Cationic Magnetoliposomes

• Separate quantities of 12 g FeCl₂ ⋅4aq and 24 g FeCl₃ ⋅6aq are dissolved in individual beakers containing 50 mL of distilled water. Afterwards, these solutions are combined in a larger 250 mL container.
• 50 mL of concentrated NH₄OH 56% (which equates to 28% NH₃) is slowly poured into the solution, coupled with prompt stirring using a rust-proof mechanical paddle. The result of this process is the creation of Fe₃O₄ cores, which, due to their instability, fall to the bottom as a dense, black sediment.
• The first stage of magnetic decantation commences by placing the beaker on a permanent magnet for an approximate duration of 15 minutes. Ensuing this, the clear liquid on the surface is discarded, while ensuring the container remains in position on the magnet.
• The sediment is cleaned using 100 mL of a solution made up of 5 mL NH₄OH_conc and 95 mL of water, and then decanted again following about 15 minutes. This phase is repeated on four subsequent occasions.
• The gel-like slurry is subsequently heated in a boiling water bath to a range of 80-90°C (temperature of the sediment, not the bath itself) and combined with 6 g of the solid-form lauric acid surfactant, with stirring accomplished via glass rod. Particle peptization results in the slurry gradually thinning and creating slight foam. Heat is maintained until foam production stops (approximately 7 minutes).
• 50 mL of water is added and the solution is centrifuged at 500 g for 7 minutes, aiding in the removal of any large clusters which may have formed. The supernatant is then gathered and preserved.

The experimental conditions detailed above yield a 114 mg Fe₃O₄/mL stock solution. Dilution is possible without causing precipitation, marking the water-adapted, lauric acid-coated iron oxide cores solution as 'dilution-insensitive' magnetic fluid.

• Phospholipid powders such as DMPC, DMPG or their mixtures, can be weighed directly in a heat-resistant sonication vial and then solubilized with a minimal volume of either chloroform or a chloroform/CH₃OH mixture. Conversely, with phospholipid stock solutions in organic solvents, the required amounts can be dispensed directly. A higher-temperature water bath boosts the solubilization process.
• The organic solvent is eliminated through evaporation within a nitrogen stream. This process results in a thin lipid film on the vial's glass wall. It is crucial to ensure total solvent removal to avoid remaining residual solvent molecules. Typically, the sonication vial is left in a desiccator under a high vacuum overnight to aid this process.
• The next step involves hydrating the deposited lipid film, and this is done in a 5 mM TES buffer with a pH of 7.0. Depending on specific experimental needs, the final phospholipid concentration generally lies between 0.1 and 20 mg/mL.
• The sonicator's transducer tip should be dipped into the suspension approximately 1 cm beneath the surface, ensuring it doesn't touch the glass vial during operation.
• Sonication takes place at 18 μm peak-to-peak power intensity (equating to approximately 75% of total power), maintained at a temperature above the phase transition temperature.
• Post-sonication, any titanium micro-debris shed from the tip is purified via centrifugation at 500 g for 10 minutes, carried out above the phase transition temperature.
• Where required, the exact phospholipid concentration can be determined utilizing the phosphate determination process.

• Commencing with a sonication process in 15 mL 5 mM TES buffer, 200 mg DMPC (alternatively DMPG or DMPC-DMPG mixtures) is utilized.
• A total of 0.35 ml of laurate-stabilized magnetic fluid is incorporated, followed by a mixture. The solution is then relocated into dialysis tubes.
• The subsequent procedure involves dialysis against 5 mM TES buffer, continuing for a minimum of 3 days accompanied by recurrent buffer alterations (minimum of 15 occurrences), surpassing the transition temperature of the applied phospholipids. During this, laurate molecules are progressively eliminated from the iron oxide surface while simultaneously being replaced by phospholipid molecules. Ultimately, a clear solution devoid of any precipitate should be achieved.

• Magnetic filter devices are placed in the 3 mm-gap present between the two electromagnet pole faces.

  Fig 1 Scheme of the consecutive steps in the preparation of MLs by HGM (Weissig, 2010)

• Each magnetic filter receives 0.75 mL of the selected sample, fed at a steady rate of 12 mL/h.
• Removing iron oxide-free vesicles is essential, achieved by washing the retentate with 0.75 mL of TES buffer, which prevents vesicle accumulation between filter wires.
• The peristaltic pump and the magnetic field are switched off.
• By deploying a high-speed buffer stream (0.5 L/h), the retentate can be flushed from the filter, thereby collecting the sample. Slight squeezing of tubing containing the magnetic filter may facilitate the release of MLs. An estimated retrieval rate of Fe₃O₄ measuring between 95–99% is typically achieved. The ML's stability is considerably enhanced when kept above the gel-to-liquid crystalline phase transition temperature of the phospholipid(s) involved.

