB was calculated, and the percentage of MB released was plotted vs. time).

Material
Note: Materials are listed in the order that they appear in procedure steps

Cell cultivation
Cells were grown at 37 °C with 5% CO 2 in a cell culture incubator.
To prepare a complete growth medium in 450 ml of DMEM/F12 following solutions are added: Ü 1% of 100x non-essential amino acids Ü 1% of 100x Penicillin / Streptomycin solution Ü 0.5% of 200 mM solution of L-alanine-L-glutamine Ü 50 ml of FBS. (Notes: Before adding, FBS is filtered through a sterile 0.2μm PES filter. This process removes any albumin polymers presented as cloudiness created by freezing, and we observed that cells grow better if FBS is filtered [9] ; all solutions used for cell growth and manipulation are preheated to 37 °C to avoid a thermal shock) 1. 9 ml of growth medium is transferred to 15 ml Falcon tube. 2. Tube with frozen cells is defrosted by partially immersing it for 2 min in the water bath heated up to 37 °C. 3. The defrosted cell suspension is transferred by sterile Pasteur pipette into a growth medium in the Falcon tube. 4. Close the Falcon tube and holding it between thumb and index finger rotate gently twice to mix cells with growth medium. 5. The cell suspension is centrifuged at room temperature at 130 g for 5 min. 6. The supernatant is discarded, and cells are resuspended in 5 ml of fresh complete growth medium. 7. Cells are counted in the Neubauer chamber [10] . 8. The final concentration of 10 0,0 0 0 cells/ml is prepared, and 7 ml of cell suspension is transferred to each 25 cm 2 flask. 9. The next day growth medium is replaced with the same volume of a fresh one. 10. Cells are grown to 85% confluency. 11. After reaching the sufficiently confluent state, the medium is removed and cells are trypsinized with 2.5 ml of preheated trypsin solution for 3 min, gently shaking until cells are visibly detached from the bottom of the flask. 12. A fresh growth medium (2.5 ml) is added to trypsin/cell suspension to stop the enzyme and everything is transferred to a 15 ml falcon tube centrifuged at room temperature at 130 g for 5 min. 13. The supernatant is discarded, and cells are resuspended in 10 ml of fresh complete growth medium. 14. Cells are counted in the Neubauer chamber. 15. The final concentration of 250 0 0 0 cells/ml is prepared, and 12 ml of cell suspension is transferred to each 100 mm Petry dish. 16. Cells are grown to 85% confluency and then harvested for further experiments.
Notes: Since the neuroblastoma cells are adherent type, seeding concentration for experiments are calculated by growth surface area; we found that the optimal concentration is 50 0 0 0 cells per square centimetre. Areas per well are the following: 2 cm 2 per well of 24 well plate; 9.6 cm 2 per well of 6 well plates. Growth medium volumes per well are the following: 500 μL for each well of 24 well plates; 20 0 0 μL for each well of 6 well plates. The final concentration of cells per ml the medium is the following: 20 0,0 0 0 cells/ml per well of 24 well plates, 240,0 0 0 cells/ml per well of 6 well plates.

Preparation of coverslips
1. Mix 1 part of 30% hydrogen peroxide with 9 parts of concentrated (96%) sulfuric acid. For 100 coverslips, 90 ml of sulfuric acid is to be mixed with 10 ml of hydrogen peroxide. The reaction is exothermic and releases elemental oxygen thus following precautions are to be made: Ü use thermic shock-resistant glassware (Duran or Pyrex glass). Ü perform mixing under a fume hood in a 300 ml wide throat Erlenmeyer flask made of Duran or Pyrex glass. Ü always use nitrile glows, latex can be easily burned through by this solution. 2. Use gentle swirl motion to mix two solutions and wait for 10 min to settle. 3. In the second 300 ml wide throat Erlenmeyer flask place 100 coverslips (15 × 15 mm square, or 12 mm round) and pour into a mixture of sulfuric acid and hydrogen peroxide. Note that the choice of coverslip's size depends on the size of the well.

Collagen type 1 growth surface coating
Collagen is applied in final concentrations of 5.5μg/cm 2 . Different volumes of diluted collagen are used for different multiwell plates to speed up the drying: 250 μL for each well of 24 well plates; 750 μL for each well of 6 well plates. Notes: Protocol is the same if coating coverslips for immunocytochemistry; before pipetting collagen solution, coverslips must be placed in each well with sterile tweezers; surface area remains unchanged since properly cleaned coverslips strongly adhere to the bottom of the well.

Cell differentiation and general experimental plan
Ü Day 0 -cells are seeded in collagenated wells and placed in an incubator. Ü Day 1 -growth medium is replaced with fresh one containing 10 μM of All-trans retinoic acid (ATRA). Ü Day 3 -growth medium is replaced with fresh one containing 10 μM of ATRA. Ü Day 7 -growth medium is replaced with fresh one containing 10 μM of ATRA. Ü Day 9 -growth medium is replaced with a fresh one with ATRA and without FBS.
Notes: Removal of FBS is necessary to remove the effect of cytokines in it. After differentiation cells change morphology. The presence of large neurite growth ( Fig. 1 .) is usually an indication of proper successful differentiation.
Ü Day 10 -growth medium is replaced with fresh one without FBS with added iron nanoparticles (see 5.2.5. chapter). Ü Day 11 -end of the experiment (in the case of 24 h incubation) and go on with further experimental procedures.

Iron nanoparticle solution preparation
A stock solution of 10 mg/ml of iron nanoparticles (INP) is prepared in a cell growth medium without FBS.  13. Dehydrate in absolute ethanol, clear in xylene and mount on a microscope slide in resinous mounting media. 14. Cover with a coverslip. (Note: Positive reaction is presented as blue staining within cells ( Fig. 2 ).)

Material
Note: Materials are listed in the order that they appear in procedure steps.

Procedure
Nanoparticles are opaque and diffract light which presents a problem for any photometric reading. Since MTT assay is based on absorbance reading we had to modify the protocol [ 12 , 13 ]. Before viability test, an MTT stock solution must be prepared.
(Note: this is enough for a total of 400 reactions performed in 24 well plates.) 2. Dissolve the powder in 20 ml of 1 × PBS.
(Notes: to speed up the process using an ultrasonic homogenizer (70W of power, 15 s duration of the continuous pulse; falcon tube must be placed in an ice bath to keep solution cold). 3. Sterilize the solution by filtering it through a 0.2 μm syringe PES filter. 4. Make 1.5 ml aliquots and freeze at -20 °C.

Procedure
Preparing cell homogenates 1. Transferee tubes with cells (see 5.2.1.) stored in a 1.5 ml Eppendorf tube to ice bloc or in the box filled with ice. (Note: Use at least 4 million cells per group/sample cells that are just collected from growth medium or previously-stored at -80 °C.) 2. Add 500 μL of ice-cold 100 mM phosphate buffer (pH 7.0) containing 1 mM EDTA in each Eppendorf tube containing cells. 3. Vortex each Eppendorf tube for 3-5 min. 4. Use tissue grinder pestle specially designed to match microtubes and homogenize cells with 20 up and down repeated motion. (Note: There should not be any pellet in the tube after homogenization. After this step cells could be also stored at -80 °C and analyzed later.)

Spectrophotometric analyses of antioxidant enzymes activities
The absorbance of all enzyme activity assays was recorded using a UV-Vis spectrophotometer. Total soluble protein concentration in cell samples used for enzymes activities calculation was measured following the protocol described by Bradford (1976), using bovine serum albumin as a standard.