Dual centrifuges that have successfully been used for the preparation of nanoparticles using horizontally orientated vials are the SpeedMixer^® DAC 150 (DAC 150, Hauschild GmbH & Co. KG, Hamm, Germany), the ZentriMix 380 R (ZM 380R, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany), and the DeltaVita 1 (DV1, Erich Netzsch GmbH & Co. Holding KG, Selb, Germany; identical in construction to ZentriMix 380 R).

The SpeedMixer^® DAC 150 (DAC 150) was initially designed for very short runs of a few seconds to minutes to mix highly viscous compounds. Thus, this device is equipped with a strong motor that requires a V-belt to couple the main and secondary rotation. Using a V-belt protects the mechanics from slippage when the dual asymmetric centrifuge starts. Despite its maximum run time being 5 min, the DAC 150 can be used to prepare nanoparticles ^[1] if the necessary run times (typically about 30 min) are reached by multiple short runs. However, standard vial adapters are only available for vertical vial orientation, which is optimal for fast mixing of highly viscous materials such as print inks, silicones, or two-component teeth filling materials ^[2]. Adapters for the horizontal orientation of small and lengthy vials are not commercially available for the DAC 150 and are all custom made for patent reasons. Since the diameter of the rotational disk to accommodate the sample vial adapter is rather small, only a few vials (typically two) can be processed simultaneously.

A special feature of the DAC 150 is that its rotor is asymmetrically constructed with only one sample holder (Figure 3). Thus, smooth running of this rotor is ensured by a fixed counterweight; as a result, the payload is also fixed to a certain weight. If very small sample quantities are processed, the necessary weight of the payload must be reached by a special (heavy) sample holder. Based on using the DAC 150 with its special asymmetric rotor for the preparation of nanoparticles, in a few publications, the DC-process is named “dual asymmetric centrifugation (DAC)”. In contrast to that, the other dual centrifuges (DV1 and ZM 380R) have symmetric dual rotors with two symmetric sample holders. Therefore, the term “dual centrifugation (DC)” refers to the fact that there are two types of rotational axes within one rotor—one axis for the main rotation of the whole DC-rotor, and a second set of axes for turning the sample vials (compare Figure 3). 

The dual centrifuges DV1 and ZM 380R are designed from the outset to produce nanoparticles, which typically requires longer run times than a few minutes. Thus, the DC-processing time is freely adjustable. Furthermore, the coupling of the main and secondary rotations is established by gear wheels, and the motor control allows a soft start to protect the mechanics. To dissipate the heat inevitably generated during DC-homogenization or -nanomilling, DV1 and ZM 380R are equipped with a powerful cooling unit. Furthermore, dual rotors of DV1 and ZM 380R have rather large rotational disks to accommodate a high number of the typically used 2 mL PP-screw cap vials (maximum 40 vial per run, arranged on 2 levels with 20 vials each), which is advantageous for formulation screening approaches, for example. Due to the frequent change in the direction of the high centrifugal acceleration during DC, all types of vials require special adapters to adequately fix the vials in place (Figure 3E,F).

However, with otherwise equally fast secondary rotation (about 850 rpm at max. speed of main rotation), the centrifugal acceleration of the ZM 380R is about 15% higher compared to the DAC 150, which is due to the larger rotor diameter that allow faster acceleration of the sample materials. Sample materials with higher viscosities thus have a better chance of reaching the end of the vials before the vial has turned again in the opposite direction. Since a significant part of the homogenization or milling processes take place at the end of the sample vials due to collisions (compare Figure 2, impact zones), reaching the end of the vials results in more effective homogenization or milling processes.

Homogenization or nanomilling is typically supported by spherical ceramic beads with a high density (ZrO-beads, above 6.0 kg/dm³) since heavy beads accumulate very high kinetic energies during acceleration and thus introduce more energy into the sample material. In the first studies, liposome preparation was performed by DC-homogenization using much lighter glass beads (approx. 2.23 kg/dm³ for borosilicate glass), which were used with satisfactory results on first glance ^[1]. However, it turned out that the numerous collisions between the glass beads resulted in glass wear, which, due to the basic surface properties of the freshly generated glass (nano)particles (containing potassium-oxide), caused degradation of the phosphatidylcholine head groups. The proposed underlying chemical reaction might be the well-known Hoffmann elimination, which started with the removal of a proton in β-position to the quaternary amino group of phosphatidylcholine-species by the basic glass (nano)particles, followed by a rearrangement of bonds. The final reaction product is trimethylamine, which can easily be detected by its fishy smell, even in small quantities ^[2]. Thus, the use of the much denser and harder ceramic beads was established and almost all studies cited in this research used those beads.

Commonly used ceramic beads are made from zirconium-oxide, which are stabilized with yttrium. An example are SiLibeads type ZY-P Pharma purchased from Sigmund Lindner GmbH (Warmensteinach Germany). Their high degree of roundness (≥0.96 (width to length ratio (x_min/x_max)) and polished surface avoids the generation of wear from the commonly used plastic vials (see below). The high hardness (microhardness: ≥1300 HV10) contributes to the avoidance of zirconia wear during bead–bead collisions as well. In a DC-nanomilling study, a zirconium amount of only 3.4 ppm was found in the resulting suspension after 90 min milling time, which was shown to be comparable with wear generated from ZrO-beads during agitator ball milling in pharmaceutical production ^[3].

For DC-homogenization, typical bead diameters range from 0.2 to 1.6 mm. However, the optimal bead size and number (bigger beads) or amounts (smaller beads) vary depending on the specific lipid blend or nanocrystal dispersion and the vial type. Smaller and thus lighter beads introduce less energy during bead–bead collisions but increase the number of bead–bead collisions. Therefore, the bead size and quantity must be optimized for each DC-process.

One important aspect of successful dual centrifugation is that the homogenization/milling vials have to be made of a slightly elastic plastic material. In most cases, disposable 2 mL screw cap polypropylene (PP) vials are in use in combination with ZrO-beads. Another important aspect of choosing the right vials for DC concerns their tightness and stability. The vials must be very tightly sealed and stable enough since during DC, the sample material and the beads are pushed with high impact and at high frequency against the lid and the bottom (compare Figure 2). Therefore, it is important to either use the exact vials described in the existing publications or carefully check the tightness of new vials that have not yet been used in test runs. As an alternative, 10 mL PP-injection vials at the same length as the screw cap vials can be used ^[2].

Considering wears from the surface of the plastic (PP) vials has not been observed or reported so far when using 2 mL screw cap vials in combination with ZrO-beads of high quality (see above). This could be explained by the high degree of roundness in combination with the smooth, polished surface of the ZrO-beads, which, even when in contact with the plastic walls of the vials, are assumed to not be able to effectively scratch the plastic material. In the case of liposome preparation by DC, the danger of scraping of the plastic walls is further reduced by the lubrication effect of the highly viscous and concentrated lipid blends. Thus far, systematic studies focusing on the generation of wear (PP or ZrO) during the preparation of liposomes within a DC do not exist.