In the scenario of handling a 14 nm-diameter iron oxide core, enveloped by an unbroken phospholipid bilayer, the anticipated phospholipid /Fe₃O₄ (mmol/g) ratio lies between 0.7 and 0.8. Detailed below are methods for assessing phosphate and iron content in ML samples:

• Phospholipid determination is done by the phosphomolybdenum blue method. All measurements (calibration+unknown samples) are done in triplicate. The following protocol is followed:
  (a) Calibration solutions are initiated from a base substance consisting of 0.700 μmol phosphate/mL. This solution is then diluted in multiples of 2.5, 5, 7.5 and 10.
  (b) 100 μL of HClO₄ is combined with both the calibration solutions and the ML samples and subjected to heat for approximately 45 minutes at a range of 180–200°C. At this juncture, white fumes swirl noticeably around within the individual tubes, signaling the ongoing chemical digestion process.
  (c) The mixtures, once cooled to room temperature, are treated with 1 mL of a devised reacting agent prepared from a sodium molybdate-infused base solution. The preparation procedure involves numerous steps: (i) mixing 400 mg of NH₂NH₂⋅2HCl dissolved in 14 mL HCl₄N with a solution of 10 g Na₂MoO₄ in 60 mL HCl4N, (ii) heating the concoction in a bath of 60°C for 20 minutes, and (iii) after cooling, Easing in 14 mL of concentrated sulphuric acid while persistent cooling and hard mixing to register a balanced reaction, (iv) adjusting the overall volume to 100 mL with water. The working reagent is prepared by combining 5.5 mL of the aforementioned base solution with 26 mL H₂SO₄ 1N, then watering it down with distilled water to achieve a final volume of 100 mL.
  (d)The reaction stage is set by pairing 1 mL of the working solution with the processed (calibration+unknown) samples in a boiling water bath for a quarter of an hour. To finish, each tube is treated with 1.5 mL H₂SO₄ 1N after it cools, and the absorption is measured at 820 nm. If the solution's absorption is too high, it's further diluted with H₂SO₄ 1N.
• Iron determination
  The method is administered via the complex formation method of Fe³⁺ with Tiron, presenting as a red color. Measurement is attained spectrophotometrically at 480 nm, employing standard 3 mL plastic tubes. Each evaluation is replicated three times for consistency.
  (a) The initial phase of this process involves developing a calibration curve. This starts with a stock solution that comprises 1 mg Fe³⁺/mL. HCl_conc (37%) and HNO_3conc (65%), along with distilled water, is used for dilution. The dilution scheme is explained in the next section. The final concentration levels of the different dilutions are 0; 10; 20; 30; 50; 70, 100, and 150 μg Fe/mL.
  (b) For both calibration and other diluted samples, 0.6 mL of a mixture is added containing 100 μL Tiron 0.25M and 0.5 mL KOH 4N to 0.5 mL of the solution. Afterwards, 1mL of 0.2M phosphate buffer at pH 9.5 is added. The appearance of red color (A^{480 nm}) is immediate, achieving maximum saturation in 15 minutes, remaining stable for several hours.
  (c) By multiplying the iron concentration of the sample, as deduced from the calibration curve, (expressed in μg Fe/ mL) by 1.38 the Fe₃O₄ concentration (μg/mL) in the ML samples is calculated.

Production of cationic MLs containing, for example, 3.33% DSTAP, proceeds according to the following consecutive steps:

• First, neutral DMPC-MLs are processed according to prior protocols. The DMPC concentration is 10.22 μmol/mL, the Fe₃O₄ is 12.07 mg/mL, the ratio of mmol DMPC/g Fe₃O₄ is 0.84, and the volume used is 15 mL.
• Following this, a zwitterionic DMPC-ML suspension of 15 mL is warmed at 37°C alongside an equally measured volume of DMPC/DSTAP (90/10 molar ratio) vesicles. These vesicles are crafted, using 93.54 mg DMPC and 10.78 mg DSTAP, through a process outlined in the Phospholipid Vesicles step.
• Upon a day's passage, the mixed solution undergoes a second magnetophoresis cycle. Following this, the cationic MLs are retrieved, with a phospholipid/magnetite ratio of 0.75 mmol/g.

Fig 1 Production of cationic MLs occurs in a two-step procedure (Weissig, 2010)