Freezone 4.5 freeze dry system

Micro particles were fabricated using a single emulsion solvent method (oil into water) described previously.^{18 (link)} The PLA based polymer with 4HTB attachment (Mw calculated: 9930 g mol^–1, 200 mg) was dissolved in of dichloromethane (DCM)(1.5mL) to form the oil phase. This solution was then added to a water phase consisting of 2.5% poly(vinyl alcohol)(8 mL) (PVA; Mowiol® 8–88; MW ~67,000; Sigma-Aldrich, St. Louis, MO) solution (w/v) dissolved in water and sonicated with a sonic dismembrator ultrasonic processor (Fisher Scientific, Pittsburgh, PA) at 40% amplitude for 30 s. The emulsified solution was then added to an external phase consisting of 2.5% PVA (w/v)(22 mL) in water. The emulsion was then stirred for 1.5–2.0 h in a fume hood to allow DCM to evaporate. The microparticle suspension was centrifuged at 657*g for 5 min to separate the larger particles that pelleted out. The supernatant was collected and then centrifuged at 8873*g for 5 min, washed twice with nanopure water, resuspended in water (5 mL), and lyophilized using a FreeZone 4.5 freeze dry system (Labconco, Kansas City, MO).

PCL and/or CUR drug-loaded mixed micelles were prepared by the thin film hydration method. PCL (1 mg/ml in 0.1% acetic acid-methanol solution) and/or CUR (2 mg/ml in 0.1% acetic acid-methanol solution) at various weight % of the polymer were added to PEG₂₀₀₀–PE and vitamin E (89:11 molar ratio) solution in chloroform. The organic solvents were removed by the rotary evaporation and a thin film of drug/micelle-forming material mixture was formed. This film was further dried overnight in freeze-dryer to remove any residuals of organic solvents (Freezone 4.5 Freeze Dry System, Labconco, Kansas City, MO). Drug-loaded mixed micelles were formed by resuspending the film in phosphate buffered saline (PBS) pH 7.4, to give the final concentration of micelle forming materials of 5 mM in all formulations. Excess non-incorporated drugs were separated by filtration through a 0.2 µm syringe filter before characterization. Micelle size was measured by the photon correlation spectroscopy (PCS) using a N4 Plus Submicron Particle System (Coulter Corporation, Miami, FL, USA). Zeta potential values of the micelles were determined by using Zeta Plus Particle Analyzer (Brookhaven Instruments Corp, Santa Barbara, CA). Critical micelle concentration (CMC) of the micelles was estimated by standard Pyrene method (Zhao et al., 1990 ) as described in (Sawant et al., 2008 (link)).

PCL and/or CUR drug-loaded mixed micelles were prepared by the thin film hydration method. Various weight % of PCL (1 mg/ml in 0.1% acetic acid methanol solution) and/or CUR (2 mg/ml in 0.1% acetic acid methanol solution) were added a to PEG₂₀₀₀-DSPE and vitamin E (89:11 molar ratio) solution in chloroform. A 5 mM concentration of micelle forming material was used in all experiments. The organic solvents were removed by the rotary evaporation to form a thin film of drug/micelle-forming material mixture. This film was further dried under high vacuum (2 × 10⁻³ mBar) for at least 12 hours to remove any remaining organic solvents (Freezone 4.5 Freeze Dry System Labconco, Kansas City, MO). Drug-loaded mixed micelles were formed by resuspending the film in phosphate buffered saline (PBS) pH 7.4. The mixture was incubated in water bath at 40°C for 10 min and then vortexed for at least 5 minutes to insure proper resuspension of the film. Excess non-incorporated drugs were separated by centrifugation (13,500 g) for 5 minutes followed by filtration through a 0.2 µm syringe filter (Nalgene, Rochester, NY) to remove any non-incorporated drug that is still present in the solution and to sterilize the solution before in vitro or in vivo use.

Approximately 14 g of frozen liver was cut into small pieces (~1 cm³). The entire quadricep muscle was removed from the femur bone while partially frozen, and cut into small pieces. For dissection of cortex and medulla, kidneys were kept partially frozen on a clean stainless steel tray placed on ice. All instruments used to separate renal cortex and medulla (scalpels and trays) were also kept at −20 °C before use. All tissues were placed into individual pre-weighed Whirl-Pak^™ bags and lyophilized for 72 hours (Labconco FreeZone 4.5^™ Freeze Dry System). Tissue mass was determined before and after lyophilization.